Superoxide Anion Generation, Its Pathological Cellular and Molecular Roles and Pharmacological Targeting in Inflammatory Pain: Lessons from the Potassium Superoxide Model

Abstract

Reactive oxygen species (ROS) are formed by the incomplete reduction of oxygen and play a crucial role in both physiological function and pathological process, being controlled by enzymatic and non-enzymatic antioxidant systems. However, excessive ROS production can exceed the body’s antioxidant capacity, resulting in oxidative stress and causing cell death and oxidation of important biomolecules. In this context, the inhibition and/or modulation of ROS has been shown to be effective in reducing pain, oxidative stress, and inflammation. Among ROS, superoxide anion (O₂^•−) is the first free radical to be formed through the mitochondrial electron transport chain (ETC) or by specific enzymes systems, such as the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) complex. O₂^•− plays a significant role in the development and maintenance of pain associated with inflammatory conditions through direct or indirect activation of primary nociceptive neurons and, consequently, peripheral and central sensitization. Experimentally, potassium superoxide (KO₂, a O₂^●− donor) is used to initiate O₂^●− mediated inflammatory and nociceptive responses, making it important for studying the mechanisms associated with ROS-induced pain and evaluating potential therapeutic molecules. This review addresses the production and regulation of O₂^•−, highlighting its biosynthesis, redox control, and its physiological and pathological roles in the development of inflammatory pain, as well as the pharmacological therapies under development aimed at its generation and/or action.

1. Introduction

Aerobic organisms evolved to use oxygen (O₂) as an electron acceptor in respiration, developing systems that could protect cellular constituents from the harmful effects of this molecule. O₂ is essential for the process of cellular respiration, as its oxidizing power enhances the conversion of energy present in the covalent bonds of carbon molecules into adenosine triphosphate (ATP) molecules. However, the high oxidizing capacity of O₂ can result in toxic effects on cellular integrity. The O₂ reduction in cells leads to the continuous formation of highly reactive intermediates that can alter the structure and function of several important biomolecules [1].

Reactive oxygen species (ROS) are free radicals, which are characterized by containing one or more unpaired electrons in their outer orbital, rendering them highly reactive. The reaction aims to donate its unpaired electron or remove it to another molecule to increase stability. ROS are carefully maintained at low concentrations by fine tuning by antioxidant systems that can be produced in the organism, obtained in the diet, or acquired by treatment. Antioxidants can be classified as enzymatic antioxidant components or non-enzymatic antioxidants (also called low molecular weight antioxidants). Among the most important antioxidant enzymes are superoxide dismutase (SODs), catalase (CAT), and glutathione peroxidase (GPx). The best-characterized non-enzymatic antioxidant agents include vitamin C (ascorbic acid), vitamin E (α-tocopherol), vitamin A (β-carotene), glutathione, and flavonoids (e.g., quercetin, hesperidin and naringenin) [2,3]. These antioxidant compounds modulation is essential for maintaining redox homeostasis, as they block the action of ROS, preventing damage to important biomolecules such as proteins, lipids, and nucleic acids. In addition, the presence of ROS at adequate levels is essential for various physiological processes that depend on the cellular redox state, such as proliferation differentiation, migration, angiogenesis, cellular senescence, and cell death [4,5].

Nevertheless, when there is an imbalance between oxidants and antioxidants in favor of oxidants, leading to a disruption of redox signaling and control, accompanied or not by cellular damage, a biological process called oxidative stress is established [6]. This process development has been associated with the pathophysiology of a wide range of diseases, such as neurodegenerative diseases (e.g., Alzheimer’s Disease and Parkinson’s Disease) [7,8,9,10], cardiovascular diseases (e.g., atherosclerosis, hypertension, and myocardial infarction) [11,12,13,14,15], metabolic diseases (e.g., type I and II diabetes) [14,16], pulmonary diseases (e.g., chronic obstructive pulmonary disease, asthma, and COVID-19) [17,18,19,20,21], inflammatory diseases (e.g., rheumatoid arthritis, lupus, and colitis) [22,23,24,25,26], aging [27,28], and cancer [29,30]. In these conditions, modulation of ROS production and/or action by antioxidant pharmacological agents is essential for rebalancing the redox state and, consequently, improving the disease [22,31,32,33,34].

In this review, we focus on the role of ROS, especially O₂^•−, in the development of pathological pain, particularly in models using potassium superoxide (KO₂), a chemical reagent that triggers nociception and pain through the donation of the O₂^•− radical. However, we strongly recommend reading other reviews that address the pain mechanisms involved in other ROS and reactive nitrogen species (RNS), such as Chávez Silva et al. (2025) [34], Salvemini et al. (2011) [35], Kallenborn-Gerhardt et al. (2022) [36], Carrasco et al. (2018) [37]. These reviews will allow for a better understanding of the role of ROS and RNS in inflammatory pain, neuropathic pain, cancer-induced pain, and chemotherapy-induced pain, for example. KO₂ is produced by burning molten potassium in an atmosphere of excess O₂. KO₂ is composed of potassium ion (K⁺) and O₂⁻ linked by an ionic bond, whose O-O. The main applications of KO₂ involve carbon dioxide (CO₂) purification, water (H₂O) dehumidification, and O₂ generation in rebreathers, spacecraft, submarines, and space suits. These applications are based on KO₂’s ability to release O₂ and absorb CO₂ simultaneously in the presence of water vapor [38]. In addition, the literature shows KO₂ to be an important inducer of inflammatory pain, since the release of O₂^•− triggers the activation of both immune cells and nociceptive neurons [33,39,40,41].

Therefore, this review will focus on the regulation of O₂^•− production, physiological and pathological roles in the development of inflammation and mainly, pain. There is an emphasis on the data obtained using the KO₂ model of inflammation and pain. We will cover aspects such as biosynthesis, antioxidant control, redox signaling, pain mechanisms, and potential pharmacological therapies that target the generation and/or action of O₂^•−.

2. ROS Biosynthesis and Physiological Function

2.1. ROS Generation Pathways

Reactive oxygen species (ROS) are generated by intracellular sources or because of the organism’s exposure to factors such as toxins, nutrients, radiation, and drugs. The main intracellular sources are the mitochondrial electron transport chain (ETC), nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX) expressed in cell membranes, cytoplasmic xanthine oxidase (XO), and flavin-centered oxidases in peroxisomes and the endoplasmic reticulum.

The acquisition of an electron by O₂ results in the generation of O₂^•−, which will be generate others ROS. The intermediates generated from O₂ are classified as free radicals or non-radicals. The free radical group includes the O₂^•−, the hydroxyl radical (OH^●), the alkoxyl (RO^●), nitric oxide (NO^●), and the peroxyl radical (ROO^●), which are characterized by the presence of an unpaired electron. The non-radical group, despite being derived from reactive O₂, does not have unpaired electrons, and among its members are H₂O₂, singlet oxygen (¹O₂), hypochlorous acid (HOCl), and peroxynitrite (ONOO⁻) [1].

ROS are mainly generated through enzymatic activity, but they can also arise from auto-oxidation reactions of glyceraldehydes [42,43,44], reduced flavin adenine dinucleotide (FADH₂) [45,46], hormones [47,48,49], and neurotransmitters [50,51]. Nevertheless, this review will focus on the enzymatic generation of ROS. In general, ROS can be generated in the mitochondria (by the ETC), the cytosol (by a set of enzymes, such as SOD, CAT, GPx, and XO), and the cell membrane (through the NOX multiprotein complex) (Figure 1).

2.1.1. Mitochondrial ROS Generation

Mitochondrial ROS (mtROS) are generated during the Krebs cycle or, above all, in the ETC, considered the main generator of cellular ROS [52]. ETC consists of four protein complexes (I–IV) and two mobile electron carriers. Among the components are complex I (reduced nicotinamide adenine dinucleotide (NADH): Ubiquinone Oxidoreductase), complex II (Succinate Dehydrogenase), Coenzyme Q (Ubiquinone, Q), complex III (Cytochrome bc1 complex), Cytochrome c, complex IV (Cytochrome c Oxidase), and complex V (ATP synthase). The process begins when NADH and FADH₂, generated in the Krebs cycle and glycolysis, donate high-energy electrons to the ETC. Sequentially, NADH donates electrons to complex I (resulting in NAD⁺) and FADH₂ donates electrons to complex II (generating FAD). The electrons then flow through the enzyme chain, passing through complexes I/II, then through coenzyme Q, reaching complex III, cytochrome, and finally complex IV, which transfers the electrons to O₂, which acts as the final electron acceptor, being reduced to H₂O. During this process, the energy released by the flow of electrons is used to pump protons (H⁺) from the mitochondrial matrix into the intermembrane space, mainly by complexes I, III, and IV. The electrochemical gradient of H⁺ (also called H⁺ motive force) stores potential energy, which is used by F1F0 ATP synthase (complex V), allowing H⁺ to return to the mitochondrial matrix, using this flow of H⁺ to synthesize ATP from adenosine diphosphate (ADP) and inorganic phosphate (Pi) [53]. During this process, electrons leak from complexes I, II, and III, resulting in partial reduction of O₂ and generating O₂^•−. In addition, mitochondrial uncoupling proteins (UCP; UCP1, UCP2, and UCP3), located in the inner membrane, are essential during oxidative phosphorylation, since they prevent the H⁺ motive force from becoming too high, these proteins facilitate the leakage of electrons from the ETC, resulting in the generation of O₂^•− [54,55].

Considering the high reactivity of ROS, mitochondria have developed antioxidant systems to prevent oxidative damage. Among the examples there is the efficient antioxidant activity of SOD enzymes, which are expressed in different isoforms in the mitochondria, cytoplasm, and cell membrane. SODs are metalloproteins belonging to the oxidoreductase class (EC1), whose shared function is the dismutation of the O₂^•− radical into H₂O₂ (more stable) [56,57]. The three isoforms of SOD are Copper (Cu)-Zinc (Zn)-SOD (SOD1), located in the cytoplasm, nucleus, and mitochondrial intermembrane space; Manganese (Mn)-SOD (SOD2), located in the mitochondrial matrix; and Cu-Zn-SOD (SOD3 or SOD-EXT), located in the extracellular membrane [58]. The main structural difference between the three isoforms is the substitution in the groups linked to the phosphate oxygen (Figure 2).

SOD1 ((C₆H₅O)₂P(S)–S–CH₂CH=CH₂) has its phosphate group bound to two phenoxy groups (–O–C₆H₅) and one allyl thioester group (–S–CH₂CH=CH₂). SOD2 ((C₆H₅CH₂O)(C₆H₅O)P(S)–S–CH₂CH=CH₂), in turn, has the phosphate group bound to a benzyloxy group (–OCH₂–C₆H₅), a phenoxy group (–O–C₆H₅), and the same allyl thioester group. Thus, the presence of one benzyloxy group, instead of two, differentiates SOD2 from SOD1. Finally, SOD3 ([(C₆H₅CH₂O)]₂P(S)–S–CH₂CH=CH₂) has the phosphate group bound to two benzyloxy groups (–OCH₂–C₆H₅) and to the allyl thioester group. This third isoform of SOD differs from the others in that it has two benzyloxy groups [55,56,57]. In the ETC, O₂^•− generation occurs mainly in complexes I and III, which promote the release of ROS into the mitochondrial matrix and interstitium. Thus, the control of mtROS levels and actions is essentially performed by SOD2, which rapidly converts O₂^•− generated by the ETC into H₂O₂. In turn, H₂O₂ can passively diffuse toward the cytosol, where it will be metabolized by SOD1 (described in Section 2.1.2) [59,60].

2.1.2. Cytoplasmic ROS Generation

The control of ROS in the cytoplasm is of paramount importance for maintaining redox homeostasis, since in this intracellular environment, ROS can interact with kinases, proteases, and transcription factors, triggering various cellular events (described in Section 2.3). The cell cytoplasm serves as the main setting for the chain reaction of free radicals, in which there is a rapid generation of highly reactive radicals, such as O₂^•−, which are sequentially converted into less reactive radicals, thus giving rise to several other ROS. Enzymatic actions and reactions involving metal ions, such as ferrous ion (Fe²⁺), are primarily responsible for generating ROS in the cytoplasm. Furthermore, the ROS actions are controlled by endogenous antioxidant systems, such as SOD1, CAT, and GPx.

Xanthine Oxidoreductase System (XOR)

Xanthine oxidoreductase (XOR) enzyme complex is a homodimeric protein that has two subunits with three independent redox centers. The modular structural domains are composed of an N-terminal domain, which contains two clusters (2Fe-S), a central domain that houses FAD and receives electrons from the Fe-S clusters, and a C-terminal domain, which houses the molybdopterin cofactor with molybdenum (Mo-co) responsible for catalyzing the hydroxylation of xanthine into the final product uric acid [61,62,63].

Initially, it is synthesized as xanthine dehydrogenase (XDH), but it can be converted to XO by the oxidation of sulfhydryl residues or by proteolysis. XOR is responsible for the catabolism of purine nucleotides, catalyzing the oxidation of hypoxanthine and xanthine to uric acid. XDH favors NAD⁺ as the main electron acceptor. On the other hand, XO has a higher affinity for O₂ as its electron acceptor. However, both enzymes can generate O₂^•− as a product of these reactions. In this reaction, two electrons are donated from the purine substrates to the Mo-co site of XOR, then can be rapidly transferred to the Fe-S redox center before being passed to NAD⁺ or O₂, resulting in O₂^•− and, subsequently, H₂O₂ [64,65].

Endoplasmic Reticulum

Endoplasmic reticulum (ER) is a complex dynamic structure that has several cellular functions, mainly related to calcium ion (Ca²⁺) storage, lipid metabolism, and protein synthesis. In this cytoplasmic organelle, ROS are released during folding and proteins since they confer the formation of disulfide bonds by the protein disulfide isomerase (PDI). During the formation of disulfide bonds, aided by the chaperone between polypeptide chain substrates, two electrons are supplied to the cysteine (Cys) residue within the active site of PDI. Then, the transfer of electrons leads to the reduction of the active site of PDI and oxidation of the substrate, generating ROS in the process [66]. Another component that assists in redox signaling in this organelle is oxidoreductase-1 in the ER (ERO1), which oxidizes PDI using FAD. This reaction also triggers the production of ROS and contributes to the development of ER stress [67].

Another important generator of ROS in the ER is the microsomal mono-oxidase (MMO) system, which contains enzymes from the cytochrome P450 family. These enzymes produce considerable levels of O₂^•− and H₂O₂, triggering the activation of redox signaling pathways. In addition, the use of p450 2E1 inhibitors reduces the concentration of ROS, highlighting the important role of ER in cellular oxidative balance [68,69].

Iron-Dependent Reactions

The genesis of ROS may depend directly on metal ions, especially iron (Fe), which acts as a catalyst for the reaction. Once in the cytosol, O₂^•− promotes the release of Fe²⁺ from Fe-S groups present in certain proteins. This increase in the cytoplasmic concentration of Fe²⁺ favors the Fenton reaction, in which H₂O₂, in the presence of Fe²⁺, is converted into OH^● and ferric ion (Fe³⁺) [70]. In turn, H₂O₂ also promotes the elevation of free Fe²⁺ from the heme groups of proteins, favoring the additional generation of OH^● [71].

Furthermore, the presence of Fe²⁺ in the cytoplasm contributes to the Haber-Weiss reaction [72]. In this reaction, Fe²⁺ acts as an indirect catalyst for the reaction involving O₂^•− and H₂O₂, resulting in hydroxide ion (OH⁻) and OH^●. Specifically, this type of reaction does not occur spontaneously, but is favored by the Fenton reaction, which provides the necessary components. Finally, for iron-dependent reactions to proceed indefinitely, Fe³⁺ can be reconverted to Fe²⁺ in a reaction mediated by O₂^•− [72].

Cytosolic Endogenous Antioxidant Enzymes

As mentioned earlier, SOD1 contains copper (Cu²⁺) and zinc (Zn²⁺) ions and catalyzes the dismutation of O₂^•− generating H₂O₂. Subsequently, H₂O₂ is decomposed by the CAT enzyme, generating two molecules of H₂O and one of O₂. CAT is an oxidoreductase located mainly in peroxisomes, which uses a heme group (Fe³⁺) as an active center as a cofactor. The reaction mediated by catalase occurs in two main steps: (1) catalase reduces the first H₂O₂ molecule accompanied by the formation of oxoferryl species (Fe⁴⁺O), (2) then, the oxoferryl complex is reduced by the second O₂ molecule, generating O₂, H₂O, and the enzyme free for a new reaction. Peroxisomes can rapidly produce and absorb O₂^•− and H₂O₂, causing dynamic fluctuations in ROS levels and allowing the activation of redox signaling pathways that modulate necessary cellular processes [73,74].

Furthermore, there is the GPx family, composed of members such as glutathione peroxidase-1 (GPx-1) [75]. These enzymes have a selenocysteine residue in the active site and have the activity of reducing lipid hydroperoxides to alcohols and H₂O₂ to H₂O. In this reaction, the tripeptide reduced glutathione (GSH) is the active form of glutathione, acting as an essential substrate for the antioxidant activity of GPx, which has a thiol group (-SH) responsible for neutralizing H₂O₂. After the reaction, oxidized glutathione (GSSG) is generated, formed by the union of two GSH molecules via a disulfide bridge (-S-S-) [76]. GSH is considered a sacrificial agent, as are vitamins A, C, and E, reacting preferentially with ROS and preventing them from reacting with important biomolecules. GSSG can be recycled back to the original compound, perpetuating cellular antioxidant reactions [3].

2.1.3. ROS Generation in the Membrane: NADPH Oxidase (NOX)

The members of the NOX family are constitutive membrane-bound enzyme complexes consisting of different subunits, whose function is the synthesis of ROS. In higher mammals, seven total members vary according to subunit composition and expression patterns: NOX 1 to 5 and the dual oxidases DUOX1 and DUOX2, which are important generators of O₂^•− and H₂O₂ by reducing O₂ [77]. In general, NOX1-4 share a certain structural homology, since all these complexes require interaction with the regulatory subunit p22^phox, which effectively produces ROS. NOX5 does not require p22^phox and, together with DUOX1 and DUOX2, is a Ca²⁺-dependent complex since they have four binding sites for calcium in their N-terminal cytoplasmic portion. Under physiological conditions, NOXs are maintained at basal levels of activation, which regulate the redox signaling of target molecules and are important for processes of cell differentiation, proliferation, and apoptosis. The following is a more detailed description of the NOX isoforms, focusing on their structure, tissue expression, and activation mechanism.

NOX1

Structurally, NOX1 requires the presence of cytosolic factors NOXO1 and NOXA1, homologous to p47^phox and p67^phox, respectively. Thus, functional activity requires transmembrane components (gp91^phox and p22^phox), in addition to NOXO1, NOXA1, and small guanosine triphosphate (GTP)ase Rac in the cytoplasm [78,79].

This isoform has been detected in a wide range of tissues, such as the adrenal gland, appendix, brain, colon, duodenum, endometrium, esophagus, adipose tissue, urinary bladder, gallbladder, heart, kidney, liver, lymph nodes, ovary, placenta, prostate, salivary gland, skin, small intestine, spleen, stomach, testis, and thyroid [80].

NOX1 activity is positively modulated by some transcription factors, such as signal transducer and activator of transcription 1 (STAT1) [81,82], signal transducer and activator of transcription 3 (STAT3) [81,83], GATA-binding protein 4 (GATA4), GATA-binding protein 6 (GATA6) [84], nuclear factor kappa B (NF-κB) [85], activator protein 1 (AP-1) [86,87], myocyte enhancer factor 2B (MEF-2B) [88], CCAAT/enhancer-binding alpha/beta/gamma (C/EBP-α/β/δ) [89,90], and activating transcription factor 1 (ATF-1) [91].

NOX2

NOX2 was the first NOX isoform to be identified and is the best characterized. This isoform is mainly expressed in phagocytic cells (macrophages and neutrophils) and has also been detected in the adrenal gland, appendix, bone marrow, brain, colon, duodenum, endometrium, esophagus, adipose tissue, urinary bladder, gallbladder, heart, kidney, liver, lymph nodes, ovary, placenta, prostate, salivary gland, skin, small intestine, spleen, stomach, testicle, and thyroid [80].

Structurally, NOX2 is composed of the catalytic protein gp91^phox and its stabilizer gp22^phox, located in the membrane (also called flavocytochrome b₅₅₈), which are supported by the cytosolic protein factors p47^phox, p67^phox, p40^phox, and small GTP-binding proteins, which can be Rac1, Rac2, and Rac3 [80,92]. In monocytes and macrophages, all Rac proteins are expressed, while in neutrophils, Rac2 predominates [93,94]. Due to its better characterization, we will use NOX2 to explain the mechanism of NOX association.

In the absence of the activation signal, the transmembrane and cytoplasmic components are kept physically dissociated in an inactive state. Rac is associated with the Rho GDP dissociation inhibitor (RhoGDI) inhibitory protein, which masks the geranylgeranyl tail of Rac within the hydrophobic pocket. However, after an activation signal, the regulatory subunits are translocated to the membrane, where they meet with flavocytochrome b₅₅₈ [95]. Phosphorylation of cytosolic factors, especially p47^phox, allows the translocation of cytoplasmic components toward transmembrane components, forming the properly functional protein complex. Similarly, Rac-guanosine diphosphate (GDP) inhibited by RhoGDI is transferred to the membrane, and its GDP is converted to GTP for final assembly with p67^phox. As a result, after NOX2 activation, there is sequential electron transfer, culminating in O₂ reduction and O₂^•− generation in the phagosome. The initial production of O₂^•− results in the formation of H₂O₂, which in turn can be converted to HOCl⁻ by the enzyme myeloperoxidase (MPO), which is very important for the microbicidal activity of phagocytes [92,96].

NOX2 can be positively upregulated by factors such as NF-κB [97,98], STAT3 [83], peroxisome proliferator-activated receptor (PPAR)-α [99], hypoxia-inducible factor 1 alpha (HIF-1α) [100], yin yang 1 (YY1) [101], E74-like factor 1 (Elfo-1), spleen focus-forming virus proviral integration oncogene (SPI1) [101], interferon consensus sequence-binding protein (ICSBP), interferon regulatory factor 1/2 (IRF-1/2) [102], homeobox A9 (HOXA9), and paired box 1 (PBX1) [103]. On the other hand, some pathways that negatively modulate its function have been identified, such as myeloid ecotropic viral integration site 1 (Meis1), and homeobox A10 (HOXA10) [103].

NOX3

NOX3 is typically expressed in components of the inner ear, such as the cochlea and vestibule. Thus, this isoform has been associated with hearing loss and perception of gravity However, it can also be detected in the adrenal gland, adipose tissue, lung, placenta, spleen, and testis [80,104]. Structurally, NOX3 is composed of the catalytic subunit (gp91^phox) and the stabilizer p22^phox, not requiring the presence of NOXO1, NOXA1, p47^phox, p67^phox, and Rac. Nevertheless, NOX3 can function in the presence of these cytosolic subunits but exhibits relatively lower constitutive activity [80].

NOX4

NOX4 has been detected in the endometrium, adipose tissue, gallbladder, urinary bladder, heart, kidneys, lungs, ovaries, placenta, and thyroid. This isoform is constitutively active, whose activation depends strictly on the membrane stabilizer p22^phox. NOX4 predominantly produces H₂O₂, even in the absence of SOD [80].

NOX4 activity is positively regulated by transcriptional factors such as NF-κB [85], AP-1 [105], STAT1/3, C/EBPα/β/δ [81], E2F transcription factor 1 (E2F1) [106], and PPARα/β/δ [107]. However, the PPAR-γ factor appears to negatively regulate NOX4 activity, resulting in lower ROS generation by this isoform [108]. Furthermore, some proteins appear to amplify the function of NOX4, such as protein 2 that interacts with polymerase delta (Poldip2), disulfide isomerase protein (PD1), and tyrosine kinase substrate 4/5 (Tks4/5) [80].

NOX5

The human NOX5 gene encodes six identified isoforms (α, β, γ, δ, and ζ) [109]. The α, β, γ, and δ isoforms of NOX5 have EF-hand-N-terminal domains, conferring Ca²⁺ activation. In addition, the α, β, and γ isoforms are functionally active and generate ROS. The δ, ε, and ζ isoforms, on the other hand, appear to be relatively inactive when it comes to O₂^•− generation [110,111].

NOX5 has an N-terminal extension containing four EF-hand motifs, and its activation is Ca²⁺-dependent. Another special feature is that this isoform does not depend on the other components common to other NOX (p22^phox, p47^phox, p67^phox, and Rac) [112]. NOX5 has been detected in the adrenal gland, appendix, brain, endometrium, esophagus, adipose tissue, gallbladder, urinary bladder, heart, kidney, ovary, placenta, prostate, salivary gland, skin, spleen, stomach, testicle, and thyroid [80].

The binding of Ca²⁺ to the extra EF-hand domain results in conformational changes that expose hydrophobic regions that bind to the catalytic core, resulting in electron transfer. Interestingly, NOX5 is expressed only in higher mammals, such as humans, and is absent in rats and mice [113]. Among the factors that positively regulate its activity are NF-κB, AP-1, STAT1/3 [114], C/EBPα/β/γ [90], and PPAR-α/β/κ/γ [107].

DUOX1/2

DUOXs are composed of the catalytic subunit gp91^phox, emblematic cytosolic domains (FAD-binging domain (FBD) and NADPH-binding domain (NBD)), an EF-hand domain that binds Ca²⁺, with two instead of four EF motifs, and an extracellular N-terminal domain like peroxidase connected to the rest of the protein by an extra TM helix. In their inactive state, DUOXs are retained in the ER. Upon activation, these isoforms migrate from the ER to the plasma membrane, where they reduce O₂, generating ROS [115,116,117].

DUOX1 predominantly produces O₂^•−, while DUOX2 generates H₂O₂. Another substantial difference concerns the expression of each, while DUOX1 is detected in the adrenal gland, bone marrow, brain, colon, endometrium, esophagus, bladder, heart, kidney, lung, ovary, placenta, prostate, salivary gland, skin, stomach, testicles, and thyroid, DUOX2 has a more restricted expression, being detected in the appendix, colon, skin, stomach, thyroid, and urinary bladder [80].

2.2. Redox Signaling

Redox signaling refers to the regulation of cellular processes by reactive oxygen species (ROS). This regulation role occurs because ROS interacts with Cys residues present in regulatory sites of enzymes, transporters, receptors, and transcription factors, triggering the expression and release of various factors that enable cellular communication.

Several proteins are sensitive to the cellular redox state, such as heat shock proteins, scaffolding proteins, ribonucleoproteins, and cytoskeletal elements [4]. Cys can be classified into two main types: those buried within a protein (with a structural function) and those exposed, expressed on the surface of the protein, and prone to redox activities. Cys embedded within a protein form intramolecular disulfide, such as Fe-S clusters. Exposed Cys residues, on the other hand, are more accessible to external signals or may be involved in the formation of mixed disulfides with binding partners. Thus, Cys may be catalytic or regulatory. In addition, a buried Cys can be exposed, becoming accessible for oxidation, as occurs with EGF [118,119,120,121].

When ROS interacts with Fe-S cluster proteins or with the ionized form of Cys residues (thiolate), modifications are generated that influence cellular processes [122,123]. H₂O₂ can oxidize thiol groups (-SH) in Cys residues, leading to the formation of sulfenic acid (-SOH), which interacts with GSH and becomes GSSG. This interaction results in disulfide bonds. The reactivity of ROS with thiols is limited to Cys residues located in regions that allow the formation of thiolate (-S). These modifications trigger changes in the activity of target proteins and signaling pathways, which trigger or suppress cellular processes. Finally, mixed disulfides may form with low molecular weight thiols, such as GSH, a process called S-glutathionylation [124].

Thus, this section aims to present some of the transcription factors that are commonly sensitive to the cellular redox state and how their activation triggers essential cellular processes, from proliferation to cell death.

2.2.1. ROS-Modulated Transcription Factors

Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2)

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of cellular responses to oxidative stress, as it increases the expression of antioxidant enzymes, modulates glucose metabolism, and suppresses pro-inflammatory genes. Moreover, this factor role contributes to proliferation and differentiation events [125].

Physiologically, Nrf2 is sequestered in the cytosol by the Kelch-like ECH-associated protein 1 (Keap1) repressor, remaining inactive. Keap1 mediates the ubiquitination of Nrf2, mainly through the cullin-3 (CUL3)-based E3 ubiquitin ligase pathway and degradation by the proteasome [126]. Keap1 has several Cys residues, such as Cys226, Cys613, Cys622, Cys624, Cys151, Cys273, and Cys288 [127,128], which undergo oxidation in the presence of ROS. The interaction of ROS with this residue leads to disulfide formation, which causes a conformational change in Keap1, preventing the ubiquitination of Nrf2 and allowing the Nrf2/Keap1 interaction to be disrupted. This process increases the stability of Nrf2, which allows its nuclear translocation and, consequently, the expression of antioxidant response elements (ARE) [129,130].

Specifically related to the nervous system, neurons have low expression of Nrf2, derived from a mechanism of epigenetic repression of the Nrf2 promoter, leading to hypoacetylation of the histone around the transcription start site of the factor [131]. Thus, these cells are highly susceptible to oxidative stress. In turn, astrocytes express high levels of Nrf2 and respond to the redox state [131]. Thus, astrocytes are cells responsible for maintaining redox homeostasis in the central nervous system (CNS), providing protection to nearby neurons through a mechanism involving the release and transport of astrocytic glutathione precursors, produced by the activation of Nrf2 [132].

NF-κB

NF-κB is an inducible transcription factor that plays a key role in immune response, cell adhesion, differentiation, proliferation, and apoptosis processes. The NF-κB family consists of five structurally similar members: p50/p105 (NF-κB1), p52/p100 (NF-κB2), RelA (p65), RelB, and c-Rel. NF-κB can be activated by two main pathways: canonical and non-canonical [133].

In resting cells, NF-κB remains in the cytoplasm bound to inhibitory regulatory protein IκB (IκBα, IκBβ, or IκBε). In the canonical pathway, the triggering stimulus leads to site-specific phosphorylation by an IκB kinase (IKK) complex, composed of IKKα and β kinases, inducing ubiquitination and subsequent proteasomal degradation of the inhibitory proteins. Thus, once free, the RelA subunit is phosphorylated and the transcription factor NF-κB is translocated to the nucleus, where it interacts with deoxyribonucleic acid (DNA) promoter regions and promotes the transcription of target genes. In the non-canonical pathway, it is activated by specific stimuli, such as CD40 ligand and CD27 ligand, for example, and depends on the precursor protein p100 (NF-κB2) rather than the degradation of IκBα [134].

ROS can trigger the activation of this pathway by triggering the phosphorylation of IκBα and IκBβ [135]. For example, H₂O₂ appears to modulate NF-κB in an ambiguous manner. On the one hand, H₂O₂ activates IKK, which phosphorylates IκBβ (S-glutathionylation of Cys179), inducing the degradation of inhibitory proteins and the continuation of the canonical pathway [136]. However, H₂O₂ has also been shown to directly oxidize NF-κB at Cys residues in the DNA-binding domain of the p50 subunit, preventing its transcriptional activity [135].

ROS generated by NOX1 modulate the nociceptive pathway via extracellular signal-regulated kinase 1/2 (ERK1/2)-NF-kB signaling and glial activation in dorsal root ganglia (DRG) and spinal cord [137]. When using a NOX1 inhibitor, ML171 (10, 20, and 30 mg/kg), there is attenuation of nociceptive behavior during acute nociception induced by formalin. In addition, ML171 reduced the nociceptive mediators p-ERK1/2, pNF-kB p65, Ionized calcium-binding adaptor molecule 1 (Iba1), and Glial Fibrillary Acidic Protein (GFAP) in the DRG and spinal cord [137].

Mitogen-Activated Proteins Kinase (MAPK)

ROS have been associated with increased Mitogen-activated proteins kinase (MAPK) activity, either by leading to sequential activation of the catalytic pathway or by oxidizing cysteine residues. MAPKs are composed of four kinases essential for cell growth, differentiation, development, and survival. The main pathways involve ERK1/ERK2, also called MAPK3/MAPK1, c-Jun N-terminal kinases (JNK) and p38 kinase [125].

ROS can act directly on Cys residues of these MAPKs, leading to reversible S-sulfenylation, resulting in their activity [138]. Furthermore, H₂O₂ can activate sensory proteins such as ASK1. In this context, thioredoxin 1 (Trx1) is oxidized, allowing ASK1 to auto-phosphorylate and then phosphorylate JNK and p38, activating the pathway [139]. ROS also oxidize MAPK phosphatases, responsible for inhibiting the pathway, which results in sustained activation of MAPKs [139]. Stimulation of some cytokine receptors also activates the MAPK pathway in a ROS-dependent manner. Activation of TLR4 and interleukin-1 receptor (IL-1R) triggers similar phosphorylation cascades, leading to the transcription of inflammatory mediators. Through induction via lipopolysaccharide (LPS), for example, these pathways transduce signals through Interleukin-1 Receptor-Associated Kinase 1/4 (IRAK1/4), which activates the TAK1-Binding Protein 1/Transforming Growth Factor-β-Activated Kinase 1 (TAB1/TAK1) complex, activating MAPKs (p38, ERK, and JNK) and, subsequently, the AP-1 factor. Furthermore, this pathway can also activate IKK, which phosphorylates the inhibitory protein IKBα, inducing its ubiquitination and proteasomal degradation, resulting in the translocation of NF-κB to the nucleus. As a result, activation of this pathway by O₂^•− leads to the expression of several pro-inflammatory mediators [cyclooxygenase (COX)-2, interleukin (IL)-6, TNF-α, and IL-1β), amplifying the innate immune response. Furthermore, IRAK1/4 can interact directly and indirectly with NOX1/2, causing Rac1 activation, p47^phox subunit migration, and direct phosphorylation of gp91^phox subunit, or promoting protein kinases C (PKC) activation, leading to O₂^•− production [89,140].

Hypoxia-Inducible Factor 1 Alpha (HIF-1 α)

Hypoxia-inducible factor 1 alpha (HIF-1α) is a heterodimer composed of the HIF-1β (constitutive) and HIF-1α (regulatory and O₂-dependent) subunits, activated in response to hypoxia and oxidative stress.

In normoxia, the tumor suppressor protein Von-Hippel-Lindau (VHL) interacts with HIF-1α by hydroxylating residues P402 and P564 through prolyl hydroxylase domain (PHD) proteins. As VHL regulates E3 ligase activity, this protein suppresses HIF-1α activity by targeting it for ubiquitination and proteasomal degradation. However, in hypoxia, the hydroxylation of proline residues of PHD proteins is inhibited, allowing HIF-1α activity. Furthermore, ROS promotes HIF-1α stability by diverting the PHD substrate 2-OG to non-enzymatic decarboxylation, oxidizing the PHD cofactor Fe²⁺ to Fe³⁺, and inhibiting HIF inhibitory factor. In turn, ROS can interact with Cys residues in the catalytic domain of the PHD2 protein (Cys520), resulting in S-glutathionlyation of this residue, which inhibits VHL ubiquitination, enabling HIF-1α activity and upregulating genes involved in the immune response, cell proliferation, angiogenesis, and energy, glucose, and Fe metabolism [141,142,143].

Forkhead Box O (FoxO) Factor

This factor is involved in cellular homeostasis in response to various oxidative stimuli, in addition to modulating cell proliferation and death processes [142,144]. In addition, Forkhead box O (FoXO) signaling is involved in insulin signaling. After the ligand binds to the insulin receptor, a phosphorylation cascade is triggered that culminates in the activation of FoxO [145].

In situations of oxidative stress, FoxO3 and FoxO4 are activated and induce the expression of antioxidant agents (e.g., CAT and SOD2), aiming to reduce cellular oxidant levels [144]. Furthermore, when ROS levels are exacerbated, FoxO also leads to increased expression of factors that lead to DNA repair, cell cycle arrest, glucose metabolism, and apoptosis. Alternatively, H₂O₂ leads to FoxO phosphorylation by the kinase AKT and exclusion from the nucleus. The formation of an intermolecular disulfide bond between FoxO and transportin (TNPO) promotes nuclear translocation. Furthermore, high H₂O₂ levels cause p300 acetylase to interact with acetylated FoxO, resulting in reduced DNA-binding capacity [145].

2.2.2. Kinases and Phosphatases Proteins

Protein kinases and phosphatases are essential in regulating cell signaling through post-translational modifications. These alterations involve the addition or removal of phosphate groups from proteins, a process called phosphorylation. Such modifications trigger the activation of pathways and the expression of various factors, leading to processes such as growth, metabolism, differentiation, and cell death. While kinases add a phosphate group (–PO₄³⁻) to the target molecule, phosphatases remove this phosphate group, reversing the action of the kinases and, in most cases, inactivating or reversing a cellular signal.

The protein kinases modulate various cellular processes, such as proliferation, differentiation, apoptosis, and cellular metabolism. ROS oxidize -SH groups of Cys in protein kinases (PK), such as A (PKA) (Cys199), PKC (Cys151, Cys153, Cys157, Tyr512, Tyr523) and D (PKD) (Cys612, Tyr463), of RTK (Cys797) and Ca²⁺/calmodulin-independent protein kinase II (CMKII) (Met281, Met282 and Cys290) [146,147]. Furthermore, PKA can undergo S-glutathionlyation or formation of disulfide bridges, the Cys199 residue is the most susceptible to this reaction, which leads to a reduction in its activity [148]. While low levels of ROS modulate PKC activity, high levels can trigger the oxidation of critical residues of this kinase, leading to the suppression of its activity [149,150]. Furthermore, some kinases, such as PKA, modulate the redox state, stimulating the production of ROS, which in turn also stimulates PKA activity [150,151]. On the other hand, kinases such as c-Src, when activated by ROS, via oxidation of residues Cys187 and Cys277, result in sulfenylation, which inhibits their enzymatic activity [152,153].

Unlike kinases, phosphatase activity is generally inhibited by oxidation. For example, protein tyrosine phosphatase 1B (PTP1B), a phosphatase that neutralizes insulin signaling, can be oxidized by ROS and have its enzymatic function eliminated. However, this enzymatic inactivation is essential for insulin signaling, as PTP1B dephosphorylates the insulin receptor and its substrates (IRS), attenuating it signaling. Thus, inactivating this enzyme activates the pathway and increases insulin sensitivity. Furthermore, H₂O₂-mediated inhibition of PTP1B accelerates proliferation and migration [154,155,156].

2.2.3. Epigenetic Redox Control

Epigenetic changes include DNA and histone methylation, as well as post-translational modifications of histone proteins, such as acetylation. Mitochondrial function can affect histone acetylation, as it provides acetyl-CoA and NAD⁺/NADH⁺, used as substrates by histone acetylases (HATs) and histone deacetylases (HDACs), respectively [157].

The tricarboxylic acid (TCA) cycle that occurs in mitochondria acts as substrates and cofactors for epigenetic modification enzymes, which modify histones and DNA, regulating gene expression. Histone acetylation, a process catalyzed by HATs, can occur reducing the protein’s positive charge, resulting in chromatin loosening and activation of gene transcription. During acetylation, acetylcoenzyme A (acetyl-CoA) is used as a substrate. Histone acetylation is catalyzed by HDACs, which remove acetyl groups from lysins, leading to chromatin condensation and transcriptional repression [158,159].

On the one hand, H₂O₂ increases the activity of HATs, such as p300/CBP or GCN5, leading to the acetylation of histones H3 and H4 [160]. On the other hand, ROS also reduce their activity by increasing the global acetylation of histones H3 and H4 and modifying the activity of HDAC1 and HDAC2 [161]. HDACs are less dependent on metabolic cofactors than HATs. HDACs are sensitive to the cellular redox state, as demonstrated by the interaction of ROS with cysteine residues of HDAC2 and HDAC4, resulting in the release and remodeling of chromatin, with subsequent activation of gene transcription [161].

2.3. ROS Physiological Actions

2.3.1. Cell Proliferations

The interaction of reactive oxygen species (ROS) with components of transcriptional pathways promotes the expression of several cellular mediators, such as cytokines and growth factors, inducing cell cycle activation. Ranjan et al. (2006) [162] demonstrated that in mouse lung epithelial cells, overexpression of NOX1 stimulates ERK1/2 phosphorylation and cyclin D1 expression (which allows the cell to enter the replication cycle), inducing cellular activation via increased ROS levels [162]. Also, synthetic CAT impairs the concentration of intracellular ROS and, consequently, impairs cyclin D1 expression, inhibiting the process of epithelial cell stimulation. Together with estrogen, ROS inhibited the activity of MAPK pathway regulatory phosphatases in primary cultures of human lung vascular smooth muscle cells (hPASMCs), resulting in an increased proliferative rate [163].

Furthermore, as previously reported, ROS can activate the NF-κB pathway, resulting in the expression of cytokines and growth factors, such as IL-8, plaque-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF), contributing to cell therapy [164]. Yan et al. (2020) [165] demonstrated that ROS, such as H₂O₂, induces the proliferation of mouse T cells, which is reversed by the administration of carbon monoxide (CO) and antioxidant agents such as N-acetylcysteine [165].

Moreover, during the wound healing process, cells such as macrophages, fibroblasts, endothelial cells, and keratinocytes utilize ROS to repair tissue [166]. During the tissue repair process, there is an intracellular increase in ROS, especially H₂O₂, in endothelial cells, fibroblasts, and keratinocytes, which triggers the entry of these cells into the cell cycle, leading to proliferation [167,168,169]. Corroborating this, O₂^•− induces proliferation and growth of the keratinocyte cell line via overexpression of the oncogenic form of the G protein v-Ha-Ras (HRAS), which is reversed by NOX inhibitors (diphenylene iodonium) or SOD overexpression [170]. These results are supported by studies that inhibit SOD, leading to an increase in O₂^•−, which stimulates cell proliferation and reduces the rate of cell apoptosis [171].

2.3.2. Cell Differentiation

Genetic deletion of NOX1 and NOX2 simultaneously disrupts ROS production, leading to inhibition of ERK and JNK [172]. Consequently, impaired differentiation of inflammatory macrophages to the tissue repair/anti-inflammatory phenotype was observed. Similarly, administration of an antioxidant drug also inhibited macrophage polarization, demonstrating the importance of ROS in this cellular process [173]. Furthermore, the absence of NOX in endothelial cells derived from murine inducible pluripotent stem cells (iPSCs) in early stages of differentiation slows the differentiation process, as demonstrated by reduced expression of endothelial markers (CD31, CD144, and eNOS) [174].

Similar physiological responses are observed in other systems. Genetic deletion of NOX2 in mouse neurons also slows neuronal differentiation [175]. In contrast, ROS production via NOX1 in undifferentiated PC12 cells inhibits nerve growth factor (NGF)-induced neurite outgrowth [176]. In bone tissue, NOX2 deficiency prevent the differentiation of osteoblasts into osteoclasts in mice, since ROS are important for Receptor Activator of Nuclear Factor κB (RANK)/RANK ligand (RANKL) signaling. Conversely, increased O₂^•− suppresses osteoblast differentiation in MC3T3-E1 cells [177]. During chondrocyte differentiation, NOX1/2 messenger RNA (mRNA) levels increase, suggesting the importance of ROS for the differentiation of this cell type. Furthermore, blocking NOX2/4 blocks ROS generation, and results in reduced differentiation and induction of apoptosis in chondrocytes [178,179].

2.3.3. Cell Migration

Cell migration is essential for several morphological changes, including immune cell migration and angiogenesis, for example. Genetic deletion of NOX1 leads to reduced activation of the ERK1/2 cascade, blocking thrombin-induced phosphorylation of Src, which reduces epidermal growth factor receptor (EGFR) transactivation and metalloproteinase 9 (MMP9) activation, inhibiting the migration process. Furthermore, due to the blockade of thrombin activity, N-cadherin release was also inhibited [180]. The same was observed in NOX4 knockout animals. In this study, the reduction in ROS levels in the ER prevented the stabilization of vascular endothelial growth receptor 2 (VEGFR2), preventing the transport of this receptor to the cell surface, which inhibited the directed migration of endothelial cells during the angiogenesis process [181]. Furthermore, the reduction in ROS by gene silencing or pharmacological inhibition of NOX1 results in reduced NF-κB activation, reducing endothelial cell migration and causing damage to the angiogenesis process [182]. Finally, the migration of glial cells, such as astrocytes and microglia, appears to be dependent on the expression of NOX1/2/4, highlighting the importance of these free radicals for this process [183].

ROS can also modulate the cytoskeleton [184,185]. Oxidation of the Cys374 residue of β-actin reduces filamentous actin polymerization and induces actomyosin disassembly, contributing to cytoskeletal contraction during cell migration. In the Nrf2-Keap1 antioxidant system, Keap1 binds to actin filaments through a double glycine repeat domain to assist in its suppressive activity on Nrf2 [186].

Another important biological process influenced by ROS is the eukocyte migration. For example, in neutrophil migration, increased ROS activate various kinases, such as c-Src and PKC, which leads to the phosphorylation of endothelial junction proteins, facilitating the migration process [187]. Furthermore, ROS increase the expression of P-selectins in endothelial cells, in addition to activating the NF-κB pathway, which culminates in the expression of Intercellular Adhesion Molecule 1 (ICAM-1), Vascular Cell Adhesion Molecule 1 (VCAM-1), and E-selectin, increasing the adhesion of neutrophils to vascular walls [188,189,190].

2.3.4. Cell Death

The types of cell death modulated by ROS include ferroptosis, necroptosis, PANoptosis, and apoptosis. We will briefly describe the mechanisms of ferroptosis, necroptosis, and PANoptosis, focusing especially on apoptosis. For more details on the concepts and mechanisms of ROS modulation on cell death by ferroptosis, necroptosis, and PANoptosis, we recommend reading some reviews in the literature, such as Fulda (2016) [191], Riuz-Pérez et al. (2023) [192], Wang and Kanneganti (2021) [193], Hsu et al. (2020) [194], Yu et al. (2021) [195] and Pandian and Kanneganti (2022) [196].

Ferroptosis is characterized by the iron-dependent intracellular accumulation of ROS and products of uncontrolled lipid peroxidation. In this type of cell death, the OH^● generated by Fenton or Haber-Weiss reactions abstracts a hydrogen from polyunsaturated fatty acids (PUFAs), forming a carbon-centered phospholipid radical. The addition of oxygen to this radical then produces a phospholipid hydroperoxide [197,198]. In turn, the resulting lipid hydroperoxides generate toxic reactive aldehydes, such as malondialdehyde (MDA), which inactivate essential proteins and enzymes, such as Glutathione Peroxidase 4 (GPX4). GPX4 uses GSH as an electron donor to reduce toxic lipid hydroperoxides [199]. However, in the presence of ferroptosis inducers, such as glutamate and erastin, there is depletion of GSH and inactivation of GPX4, resulting in a substantial increase in intracellular ROS levels and lipid peroxidation, resulting in necroptosis with compromised cell membrane integrity [197].

Necroptosis is a regulated form of necrosis characterized by cell rounding, cytoplasmic swelling, and membrane rupture. The mechanism involves different cell death receptors belonging to the Tumor Necrosis Factor Receptor (TNFR) superfamily, such as Fas and Tumor Necrosis Factor Receptor 1 (TNFR1). In general, when TNF-α interacts with TNFR1, there is recruitment of TNF Receptor-Associated Death Domain (TRADD), TNF Receptor-Associated Factor 2 (TRAF2), Cylindromatosis (CYLD), Cellular Inhibitor of Apoptosis Protein (cIAP) 1 and 2, and Receptor-Interacting Protein 1 (RIP1). RIP1 is deubiquitinated by CYLD, leading to the formation of complex IIb, also called necrossome, which consists of Receptor-Interacting Protein Kinase (RIPK) 1 and 3, and FADD. In situations of caspase 8 inactivity, Mixed Lineage Kinase Domain-Like protein (MLKL) is phosphorylated by RIPK3, which will be translocated to the plasma membrane, increasing permeability and resulting in cell death by necroptosis [200,201]. In the mechanism involving Fas, Fas Ligand (FasL) interacts with Fas, recruiting FADD, RIPK1, pro-caspase-8, and cellular FLICE-like Inhibitory Protein (cFLIP) to form Death-Inducing Signaling Complex (DISC). A necrosome is formed by a dysfunctional caspase, resulting in the phosphorylation of RIPK3 with subsequent phosphorylation of MLKL, causing necroptosis [202,203,204]. Furthermore, ROS can interact with Cys residues of RIPK1, inducing autophosphorylation and facilitating necrosome formation, thereby aiding in the process of necroptosis. In turn, RIPK1 and RIPK3 enhance mtROS production, creating a positive feedback loop [205].

PANoptosis is defined as an inflammatory pathway of programmed cell death with characteristics similar to pyroptosis, apoptosis, and/or necroptosis, but which cannot be explained by any of these pathways alone. This type of cell death is observed during viral, bacterial, and fungal infections, as well as autoimmune diseases and cancer, for example [206,207,208,209]. Thus, through Pathogen-Associated Molecular Patterns (PAMPs) or Damage-Associated Molecular Patterns (DAMPs), the mechanisms of this type of cell death are initiated. Initially, multiprotein complexes called PANoptosomes are formed, composed of caspase 8, FADD, RIPK1, RIPK3, MLKL, caspase 1, ASC, and gasedermin D. Next, the PANoptosome simultaneously activates caspase 8, RIPK1/RIPK3, and caspase 1, resulting in the activation of apoptosis, necroptosis, and pyroptosis pathways. As a result, there is rupture and pore formation in the membrane, in addition to the release of pro-inflammatory mediators that induce the activation of immune cells [196,210,211]. Thus, considering the role of ROS individually in the mechanisms of apoptosis and necroptosis, especially because they interact with Cys residues of kinases and caspases, they are also important modulators of PANoptosis.

Apoptosis is a genetically programmed cell death mechanism induced by intrinsic or extrinsic signals. The process involves chromatin condensation, DNA fragmentation, the formation of apoptotic bodies, and caspase activation, all without a sufficient stimulus to trigger an actual inflammatory response. The apoptosis process can occur through two pathways: intrinsic (or mitochondrial) and extrinsic [212,213].

The intrinsic pathway can be triggered by cellular stress, which includes excess ROS, for example. Altered ROS levels lead to an imbalance between pro-apoptotic (such as Bax and Bak) and anti-apoptotic (such as Bcl-2 and Bcl-xL) proteins of the Bcl-2 family, leading to increased mitochondrial outer membrane permeability (MOMP). Consequently, cytochrome c is released into the cytosol and associates with the protein apoptotic protease activating factor 1 (Apaf) and ATP/deoxyadenosine triphosphate (dATP), forming the apoptosome. In turn, the apoptosome recruits and activates caspase 9, which then activates executioner caspase 3. Once activated, caspase 3 promotes the degradation of essential cellular components [212].

The extrinsic pathway is activated by external stimuli and involves death ligands such as FasL, TNF-α, and TNF-related apoptosis-inducing ligand (TRAIL). These molecules bind to specific receptors on the target cell’s plasma membrane, such as Fas (CD95), TNFR1, and DR4/DR5. The ligand-receptor interaction promotes the recruitment of the adaptor protein Fas-associated death domain (FADD), followed by the formation of the DISC, which facilitates the activation of caspase-8. In turn, caspase-8 cleaves and activates effector caspases, such as caspase-3, which are responsible for degrading the cell’s structural and functional proteins. In this pathway, in certain situations, caspase-8 can also cleave the Bid protein, converting it into tBid, which can activate the intrinsic pathway, interconnecting the two pathways [213].

However, ROS appear to have a dual effect in the context of apoptosis. High levels of ROS can induce mitochondrial damage and increase mitochondrial permeability, consequently resulting in the release of cytochrome c and the subsequent activation of caspases 9 and 3 [214]. Mitochondrial dysfunction appears to be the main mechanism by which the intrinsic pathway is triggered by ROS. p66Shc is a mitochondrial intermembrane enzyme that, when active, interacts with complex III to subtract electrons. In turn, p66Shc oxidizes cytochrome c, resulting in H₂O₂, which induces the opening of the permeability transition pore, triggering cellular apoptosis. During mitochondrial dysfunction, there is oxidation of essential mitochondrial components, such as cardiopilin, a dimeric phospholipid high in unsaturated fatty acids that makes up the mitochondrial inner membrane [215]. Cardiopilin oxidation alters mitochondrial permeability, resulting in the formation of a pore that allows the release of ROS and cytochrome c into the cytosol. However, ROS induce the activation of ASK1, activating JNK and p38 MAPK, which can lead to the phosphorylation of anti-apoptotic proteins such as Bcl-2 [216]. ROS increase the expression of Fas and TRAIL through direct oxidative interactions, which can also occur with caspases important for the apoptosis pathways (caspase-8, caspase-9, and caspase-3) [216,217]. Furthermore, O₂^•− seems necessary for the induction of cellular apoptosis, since complete inhibition of SOD or excessive increase in stretch stress in cardiomyocytes resulted in apoptosis via ERK and JNK, with an increase in the level of the pro-apoptotic protein Bax. On the other hand, moderate SOD inhibition and lower stretch stress lead to cell proliferation and differentiation, allowing us to conclude that the induction of cell death depends mainly on the amount of intracellular ROS [218].

In turn, some studies show that low levels of ROS can have an anti-apoptotic effect by positively modulating survival pathways, such as NF-κB. Furthermore, the presence of O₂^•− inhibits FasL, preventing its association with CD95 and, consequently, interrupting the process of cell death by apoptosis through the extrinsic pathway [219,220].

2.3.5. Immune Response

ROS are essential for host defense, as illustrated by chronic granulomatous disease (CGD). In this immunodeficiency condition, individuals have mutations in the CYBB gene that encodes NOX2 on the X chromosome, resulting in an inactive NOX2. Consequently, this results in the loss of efficient microbicidal activity of phagocytes, and thus, individuals tend to have recurrent infections, granuloma formation, and a greater likelihood of developing autoimmune diseases [221,222].

In the inflammatory response, during the initial phase, increased O₂^•− production by resident and tissue cells at the injury site promotes increased vascular permeability and endothelial activation, contributing to the leukocyte recruitment process, as previously mentioned [89,140,223]. In turn, recruited neutrophils and macrophages produce large amounts of O₂^•− via NOX2 [60,224]. NOX2 can be induced by histamine, IL-1β, TNF-α, VEGF, for example, which induce the activation of Rac1, which promotes the binding of p67^phox to flavocytochrome b₅₅₈, resulting in the functional formation of NOX2 [60]. During the respiratory burst of activated neutrophils, H₂O₂, generated from O₂^•−, can be converted to HOCl⁻ in the presence of MPO, a reaction essential for the microbial function of these phagocytes [225]. Furthermore, phagocyte NOX2 is essential for phagocytosis and microbial clearance. For example, early in a bacterial infection, chemotactic compounds [bacterially derived N-formyl-Met-Leu-Phe (fMLF) and host complement-derived C5a] or local IL-8 release trigger NOX2 activation [226]. Furthermore, ROS generated in the phagosome, such as HOCl⁻, can selectively inactivate bacterial virulence factors, such as quorum sensing peptides of some Staphylococcus aureus strains [227].

Specifically in neutrophils, ROS participate in the formation of neutrophil extracellular traps (NETs) [228]. NETs are networks of extracellular fibers composed primarily of DNA and granular proteins that aim to capture microorganisms and limit infectious spread. Specifically, ROS promotes chromatin decondensation in the early stages of NET formation. Furthermore, NOX2 activation, with subsequent ROS production and associated Ca²⁺ increases, drives the phosphorylation of kinases such as PKC, contributing to the efficient formation of NETs [228,229,230,231].

ROS are essential for the activation of cytotoxic cells, such as natural killer (NK) cells and CD8⁺ T lymphocytes. In CD8⁺ T lymphocytes, ROS appear to enhance cellular activity. For example, low and sustained levels of ROS produced via NOX2 aid in the cross-presentation of antigens by dendritic cells (DCs) to CD8⁺ T lymphocytes. Concomitantly with NOX2 activation, Rac2, which migrates along with the other cytoplasmic subunits of the complex, leads to lipid oxidation of the endosomal membrane, aiding in the release of endosomal antigens to the cytosol and, thus, facilitating cross-presentation [232,233]. The primary role of ROS in CD8⁺ T cell-mediated immune defense is also demonstrated by intracellular infection models. p47^phox knockout mice have lower CD8⁺ T cell counts and higher Trypanosoma cruzi burden compared with wide-type (WT) mice [234]. Conversely, p45^pho−/⁻ mice infected with choriomeningitis virus have reduced viral titer and increased survival, demonstrating that ROS can mediate the amplification of inflammation and potential tissue-damaging effects of CD8⁺ T cells [235]. In NK cells, ROS inhibits the release of cytotoxic granules, decreases the expression of essential NK activating receptors (NKG2D, NKp46, and CD16ζ), and inhibits the phosphorylation of factors such as eIF2B, which results in lower cytotoxic activity and interferon (IFN)-γ production [236,237].

In the gastrointestinal and respiratory tracts, the expression of DUOX1 and DUOX2 ensures adequate ROS levels, contributing to mucosal immunity. In the airways, the production of H₂O₂ by these NOX isoforms results in the generation of microbicidal compounds through the oxidation of thiocyanate and iodide, modulated by lactoperoxidase. Furthermore, DUOXs silencing leads to increased intestinal infection by gut bacteria, which is reversed upon enzyme reintroduction [238,239,240].

Furthermore, in a setting of sterile inflammation, ROS can regulate transcriptional pathways that lead to the expression of IL-1α and granulocyte colony-stimulating factor (G-CSF), resulting in neutrophil migration to tissue lesions. XO can also activate the inflammasome pathway, generating both ROS and uric acid, a product of its catalytic reaction. In general, uric acid crystals generated by XO can act as alarmins when released into the extracellular medium due to a necrotic event, for example, and thus activate the TLR4/myeloid differentiation primary response 88 (MyD88)/TRAF6/NF-κB pathway, resulting in the production of pro-inflammatory cytokines, such as IL-1β [241]. Furthermore, the internalization of uric acid crystals through phagocytosis may be inefficient, as the crystals induce physical destabilization of lysosomal membranes during fusion between the phagocytic vacuole and lysosome. This destabilization leads to rupture of the lysosomal membrane and release of proteases into the intracellular environment, such as cathepsin B, which, associated with K⁺ efflux and the presence of ROS in the cytosol, promotes the oligomerization and assembly of the NLR-family pyrin domain containing 3 (NLRP3) inflammasome. Thus, NLRP3 activation leads to the cleavage of pro-caspase 1 into active caspase 1, responsible for generating active IL-1β and IL-18 [241]. XDH also contributes to the inflammatory response by promoting an increase in pro-inflammatory cytokines (IL-1β, TNF-α, and IFN-γ) [242,243]. Interestingly, XO/XDH activity can be induced by IFN-γ and is directly related to NF-κB activation, polymorphonuclear cell migration, and expression of pro-inflammatory cytokines (TNF-α and IL-1β) [244], contributing to oxidative stress.

Mitochondrial dysfunction resulting from an exacerbated increase in ROS production leads to mitochondrial DNA (mtDNA) leakage into the cytoplasm and culminates in the activation of pro-inflammatory signaling pathways. For example, mtDNA can activate cGAS and the STING-NFκB pathway, resulting in the production of pro-inflammatory cytokines. Furthermore, the hypermethylated CpG sequence in mtDNA is recognized by endosomal TLR9, which mediates the NF-κB pathway, leading to the expression of IL-1, IL-6, and TNF-α [215]. Another interesting point is that the oxidation of mtDNA (OX-mtDNA) in the cytosol can promote the activation of AIM2, which, together with the presence of cytoplasmic ROS, promotes the formation of NLRP3-ASC/Caspase-1 inflammasome complexes, culminating in the cleavage and activation of mature caspase-1, with subsequent generation of IL-1β and IL-18 [245].

3. Molecular Mechanisms and Signaling Pathways Involved in Superoxide Anion-Induced Pain

Reactive oxygen species (ROS) are critically important in the development of pain in several etiologies and O₂^•− plays an important role in the pathophysiology of pain, especially in inflammatory and neuropathic conditions. O₂^•−-induced pain involves cellular and molecular mechanisms, including activation of various signaling pathways described in Figure 3.

O₂^•− induce inflammation and pain by triggering the release a variety of pro-inflammatory and pronociceptive mediators, such as bradykinin (BK), serotonin (5-HT), histamine, NO, prostaglandins (PGs), and cytokines sensitizing neurons and activating ions channels [246]. In this regard, initially there is an increase in vascular permeability and endothelial activation, contributing to the activation of immune cells. Leukocyte recruitment, in addition to producing large amounts of O₂^•− via NOX2, are responsible for the release of pro-inflammatory cytokines (e.g., TNF-α, IL-1β, and IL-6) responsible for play a major role in the development of pain through direct peripheral sensitization [60].

Pain modulation by O₂^•− also occurs through the activation of inflammatory transcription factors, such as NF-κB that plays an important role in the regulation of redox-sensitive genes which are related to the pathogenesis of various diseases and leads to the expression of several pro-inflammatory mediators (COX-2, IL-6, TNF-α and IL-1β), amplifying the innate immune response [247]. As mentioned previously, mitochondria play a crucial role in the generation of ROS, and their dysfunction leads to the leakage of mtDNA into culminates in the activation of cGAS and consequent activation of the STING-NF-κB pathway, resulting in the production of pro-inflammatory cytokines and its oxidation can promote the activation of AIM2, which can promote the formation of NLRP3-Apoptosis-associated speck-like protein containing a CARD (ASC)/Caspase-1 inflammasome complexes [215,245]. In this sense, uric acid generated by the catalytic reaction of XO activation can activate the inflammasome pathway through the TLR4/MyD88/TRAF6/NF-κB pathway with subsequent generation of IL-1β and IL-18 [241].

PK influences the development of peripheral sensibilization (depolarize unmyelinated afferent neurons, sensitize afferent neurons, enhance currents in afferent neurons, and when inhibited can block sensitization in afferent neurons) and central sensibilization (activated both in the neurons and astrocytes) [248]. The increase in ROS enhances directly or indirectly the activity of PK suppression of dephosphorylation by one or more of the protein phosphatases (PP) 1, PP2A, and PP2B, a negative regulation that resets the steady state of target kinases at a higher level [249]. Hongpaisan et al., 2004 [250] found that Ca²⁺-dependent upregulation of mitochondrial O₂^•− production may be a general mechanism for linking Ca²⁺ entry to enhanced kinase activity, which is critical for sensitization of spinal neurons and persistent pain [250,251]. Overproduction of O₂^•− significantly contribute to intracellular activation of the calcium-dependent protein kinase Ca²⁺/calmodulin-dependent protein kinase II (pCamKII), which contributes to neuropathic pain and spinal dorsal horn neuronal hyperexcitability [252].

In neuropathic pain model, the O₂^•− -induces hyperalgesia amenable by intrathecal PKC inhibitor, indicating that spinal O₂^•− can also contribute to pain via the O₂^•−-PKC pathway [250]. Moreover, PKC is involved in the O₂^•−-induced NF-κB activation in human umbilical vein endothelial cells (hUVECs) [247]. Further, the translocation of PKCε to the plasma membrane, positively modulating signaling via transient receptor potential subfamily vanilloid 1 (TRPV1) and leading to increased activity contributing to the maintenance of nociceptive pain [37].

Through the activation and sensitization of nociceptors, O₂^•− acts as a key component in nociceptive pain being able to perform redox modifications on free thiol groups in ion channels, such as transient receptor potential (TRP) channels [37]. Cysteine residues located intracellularly in TRPV1 channels are affected by the oxidizing action of ROS, leading to the formation of intercysteine disulfide bonds that facilitate the channel activation [253]. Activation of intracellular pathways leads to increased transcription of the NGF gene acting crucially in the maintenance of chronic pain. It induces an increase in NOX expression, and this activates Rac1 and the expression of gp91^phox, resulting in increased TRPV1 expression via NOX through activation of PKC in neurons and astrocytes and MAPK downstream of peripheral nociceptive fibers, for example, via TrkA activation, inducing additional TRPV1 expression and subsequent increased mechanical [254].

The hyperalgesia results from a persistent state of peripheral afferent sensitization mediated by O₂^•−, which causes spinal sensitization through the release of the excitatory amino acid glutamate. O₂^•− also reacts with NO to form ONOO⁻ promoting inflammation, and favoring nitration of endogenous SOD2 in the spinal cord. Consequently, levels of O₂^•− remain elevated, favoring the maintenance of nociceptive signaling [246].

PARP activation has been implicated in the development of hyperalgesia, such as with the chronic use of opioids. Its activation occurs through DNA single-strand damage induced by O₂^•− and ONOO⁻ resulting in the depletion of its substrate NAD⁺ in vitro and a reduction in the rate of glycolysis triggers a rapid fall in intracellular ATP and cell injury [246]. Wang et al., 2004, shows that the development of pain by s O₂^•− through PARP activation in a Carrageenan injection leads to nitration of proteins, as detected in the periphery and in the spinal cord [246].

Glutamate released at the level of spinal cord and subsequent activation of the N-methyl-D-aspartate (NMDA) receptor, a subclass of excitatory amino acid receptor, releases O₂^•− and causes the spinal cord neuron to become more responsive to all its inputs, resulting in central sensitization being fundamental in the development of hyperalgesic responses associated with pain of several etiologies [255]. Indeed, enhanced glutamatergic signaling is also associated with TRPV1 activation and facilitation of long-term potentiation, which are important contributors to central sensitization [35].

Upon such glutamatergic receptor stimulation, PKC can be activated, and PKC activity leads to phosphorylation and activation of receptors, such as the NMDA receptor leading to enhanced NR1 phosphorylation, as well as increased COX-2 protein expression [256]. Modulation of COX-1 and activation/induction of COX-2 influences central sensitization through PGE2 that contribute to the development of peripheral sensitization associated with inflammation [257]. Furthermore, O₂^•− increase the production of PGs from macrophages by acting post transcriptionally or translationally to increase COX-2 protein levels or to increase its mRNA stability [258].

Taken together some of the hall toks, O₂^•− has been shown to be involved in signaling pathways modulates PK, ion channels, alters glutamatergic neurotransmission and neuroinflammation contributing to the development of central sensitization. In this review, we will also briefly discuss in the next section the current therapeutics to contribute to future directions for the targeting of these important species in different contexts of pain and inflammation.

4. Molecular Mechanisms and Signaling Pathways Involved in Potassium Superoxide (A O₂^•− Anion Donor)-Induced Pain

KO₂ is an O₂^•− donor widely used in murine models to induce inflammatory responses and trigger reactive oxygen species (ROS)-mediated nociceptive responses. In this context, KO₂ administration promotes a pro-oxidant and inflammatory environment capable of sensitizing peripheral nerve endings and CNS cells, triggering intense nociceptive responses. Several intracellular signaling pathways participate in the mediation and amplification of these effects, including classical inflammatory pathways and mechanisms associated with oxidative stress, pro-inflammatory cytokine expression, and glial activation [32,33,39,41,259,260,261,262,263]. Below, in this section and in Figure 4, the main molecular pathways involved in the modulation of pain and inflammation induced by KO₂ are described, with emphasis placed on their mediators and functional interactions.

Directly, KO₂ administration can trigger nociceptive responses, such as mechanical and thermal hyperalgesia, and overt pain behaviors (e.g., abdominal contortions, flinches, and paw licking), as well as acts on the development of edema. In addition, KO₂ induces inflammatory molecular patterns, including increased recruitment of mononuclear and polymorphonuclear leukocytes, intensification of oxidative stress, reduction in local antioxidant defenses, and increased production of pro-inflammatory cytokines such as IL-1β, IL-33, and TNF-α [32,33,39,41,259,260,261,262,263].

The generation of oxidative stress is one of the main pathways by which KO₂ induces nociceptive and inflammatory responses. As seen previously, oxidative stress is an imbalance between oxidants and antioxidants that can cause cellular damage, such as cell death and oxidation of lipids, proteins, and DNA [6]. Different studies have shown that KO₂ administration is responsible for increasing O₂^•− levels, ONOO⁻ production, lipid peroxidation, and gp91^phox mRNA expression (a subunit of NADPH oxidase that catalyzes the production of O₂^•−). In contrast, KO₂ induces the depletion of endogenous antioxidants and the mRNA expression of Nrf2. Nrf2 is a transcription factor responsible for activating the expression of antioxidant enzymes, such as heme-oxygenase (HO-1). Thus, one of the consequences of Nrf2 depletion is the decrease in HO-1 expression, also observed after KO₂ administration [32,33,39,41,260,261,262,263].

Studies have shown that KO₂-induced nociception is also dependent on the generation of inflammatory mediators, such as cytokines. Pro-hyperalgesic cytokines, such as IL-1β and TNF-α, participate in pain modulation by binding to cytokine receptors present in nociceptive neurons and sensitizing them. During inflammatory processes, resident and recruited cells release inflammatory cytokines (e.g., IL-1β and TNF-α), which interact with nociceptive neurons and trigger intracellular signaling pathways, leading to ion channel opening (e.g., Nav1.7, Nav1.8, Nav1.9, TRPV1, and TRPA1), depolarization, and increased neuronal excitability [32]. Cytokines also stimulate the production of PGE2 through COX-2, which, in addition to generating PG, produces O₂^•−. This, in turn, induces the expression of more COX-2 and intensifies hyperalgesia [257]. In this context, different studies confirm that the administration of KO₂ is responsible for increasing the expression of mRNA and the production of pro-inflammatory cytokines, such as IL-1β and TNF-α, in the periphery and spinal cord [32,33,39,260,261,262,263].

Furthermore, recent studies demonstrate that the IL-33/ST2 signaling pathway plays a crucial role in modulating pain and inflammation triggered by KO₂. IL-33 is a pleiotropic cytokine of the IL-1 family that acts as a key mediator in inflammation and regulation of innate and adaptive immune responses. It is produced by immune, endothelial, and glial cells (mainly oligodendrocytes in the spinal cord) [264] and exerts its effects by binding to the ST2 receptor [259]. Borghi et al., 2025 [259], observed that KO₂ administration induced mechanical and thermal hyperalgesia, edema, neutrophil recruitment, overt pain behaviors, and glial (astrocytes, microglia and oligodendrocytes) and neuronal activation in the spinal cord. These effects were associated with increased IL-33 levels in the plantar tissue and spinal cord of the animals. Taken together, mice with ST2 receptor deficiency (ST2^−/−) showed a significant reduction in these effects. Thus, these findings reinforce the idea that the IL-33/ST2 pathway modulates both local and spinal cord inflammatory events, contributing significantly to the O₂^•−-induced pain response [259].

The increase in cytokine levels observed after KO₂ administration may be due to increased activation of the NF-κB transcription factor. NF-κB is a redox-sensitive component essential for the development of inflammation, acting mainly in the modulation of inflammatory mediators [257]. Pinho-Ribeiro et al., 2016 [263], demonstrated that KO₂ administration induces the degradation of IκBα, a cytoplasmic inhibitor of NF-κB, in the periphery, with a more pronounced degradation 3 h after stimulation with KO₂. In addition, it was possible to observe that KO₂ increased the phosphorylation of the p65 subunit of NF-κB in the periphery and spinal cord. These findings indicate that KO₂ activates the classical NF-κB pathway in a time-dependent manner, both peripherally and in the spinal cord, directly contributing to the increased expression of inflammatory cytokines and, consequently, to the intensification of the O₂^•−-induced inflammatory and nociceptive response [263]. Similar effects have been demonstrated in other studies [32,261].

KO₂-induced nociception is also dependent on COX-2 and endothelin 1 (ET-1). COX-2 is an enzyme that plays an important role in pain and inflammation, mainly through the production of PG [32,33]. Maioli et al., 2015 [41], demonstrated that the administration of KO₂ induces a significant increase in COX-2 expression in the plantar tissue of mice. The increase in the expression of this enzyme is one of the main mechanisms responsible for the hyperalgesia and inflammation observed in the model. The pro-nociceptive effect of KO₂ was reversed by treatment with celecoxib, a selective COX-2 inhibitor, indicating that this is an important pathway in the modulation of O₂^•−-induced pain [41]. Other studies reaffirm that the pronociceptive effects of KO₂ are dependent on COX-2 production [32,33,262].

ET-1 is a potent vasoconstrictor peptide involved in cardiovascular and inflammatory diseases. In pain and inflammation, it promotes neutrophil activation, increases the expression of adhesion molecules, cytokine production, vascular permeability, and nociceptive sensitivity. ET-1 acts directly on neurons and indirectly through mediators such as cytokines, prostanoids, and ROS [39]. Serafim et al., 2015 [30], explored the role of ET-1 in KO₂-induced pain and inflammation. The authors observed that administration of KO₂ induces the expression of preproET-1 mRNA in plantar tissue and spinal cord. In contrast, the administration of an ET-1 receptor antagonist was responsible for inhibiting mechanical and thermal hyperalgesia, overt pain behaviors, edema, production of pro-inflammatory cytokines, and neutrophil recruitment. These findings suggest that the ET-1/receptor pathway plays an essential role in modulating O₂^•−-induced pain and inflammation [39]. Other studies reaffirm that the pro-nociceptive effects of KO₂ are dependent on the production of ET-1 [32,33,260,262].

Taken together, studies demonstrate that the nociceptive effects of KO₂ may be related to the TNF-α signaling pathway and TNF receptors 1 and 2 (TNFR1 and TNFR2, respectively) [40]. TNF-α, a pro-inflammatory cytokine, can sensitize nociceptive neurons indirectly, by stimulating the production of PGE2, or directly, through the activation of TNFR present in the neurons themselves. In addition, TNF-α is capable of modulating neutrophil recruitment and O₂^•−-generating systems. These pathways of action contribute to increased pain sensitivity and oxidative stress, resulting in hyperalgesia [265,266,267].

Yamacita-Borin et al., 2015 [40], demonstrated that TNFR1 deficiency (TNFR1^−/−) and treatment of wild-type mice with etanercept (a soluble TNFR2 receptor) inhibit KO₂-induced nociceptive behaviors. Together, TNFR1⁻/⁻ mice were protected from oxidative stress, and treatment of wild animals with apocynin (a NADPH oxidase inhibitor) or 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) (a mimetic of SOD) inhibited TNF-α-induced mechanical hyperalgesia and neutrophil recruitment. These findings support the idea that TNFα/TNFR1/TNFR2 significantly signaling contributes to the mechanisms of oxidative stress and pain perpetuation in a KO₂-induced model. This signaling establishes a positive feedback loop in which the O₂^•−, donated by KO₂, stimulates the release of TNF-α, and this, in turn, activates cellular mechanisms that intensify the endogenous production of O₂^•−, amplifying pain and inflammation [40].

Finally, the detection of nociceptive stimuli in the periphery by primary sensory neurons initiates the signaling of the painful sensation, which propagates to the spinal cord. At this site, glial cells are activated, which begin to release inflammatory mediators capable of sensitizing nociceptors and promoting neuroinflammation [33]. In this context, studies have shown that KO₂ also performs its functions at the central level. Bernardy et al., 2017 [33], demonstrated that the administration of KO₂ increases the expression of GFAP (marker of astrocytic activity) and Iba-1 (marker of microglial activity) mRNA in the spinal cord. This increase directly contributes to the hyperalgesia observed in the periphery. These data reinforce that KO₂ not only acts peripherally but also exerts central effects by inducing the activation of astrocytes and microglia in the spinal cord, contributing to pain maintenance and amplification through glial neuroinflammation [33].

In summary, the data presented demonstrate that KO₂ induces intense nociceptive responses through interconnected mechanisms of oxidative stress, inflammation, and glial activation. The generation of O₂^•− triggers cellular damage and activates pro-inflammatory signaling pathways, such as NF-κB, as well as promoting the increase in cytokines, COX-2 and ET-1, all elements that amplify pain and inflammation. Additionally, the involvement of the IL-33/ST2 pathway and TNFα/TNFR signaling highlights the central role of neuroimmune interactions in this model. Thus, KO₂ constitutes a robust experimental tool to study pain associated with oxidative stress and offers important insights for the development of more effective and targeted analgesic therapies.

5. Therapeutic Approaches

In view of the central role of O₂^•− in the genesis of inflammatory pain, therapeutic strategies have been directed to neutralize its synthesis, modulating the metabolism and actions triggered by this radical. In preclinical models, a wide range of therapeutic agents have been investigated in different disease models, which there is the presence and participation of O₂^•− in the development of pain. These experimental treatments seek to reduce oxidative stress and nociception through different mechanisms, offering promising prospects for the management of pain associated with ROS. In this section, we present the main therapeutic approaches in the preclinical phase of development directed at O₂^•−, potassium superoxide, and the mechanisms triggered by the presence of this radical. Figure 5 summarizes the general mechanisms by which the treatments act (the specifications of each treatment and experimental model are found in the sections below).

5.1. Superoxide Anion-Induced Pain

The generation of O₂^•− is an initial step in the production of other reactive oxygen species (ROS) and RNS and consequently influences several cellular signaling pathways, contributing significantly to the pathophysiology of several inflammatory diseases. In view of this, studies show that O₂^•− inhibition promotes the reduction in pain and inflammation (

Table 1. Therapeutic Approaches Targeting Superoxide Anion-Induced Pain.

Treatment	Experimental Model	Route of Administration and Dose	Model	Analgesic Effects	Mechanisms of Action	Reference
Apocynin	Rats	Intratecal 0.1–0.6 nmol/day	Morphine tolerance	Inhibited thermal hyperalgesia	Blocking NADPH oxidase activation	[268]
Apocynin	Rats	Intraperitoneal 100 mg/kg	Streptozotocin-induced diabetes	Ameliorated hyperglycemia-induced hyperalgesia and prevented sciatic nerve damage	NADPH oxidase inhibition	[269]
Allopurinol, SOD L-NAME	Rats	Intraperitoneal 40 mg/kg 4000 U/kg 10 mg/kg	CRPS	Mechanical allodynia reduction	Activation of NMDA receptor phosphorylation in the spinal dorsal horn	[270]
Allopurinol, SOD L-NAME	Rats	Intraperitoneal 40 mg/kg	CIPIP	Alleviation in mechanical and cold allodynia	XO inhibition	[271]
Sulforaphane	Mice (C57BL/6)	Intrathecal 10 mg/kg or 50 mg/kg Intraperitoneal 50 μmol/kg or 50 mg/kg	SNT	Allodynia and inflammatory hyperalgesia reduction	Inducing nuclear translocation of Nrf-2 with subsequent HO-1 expression in myeloid cells, including the microglia	[272]
PBN TEMPOL	Mice (C57BL/6)	Intraperitoneal 100 mg/kg 300 mg/kg	Antimycin A	Mechanical hyperalgesia reduction	Transient antinociception	[273]
TEMPOL	Rats	60 mg/kg	Carrageenan	Inhibition of edema and hyperalgesia	SOD mimetics	[274]
TEMPOL	Rats	15, 30, and 60 mg/kg	Carrageenan DETCA	Attenuates paw edema and thermal hyperalgesia	SOD mimetics	[274]
MnL4	Rats	Intraperitoneal or subcutaneously 15 mg kg−1	Carrageenan, CFA, and MIA	Decrease mechanical hypersensitivity and prevent the hind limb weight bearing alterations	SOD mimetic inhibition of PARP activation	[275]
M40403	Rats	1–10 mg/kg	Carrageenan	inhibition of edema and hyperalgesia	Antinociceptive effect	[246]
SOD-loaded nanoparticles	Mice (C57BL/6)	Intra-articular 500 U/mL	OA DMM	Allodynia reduction	Reduced ROS production and the synthesis of catabolic proteases	[276]
TEMPOL	Rats	Intrathecal 1 mg/kg	SCI	Mechanical sensitivity decrease	SOD mimetics	[252]
Porphyrin PNDCs	Rats	Intrathecal	Morphine	Attenuates morphine hyperalgesia and antinociceptive tolerance	Reduces PN-mediated mitochondrial nitroxidative stress in the spinal cord	[277]
Apocynin and DPI	Rats	Intrathecal 100 mg/kg 1 mg/kg	Morphine	Blocked spinal NADPH oxidase activation	NADPH oxidase inhibition	[278]

The table presents the main therapeutic approaches investigated for the management of O₂^•−-induced pain. For each intervention, the experimental animal model, the route of administration and dose of the treatment, the experimental disease model, the analgesic effects, and the mechanisms of action are described. Abbreviations: NADPH (nicotinamide adenine dinucleotide phosphate); SOD (superoxide dismutase); CRPS (Complex Regional Pain Syndrome); CIPIP (chronic post-ischemia pain); NMDA (N-methyl-D-aspartate); SNT (spinal nerve transection); Nrf2 (nuclear factor erythroid 2-related factor 2); HO-1 (heme oxygenase 1); PBN (phenyl-N-tert-butylnitrone); TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl); DETCA (SOD inhibitor diethylthiocarbamate); MnL4 (Manganese transporter protein MntH-like 4); CFA (complete Freund’s adjuvant); MIA (Melanoma Inhibitory Activity protein); PARP (poly(ADP-ribose) polymerase); OA (osteoarthritis); DMM (Destabilization of the Medial Meniscus); ROS (reactive oxygen species); XDH (xanthine dehydrogenase); SCI (spinal thoracic contusion injury); PNDCs (peroxynitrite decomposition catalysts); DPI (diphenyleneiodonium).

), highlighting the importance of this reactive species for the development of inflammatory pain.

Considering the role of NADPH oxidase in pain generation, several studies aim to inhibit it as a mechanism for analgesia. Thus, Apocynin, an NADPH oxidase inhibitor, has been explored in diverse experimental models of oxidative stress. Doyle et al., 2010 [269], demonstrated that activation of spinal NADPH oxidase contributes to the development of morphine-induced hyperalgesia and antinociceptive tolerance from the intrathecal administration of apocynin that blocks these events inhibiting thermal hyperalgesia [278]. In addition, intraperitoneal administration of Apocynin also contributes to the control of hyperglycemia-induced hyperalgesia and prevented sciatic nerve damage developed from O₂^•− by acting both at spinal and supraspinal sites [269].

During morphine-induced hyperalgesia and antinociceptive, intrathecal injections of PNDCs (peroxynitrite decomposition catalysts) that accumulate within mitochondria, MnTE-2-PyP⁵⁺ and MnTnHex-2-PyP⁵⁺, blocks hyperalgesia and tolerance as well as activation of transcription pathways that play an essential role in the development of neuroimmune activation such as NF-κB, p38, and ERK [277].

Microglia activation and subsequent pro-inflammatory cytokine expression are responsible for enhanced pain hypersensitivity. In peripheral nerve injury the NOX2-mediated O₂^•− generation is involved in spinal nerve transection (SNT)-induced spinal cord microglia activation and pro-inflammatory cytokine expression. Despite this, Sulforaphane, an isothiocyanate molecule with antioxidant effects by increasing HO-1 expression via Nrf2 activation was able to reduce allodynia and inflammatory hyperalgesia acting as a strong suppressor of ROS production [272].

ROS, such as O₂^•−, increase the phosphorylation of several receptors to induce central sensitization including the NMDA receptor that plays an important role in the regulation of ischemic brain injury [279]. Ryu et al., 2010 [270], suggest that the use of ROS scavengers, such as SOD and L-NAME and allopurinol blocking O₂^•− as effective therapeutics for patients of chronic pain related to ischemic injury for reducing mechanical allodynia in a model of chronic post-ischemia pain (CPIP) [270]. Kwak et al., 2009 [271], reported allopurinol as xanthine oxidase inhibition and demonstrate significantly reduces pain in CPIP model [271].

In fact, ROS scavengers have been widely explored due to their antinociceptive effects in pain control [276]. In mice treated with antimycin A (a complex III inhibitor), phenyl-N-tert-butylnitrone (PBN) and TEMPOL were able to reduce mechanical hyperalgesia demonstrating their effects in reducing pain perception [273]. Lee et al., 2021 [252], examined O₂^•−-mediated excitatory nociceptive transmission on at-level neuropathic pain following spinal thoracic contusion injury (SCI) and the intrathecal administration of TEMPOL decreased mechanical sensitivity with reduction in O₂^•− and consequent decreases the mechanical hypersensitivity and pCamKII-induced hyperexcitability of spinal neurons in the spinal dorsal horn [252]. In addition, TEMPOL injection prohibited carrageenan-induced paw edema in rats even with stress produced by endogenous O₂^•− via SOD inhibition and prevented the intensification of such hyperalgesia by O₂^•− [274]. Unfortunately, because they are non-selective scavengers of nitroxidative species, the use of antioxidants such as PBN or TEMPOL cannot be used to assess the specific contribution of O₂^•− [245].

The first compound studied as an SOD mimetic (SODm) was a metal complex with SOD enzymes (FeSOD, MnSOD, NiSOD, Cu, ZnSOD). The catalysis occurs at the redox active metal site, which can efficiently catalyze the dismutation of O₂^•− and accumulate in mitochondria [280]. While MnSOD immunoreactivity occurs strongly in neurons and in glia surrounding blood vessels, the expression of Cu, ZnSOD is found predominantly in astrocytes; nevertheless, both exert effects at the cellular level within the nervous system [245]. In addition, M40403 [manganese (II) complex with a bis(cyclo-hexylpyridine-substituted) macrocyclic ligand] has been shown to lessen undesired side effects of inflammation and blocked parameters of hyperalgesia highlighted that O₂^•− is formed and plays a major role in the development of pain through direct peripheral sensitization by blocks cytokine release is by preventing the activation of redox-sensitive transcription factors, including NF-κB and AP-1, by O₂^•−, which in turn regulates the genes that encode pro-inflammatory and pronociceptive cytokines [246].

Given the importance of SOD in reducing anion toxicity, the activity of some polyamine-polycarboxylate-Mn^II complexes such as MnL4 have been shown reduce O₂^•− generated enzymatically (xanthine/XO) or by formyl-methionyl-leucyl-phenylalanine- (fMLP-) activated macrophages via a direct antioxidant mechanism and decreasing the nociceptive nervous fiber activation [275]. Gui et al., 2022 [276], tested an antioxidative nanoparticle based on SOD-loaded porous polymersome nanoparticles (SOD-NPs) for delivery of SOD to mouse knee joints attenuating allodynia in osteoarthritis (OA) model, thus, the direct use of antioxidant enzymes characterized as an effective drug delivery system to knee joints [276].

It is well known that removing O₂^•− attenuates diverse pain conditions. Herein, treatment with the ROS scavenger, SOD mimetic, antioxidant molecules, blocking the sensation of pain, NADPH inhibition, rapidly inhibited neuronal discharge in response to a different stimulation, demonstrating the importance in maintaining pain and the beneficial effects of controlling the generation of ROS through efficient attenuation of neuronal hyperexcitability and consequent control of pain perception.

5.2. Therapeutic Approaches Targeting Potassium Superoxide-Induced Pain

ROS-induced pain, such as KO₂, represents a significant challenge in understanding the nociceptive mechanisms associated with oxidative stress. As previously seen, KO₂ acts as an O₂^•− donor, promoting an inflammatory and oxidative state that sensitizes peripheral and central nerve endings. In view of this, several therapeutic approaches have been investigated with the aim of mitigating the pro-nociceptive effects triggered by this molecule.

Table 2. Therapeutic approaches targeting potassium superoxide-induced pain.

Treatment	Experimental Model	Route of Administration and Dose	Analgesic Effects	Mechanisms of Action	Reference
Morphine	Mice (Swiss)	Intraplantar 12 µg/paw	Reduction in mechanical and thermal hyperalgesia, and inhibition of overt pain behaviors	-	[41]
Quercetin	Mice (Swiss)	Intraperitoneal 100 mg/kg	Inhibited mechanical and thermal hyperalgesia, paw edema, and overt pain behaviors	Inhibited the recruitment of total leukocytes, mononuclear and polymorphonuclear leukocytes into the peritoneal cavity. Reduced oxidative stress in plantar tissue	[41]
Celecoxib	Mice (Swiss)	Intraperitoneal 30 mg/kg	Inhibited mechanical and thermal hyperalgesia, and overt pain behaviors	Inhibited COX-2 mRNA expression in plantar tissue	[41]
Bosentan	Mice (Swiss)	Oral 100 mg/kg	Inhibited mechanical and thermal hyperalgesia, overt pain behavior, and paw edema	Inhibited MPO activity in plantar tissue, leukocyte recruitment into the peritoneal cavity, and pro-inflammatory cytokine production. Increased anti-inflammatory cytokine production and antioxidant capacity. Induced prepro-ET-1 mRNA expression	[39]
TEMPOL	Mice (Balb/c)	Intraperitoneal 100 mg/kg	Inhibited mechanical and thermal hyperalgesia, and paw edema	Inhibited the mRNA expression of pro-inflammatory cytokines, COX-2 and prepro-ET-1. Improved the depletion of antioxidant capacity, and inhibited oxidative stress and glial cell activation	[33]
Pyrrolidine Dithiocarbamate	Mice (Swiss)	Subcutaneous 100 mg/kg	Inhibits mechanical and thermal hyperalgesia, and paw edema	Inhibits activity of MPO and NAG in plantar tissue, the recruitment of leukocytes into the peritoneal cavity, the degradation of IkBa, the activation of NF-kB, the production of pro-inflammatory cytokines and oxidative stress.	[263]
		Intrathecal 300 µg/animal		Inhibits activity of MPO in plantar tissue
Vinpocetine	Mice (Swiss and LysM-eGFP)	Oral 30 mg/kg	Inhibited mechanical and thermal hyperalgesia, overt pain behavior, and paw edema	Restored local antioxidant capacity, increased Nrf2 and HO-1 expression. Reduced leukocyte recruitment in plantar tissue and peritoneal cavity, oxidative stress, pro-inflammatory cytokine production, ET-1 and COX-2 expression, and NF-κB activation	[32]
Curcumin	Mice (Swiss)	Subcutaneous 10 mg/kg	Inhibited mechanical and thermal hyperalgesia, and overt pain behaviors	Inhibited leukocyte recruitment, MPO activity, pro-inflammatory cytokine production, NF-kB activation, and oxidative stress. Increased Nrf2 and HO-1 mRNA expression	[261]
Naringenin	Mice (Swiss)	Oral 50 mg/kg	Inhibited mechanical and thermal hyperalgesia, and overt pain behaviors	Inhibited MPO activity, oxidative stress, pro-inflammatory cytokine production, and gp91phox, COX-2, and preproET-1 mRNA expression. Increased antioxidant capacity and Nrf2 and HO-1 mRNA expression. Activated NO-cGMP-PKG-ATP (KATP)-sensitive potassium channels	[262]
Clazosentan	Mice (Swiss)	Intraplantar 30 nmol/animal	Inhibited mechanical and thermal hyperalgesia, overt pain behaviors, and edema	Inhibited MPO activity, oxidative stress and pro-inflammatory cytokine production	[260]
		Intraperitoneal 30 nm/animal	Inhibited overt pain behavior	Inhibited the recruitment of total leukocytes
BQ-788	Mice (Swiss)	Intraplantar 30 nmol/animal	Inhibited mechanical and thermal hyperalgesia, overt pain behaviors, and edema	Inhibited MPO activity and oxidative stress	[260]
		Intraperitoneal 30 nm/animal	Inhibited overt pain behavior	Inhibited the recruitment of total leukocytes
Etanercept	Mice (C57BL/6)	Intraperitoneal 10 mg/kg	Inhibited mechanical hyperalgesia and overt pain behaviors	Inhibited MPO activity	[40]
Sodium Propionate	Rats (Sprague-Dawley)	Intraplantar 30 and 100 mg/kg	Inhibited paw edema, nociceptive stimulation and overt pain behaviors	Reduced the recruitment of inflammatory cells, tissue morphological changes, and MPO activity. Increased the expression of SOD2, HO-1, and GSH.	[281]

The table presents the main therapeutic approaches investigated for the management of KO₂-induced pain. For each intervention, the experimental model, route of administration and treatment dose, analgesic effects, and mechanisms of action are described. Abbreviations: COX-2 (cyclooxygenase-2); mRNA (messenger ribonucleic acid); MPO (myeloperoxidase); ET-1 (endothelin 1); NAG (N-acetylglucosaminidase); NF-kB (nuclear factor kappa B); LysM (lysozyme M); eGFP (enhanced green fluorescent protein); Nrf2 (nuclear factor erythroid 2-related factor 2); HO-1 (heme oxygenase 1); TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl); SOD (superoxide dismutase); GSH (reduced glutathione).

summarizes the main strategies under study, as well as their analgesic effects and mechanisms of action.

Maioli et al., 2015 [41], evaluated the analgesic and anti-inflammatory action of three different treatments in the murine model of pain and inflammation induced by KO₂, namely morphine (analgesic opioid), quercetin (antioxidant flavonoid), and celecoxib (anti-inflammatory COX-2 inhibitor). Morphine treatment was effective in reducing mechanical and thermal hyperalgesia, and overt pain behaviors (flinches and paw licking) in a dose-dependent manner. This analgesic effect was reversed when morphine was administered together with naloxone, which confirms that its action was mediated by classical opioid receptors and that this pathway participates in the modulation of KO₂-induced pain [41].

Quercetin treatment also inhibited mechanical and thermal hyperalgesia, paw edema, and overt pain behaviors (abdominal contortions, flinches, and paw licking) in a dose-dependent manner. These effects were mediated by a reduction in total leukocyte recruitment (mononuclear and polymorphonuclear) and by an increase in antioxidant capacity and, consequently, inhibition of oxidative stress at the site of inflammation. Similarly, celecoxib treatment significantly inhibited mechanical and thermal hyperalgesia and overt pain behaviors (abdominal contortions, flinches, and paw licking). The analgesic effect of the treatment was due to the suppression of COX-2 mRNA expression, indicating that the COX-2 pathway is determinant in the amplification of pain and inflammation induced by KO₂ [41].

Serafim et al., 2015 [39], evaluated the effects of bosentan, a mixed antagonist of ET-1 and endothelin 2 (ET-2) receptors, in the KO₂-induced of pain and inflammation model. As a result, they observed that the treatment was efficient in inhibiting mechanical and thermal hyperalgesia, overt pain behaviors (abdominal contortions, flinches and paw licking) and edema. These effects were mediated by reduction leukocyte recruitment both in the paw and in the peritoneal cavity. In addition, bosentan inhibited oxidative stress and increased antioxidant capacity at the site of inflammation, decreased the production of pro-inflammatory cytokines (IL-1β and TNF-α), and increased the production of anti-inflammatory cytokines (IL-10) in the paw and spinal cord. Finally, it was observed that the treatment blocks the increased expression of preproET-1 mRNA in both the paw and spinal cord, indicating that endothelin plays a central role in amplifying pain, inflammation, and oxidative stress in this model [39].

Other studies have also evaluated the analgesic and anti-inflammatory effects of molecules in the context of KO₂-induced pain and inflammation. Bernardy et al., 2017 [33], evaluated the effects of TEMPOL, a SOD mimetic. Like previous studies, they observed that TEMPOL administration significantly reduced mechanical and thermal hyperalgesia, and paw edema. Furthermore, the treatment inhibited increased mRNA expression of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-10), COX-2, preproET-1, and Nrf2 in plantar tissue and spinal cord, and inhibited increased mRNA expression of glial cells in the spinal cord. They also observed that TEMPOL has an antioxidant and cellular protection action, as it was responsible for blocking the depletion of local antioxidants, increased expression of gp91^phox, and oxidative stress [33].

Pinho-Ribeiro et al., 2016 [263], evaluated the effects of treatment with pyrrolidine dithiocarbonate (PDTC), a chemical compound used as a metal chelating agent. Initially, it was observed that subcutaneous treatment with PDTC attenuated mechanical and thermal hyperalgesia, and paw edema. This effect was due to a reduction in neutrophil and macrophage recruitment at the site of inflammation. Together, the treatment prevented the activation of NF-κB, both locally and centrally, interrupting the pro-inflammatory pathway. Because of the blockade of this pathway, a significant decrease in pro-inflammatory cytokines (IL-1β, TNF-α, and IL-10), NO levels and lipid peroxidation in plantar tissues and spinal cord was observed. Similar analgesic effects have been observed with intrathecal treatment with PDTC [263].

The effects of treatment with vinpocetine, a drug of the nootropic class, have also been investigated in this context. Lourenço-Gonzalez et al., 2019 [32], demonstrate that treatment with vinpocetine reduces mechanical and thermal hyperalgesia, overt pain behaviors (abdominal contortions, flinches, and paw licking), and edema. This drug also showed anti-inflammatory activities by reducing the recruitment of neutrophils and macrophages to the plantar tissue and peritoneal cavity. Local antioxidant capacity was restored after treatment and there was an increase in gene expression of protective factors such as Nrf2 and HO-1, together with a reduction in O₂^•− production and gp91phox expression. Finally, vinpocetine reduced ET-1 and COX-2 mRNA expression, pro-inflammatory cytokine levels (IL-1β, TNF-α, and IL-33), and activation of transcription factor NF-κB. These data demonstrate that vinpocetine, in addition to being a nootropic, can be used as an analgesic and anti-inflammatory alternative, especially in conditions of pain associated with oxidative stress [32].

Similar effects were observed in the murine model of KO₂-induced pain and inflammation after curcumin treatment. Fattori et al., 2015 [261], demonstrated that curcumin has potent analgesic effects by inhibiting mechanical and thermal hyperalgesia, and overt pain behaviors (abdominal contortions, flinches and paw licking). Regarding anti-inflammatory effects, curcumin inhibited the recruitment of total leukocytes (mononuclear and polymorphonuclear), and MPO activity. Together, the treatment reduced the activation of the transcription factor NF-κB and, consequently, the levels of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-10) and oxidative stress. On the other hand, curcumin acted by activating antioxidant defenses by increasing the expression of Nrf2 and HO-1 mRNA [261].

Manchope et al., 2016 [262], sought to evaluate the effect and mechanism of action of the flavonoid naringenin in a murine model of KO₂-induced pain and inflammation. As a result, it was observed that treatment with naringenin reduced, in a dose-dependent manner, mechanical and thermal hyperalgesia, and overt pain behaviors (abdominal contortions, flinches, and paw licking). The analgesic effect was abolished in the presence of inhibitors of the NO-cGMP-PKG-ATP-ATP-sensitive potassium channel signaling pathway, indicating that their action depends on this mechanism of action. In addition, a decrease in neutrophil recruitment (assessed through MPO activity) was observed, as well as a reduction in tissue oxidative stress, pro-inflammatory cytokine production (TNF-α, IL-10, and IL-33), and mRNA expression of gp91^phox, COX-2, and preproET-1. In parallel, naringenin activated the antioxidant response by restoring GSH levels and increasing Nrf2 and HO-1 mRNA expression. These results highlight the potential of naringenin as a multimodal agent with analgesic, anti-inflammatory, and antioxidant properties in the context of oxidative-stress-mediated pain [262].

Fattori et al., 2017 [260], evaluated the effect of treatment with clazosentan and BQ-788, selective antagonists of endothelin (ET) A e ETB receptors, respectively, in a murine model of pain and inflammation induced by KO₂. Treatment with clazosentan significantly reduced mechanical and thermal hyperalgesia, overt pain behaviors (abdominal contortions, flinches, and paw licking), and paw edema. These effects were due to a reduction in MPO activity in plantar tissue, the recruitment of total leukocytes (mononuclear and polymorphonuclear) to the peritoneal cavity, oxidative stress, and the production of pro-inflammatory cytokines (IL-1β and TNF-α) at the peripheral and central levels. Similarly, treatment with BQ-788 significantly reduced mechanical and thermal hyperalgesia, overt pain behaviors (abdominal contortions, flinches, and paw licking), and paw edema. These effects were due to a reduction in MPO activity in plantar tissue, the recruitment of total leukocytes (mononuclear and polymorphonuclear) to the peritoneal cavity, and oxidative stress at the peripheral and central levels. However, treatment with BQ-788 was not efficient in reducing the levels of pro-inflammatory cytokines [260].

Yamacita-Borin et al., 2015 [40], demonstrated that treatment with etanercept, a soluble TNFR2 receptor that inhibits TNF-α actions, was responsible for inhibiting mechanical hyperalgesia, overt pain behaviors (abdominal contortions, flinches, and paw licking), and MPO activity. These findings show that TNFR2 receptors and TNF-α participate in pain and inflammation induced by KO₂ [40]. Finally, Filippone et al., 2020 [281], demonstrated that treatment with sodium propionate (SP) was efficient in reducing paw edema, nociceptive stimulus, and manifest pain behaviors (flinches). This analgesic effect was due to a significant reduction in the recruitment of inflammatory cells and changes in the tissue architecture of the paw caused by KO₂. In addition, administration of SP significantly reduced MPO activity and increased expression of the antioxidant enzymes SOD2, HO-1, and GSH, protecting tissue from oxidative stress [281].

Together, the studies presented demonstrate that several molecules with analgesic, anti-inflammatory and antioxidant properties can attenuate the nociceptive and inflammatory effects induced by KO₂. Although they act through different pathways, they all converge in the modulation of targets related to oxidative stress, inflammatory response, and glial activation, central elements in the genesis and modulation of pain in this model. These findings not only validate KO₂ as a relevant experimental tool for the study of pain associated with ROS but also reinforce the therapeutic potential of these approaches for the development of new pharmacological strategies in the management of inflammatory pain.

6. Clinical Prospects and Challenges

The involvement of ROS is critical in the development and maintenance of pain across diverse etiologies. Despite this fact, there are not medications currently available in clinical practice specifically targeting O₂^•−, and this absence reflects the complexity of precisely defining the timing and context in which O₂^•− plays a decisive role in inducing inflammatory pain.

Another point is that some of the compounds discussed in the article are not intended to directly target O₂^•−, but they target molecules that are produced upon the stimulation by O₂^•−. Bosentan, for example, has been approved for clinical use to treat pulmonary hypertension [282,283]. In this case, endothelin-1 is the primary target of this ETA/ETB receptor antagonist. O₂^•− can induce endothelin-1 production [39] and also participates in pulmonary hypertension development [284,285]. However, the extend that endothelin-1 production depends on O₂^•− in pulmonary hypertension is less clear. The description of bosentan activity as an ETA/ETB receptor antagonist is correct and we do not challenge that. The point is that determining the contribution of O₂^•− to endothelin-1 production and the relevance of this cascade to bosentan clinical application would bring a clearer view of O₂^•− relevance as a target and potentially novel pharmacological approaches to be investigated. Thus, a better understanding on the contribution of O₂^•− via the production of other molecules that are drug targets is essential both to improve treatment as well as to rational repurposing of drugs to other diseases in which O₂^•− is relevant. Furthermore, the involvement of O₂^•− in different diseases and contexts, and its action on different cells, still requires detailed studies and this is an area that needs further exploration.

7. Conclusions

O₂^•− plays important roles in both cellular homeostasis and the pathogenesis of several diseases. In a physiological context, it is essential for activating transcriptional pathways and driving fundamental cellular processes, such as proliferation, migration, differentiation, and cell death. It is also essential for adequate immune response. However, dysregulation in reactive oxygen species (ROS) production pathways can contribute to a dysregulated inflammatory response and pain. In the context of pain induced by O₂^•− or its donor, KO₂, the activation of redox-sensitive signaling pathways and the intensification of inflammation promote the sensitization of nociceptive neurons and the amplification/transmission of the nociceptive response. We summarized and discussed the pharmacological approaches that have been tested as analgesics to target O₂^•−-triggered pain.

Advances in the understanding of these mechanisms have contributed to the development of therapeutic approaches, including antioxidants, signaling pathway modulators, and enzyme inhibitors capable of reducing the production and/or effects of O₂^•−. Thus, it can be concluded that the integration of knowledge about the biosynthesis and physiological functions of O₂^•−, as well as advances in the understanding of the molecular mechanisms involved in ROS-induced pain conditions, represents a promising path for the development of more specific and effective pharmacological approaches for pain management in this context.