Flavonoids Alleviate Peripheral Neuropathy Induced by Anticancer Drugs

Simple Summary Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating condition that severely reduces the quality of life of a considerable proportion of cancer patients. There is no cure for CIPN to date. Here, we explore the potential of flavonoids as pharmacological agents in combating CIPN. Flavonoids alleviate CIPN by reducing oxidative stress, inflammation, and neuronal damage, among other mechanisms. Future research should evaluate the efficacy and side effects of flavonoids in human models of CIPN. Abstract Purpose: This study aimed to assess the potential of flavonoids in combating CIPN. Methods: PubMed and Google Scholar were used, and studies that investigated flavonoids in models of CIPN and models of neuropathic pain similar to CIPN were included. Only studies investigating peripheral mechanisms of CIPN were used. Results: Flavonoids inhibit several essential mechanisms of CIPN, such as proinflammatory cytokine release, astrocyte and microglial activation, oxidative stress, neuronal damage and apoptosis, mitochondrial damage, ectopic discharge, and ion channel activation. They decreased the severity of certain CIPN symptoms, such as thermal hyperalgesia and mechanical, tactile, and cold allodynia. Conclusions: Flavonoids hold immense promise in treating CIPN; thus, future research should investigate their effects in humans. Specifically, precise pharmacological mechanisms and side effects need to be elucidated in human models before clinical benefits can be achieved.

Several studies investigated flavonoids' role in counteracting CIPN and reversing related oxidative stress and neuronal damage. The growing burden of CIPN and the emerging potential of flavonoids necessitate an analysis of the mechanisms by which flavonoids counter CIPN. Here, we review these mechanisms (see Figure 2) in the periphery, dorsal root ganglion, and spinal cord dorsal horn synapse, as well as in astrocytes and microglial cells.

Materials and Methods
PubMed and Google Scholar were searched using the following keywords: "flavonoids", "CIPN", "neuropathic pain", and "peripheral neuropathy". One hundred thirty-four results were obtained for the combination "flavonoids" AND "CIPN", 6620 for "flavonoids" AND "neuropathic pain", and 4909 for "flavonoids" AND "peripheral neuropathy". We included studies that investigated the effects of flavonoids on models of CIPN, sciatic nerve chronic constriction injury (CCI), partial sciatic nerve ligation (PNL), spared nerve injury (SNI), and spinal nerve ligation (SNL); the latter four share mechanisms with CIPN. We included studies with nerve injury models only if their findings were likely to be generalizable to CIPN (i.e., they investigated a mechanism in common with CIPN). Studies on diabetic, herpetic, or other clinically manifesting peripheral neuropathies apart from CIPN were excluded. We only included studies discussing peripheral nervous system mechanisms. These inclusion criteria encompassed 8 studies on CIPN and flavonoids; we selected seven. Similarly, we found 18 studies investigating flavonoids with CCI, SNL, PNL, and SNI, and included four.

General Effects of Anticancer Drugs and Flavonoids
Oxaliplatin, paclitaxel, and bortezomib destabilize the nociceptor membrane by elevating the resting membrane potential toward the threshold; this increases the likelihood of an action potential. Oxaliplatin activates ion channels involved in action potential initiation and propagation [10,16]. Paclitaxel and bortezomib enhance the release of proinflammatory cytokines, which sensitize peripheral nociceptors [1,2,19] (Figure 3a). Icariin, trimethoxyflavones, and dimethoxyflavones decrease proinflammatory cytokine release and reduce mechanical allodynia [13][14][15]. Moreover, quercetin inhibits paclitaxel-induced mast cell degranulation, reducing the release of inflammatory mediators and decreasing the thermal hyperalgesia and mechanical allodynia thresholds [12].  Flavonoids counter anticancer drug induced nociceptor sensitization. Anticancer drugs, via the enhanced release of proinflammatory cytokines and activation of ion channels, sensitize the nociceptor, increasing the likelihood that the membrane potential will reach the threshold potential. Thus, there is a greater chance that action potentials will result from weak stimuli that under normal conditions would not reach the threshold potential. (b) Effects of anticancer drugs on the peripheral nociceptor. Anticancer drugs increase the release of proinflammatory mediators such as TNF-α, IL-1β, IL-6, and histamine. These mediators directly sensitize ion channels and activate the ERK/JNK and p38/MAPK pathways, resulting in the activation of Na +1.8 and Na +1.9 and the inhibition of K +v . Histamine, prostaglandins, and tryptase bind to their respective receptors and activate the PKA and PKC pathways, which increase the membrane density of TRPV1 and Na + v channels. These events ultimately elevate the membrane potential to the threshold value, increasing the likelihood of an action potential; (c) Actions of flavonoids counter anticancer drugs' effects. Icariin and trimethoxy and dimethoxy flavones decrease the release of TNF-α, IL-1β, and IL-6 from astrocytes, microglia, and the DRG, and thereby downregulate the ERK/JNK and p38/MAPK pathways. This reduces the membrane density of ion channels, and consequently decreases the likelihood of reaching the threshold potential and reduces pain signal transmission; (d) Activation of PKA and PKC derivatives by anticancer drugs. Paclitaxel induces the degranulation of mast cells, which release tryptase and other proinflammatory mediators. Tryptase acts on PAR2, leading to PKA activation, the sensitization of TRPV1, TRPA1, and TRPV4, and the increased membrane fusion of Na + v . The sensitization of TRP channels increases Na + and Ca 2+ inflow into the nociceptor. Ca 2+ causes vesicles to fuse with the membrane and activates PKCδ, CaMKII, and PKC∈, which activate TRPV4 and TRPV1, further increasing ion inflow. These events increase the likelihood that the membrane potential will reach the threshold potential; (e) Flavonoids prevent neuropathic pain, affecting PKA and PKC derivatives activation. Quercetin inhibits the translocation of PKC∈ from the cytoplasm to the membrane and prevents paclitaxel-induced mast cell degranulation. Thus, there is less activation of PKA and PKC derivatives, which leads to decreased channel activation, less ionic influx, and a reduced likelihood that the membrane potential will reach threshold potential; (f) Anticancer drugs lead to the release of proinflammatory cytokines, which sensitize the nociceptor. Proinflammatory cytokines act directly on and sensitize TRP channels. They also increase the phosphorylation of transcription factors in the DRG via the p38/MAPK, ERK, and JNK pathways, increasing the synthesis of primary afferent channels; (g) Flavonoids (icariin and trimethoxy and dimethoxy flavones) reduce the release of proinflammatory cytokines by astrocytes, microglia, and the DRG. Thus, they downregulate the p38/MAPK, ERK, and JNK pathways, decrease ion channel density and phosphorylation, and consequently decrease the likelihood that the membrane potential will reach the threshold potential.

Ion Channel Activation
Oxaliplatin upregulates the expression of transient receptor potential melastatin 8 (TRPM8) [10,16], causing cold allodynia. It also activates TTX-R Na + 1.8 (involved in action potential initiation), TTX-R Na +1.6 , and HCN (hyperpolarization-activated channels involved in action potential propagation) while inhibiting TREK1 and TRAAK (potassium channels which restore the membrane potential to the resting state) [11]. Furthermore, nerve growth factor (NGF) and ATP activate protein kinase C (PKC) through their actions on TrkA1 and P2X3, respectively; PKC, in turn, increases the membrane fusion of transient receptor potential vanilloid 1 (TRPV1) vesicles [17,18]. These increases in ion channel activity and density destabilize the nociceptor membrane and cause the neuron to exhibit oscillatory behavior.

Icariin, Trimethoxy-and Dimethoxyflavones
In paclitaxel-induced models of CIPN, the flavonoid icariin inhibited the release of IL-1β, TNF-∝, and IL-6 from the DRG, astrocytes, and microglia [13], while trimethoxy and dimethoxy flavones inhibited the release of IL-1β, TNF-∝, and free radicals [14,15] ( Table 2). Decreased action of proinflammatory cytokines on the nociceptor would render p38/MAPK, ERK, and JNK less active, decreasing the activation and synthesis of TTX-Resistant Na + v channels ( Figure 3g). Thus, the membrane potential would be less likely to reach the threshold potential ( Figure 3c). These factors may account for the reduction of tactile allodynia and thermal hyperalgesia by trimethoxy and dimethoxy flavones and of mechanical allodynia by icariin [13][14][15].

Quercetin
In a paclitaxel-induced model of CIPN, quercetin inhibited the degranulation of mast cells and thereby reduced histamine release [12]. Decreased histamine-HIR pathway activation reduced HPETE release, IP3 activation, TRPV1 activation, and intracellular Ca 2+ release. Quercetin also inhibits PKC epsilon movement from the cytoplasm to the membrane [12]; thus, there is lesser activation of TRPV1 and TRPV4 [6]. Overall, quercetin decreases the intraneuronal concentrations of Ca 2+ and Na + , decreasing the likelihood that the membrane potential will reach the threshold potential. This explains the decrease in thermal hyperalgesia and mechanical allodynia thresholds observed by Gao et al. [12]. Mechanisms of CIPN at the peripheral nociceptor induced by anticancer therapy and the role of flavonoids that counter these effects are summarized in Table 1.

General Effects of Anticancer Drugs and Flavonoids
Anticancer drugs exert various effects on the dorsal root ganglion that eventually lead to receptor sensitization, altered channel expression, inflammation, increased likelihood of an action potential and NT release, and oxidative damage (Figure 4a). Inflammation is mediated by increased proinflammatory cytokine production, mainly through the activation of the transcription factor NF-κB ( Figure 4b). Flavonoids interfere with different pathways to decrease chemotherapy-induced inflammation ( Figure 4c). Icariin, 7,2 ,3 /7,2 ,4 /-, 7, 3 , 4 /7,5,4 -trimethoxyflavones and 3 ,4 /6,3 /7,2 /7,3 -dimethoxy-flavanol reduce paclitaxel-induced inflammation and allodynia [33][34][35], whereas 6-methoxyflavone mitigates cisplatin-induced static and dynamic allodynia [38]. Moreover, quercetin and rutin restore the mechanical and cold nociceptive thresholds decreased by oxaliplatin [45].  Flavonoids counteract anticancer drugs and increase mitochondrial damage, proinflammatory cytokine production, receptor sensitization, and the likelihood of an action potential. Also depicted is how anticancer drugs increase satellite glial cell coupling and excitability. (b) Anticancer Drugs on Proinflammatory Cytokine Production at the DRG. Anticancer drugs increase the translation of proinflammatory cytokines, prostaglandins, and NO through SIRT1 pathway downregulation and NF-κB activation. This leads to receptor sensitization and an increased likelihood of an action potential firing; (c) Flavonoids on Anticancer Drug-Induced Proinflammatory Cytokine Production in the DRG. Flavonoids counteract anticancer drugs' actions at the DRG by inhibiting the translocation of NF-κB, upregulating the SIRT1 pathway, and inhibiting the production of proinflammatory cytokines and prostaglandins. These actions decrease inflammation and consequently decrease the likelihood of AP firing and receptor sensitization; (d) Anticancer Drugs on Ion Channels and NT Production at the DRG. Anticancer drugs increase the expression of ion channels such as TRPV1 and N-type VGCC, increasing intracellular Ca 2+ concentrations and consequently increasing NT release. It also results in a dorsal root reflex; (e) Flavonoids on Anticancer Drug-Induced Increases in NT Production and Ion Channel Expression. 6-MeOF triggers GABAA receptors, decreasing an AP's likelihood and consequently reducing NT release and alleviating pain; (f) Anticancer Drugs on the DRG's Oxidative Stress Level. Anticancer drugs increase mitochondrial oxidant production, resulting in lipid peroxidation and tyrosine nitrosylation, lowering GSH levels in the cell and increases oxidative stress, causing neuronal damage; (g) Flavonoids on Anticancer Drug-Induced Increases in DRG Oxidative Stress. Flavonoids counteract the increase in oxidative stress by increasing the translation of GSH and directly scavenging reactive oxygen species; (h) Anticancer Drugs on Satellite Glial Cells. Anticancer drugs increase the expression of gap junctions that connect astrocytes surrounding the same and different DRGs. They also connect SGCs and neurons. Ca 2+ waves travel through these gap junctions, reducing the membrane potential of SGCs and increasing the likelihood of AP firing in neurons. The Ca 2+ waves are hypothetically generated by the anticancer drug-induced increase in P2X levels following inflammation. Oxaliplatin blocks the production of Kir4.1, disrupting the extracellular concentration of K + and leading to an increased likelihood of an AP; (i) Flavonoids on Anticancer Drug Effects in Satellite Glial Cells. No relevant research was found on how flavonoids counteract anticancer drug effects on SGCs.

Upregulation of the NF-κB Pathway
Paclitaxel activates NF-κB by stimulating TLR4 and upregulating ERK/JNK signaling [46]. These events lead to the phosphorylation and nuclear translocation of NF-κB, increasing the acetylation of the histone H4 and resulting in the transcription of various proinflammatory factors such as CX3CL1, TNF-α, IL-1β, and IL-6 [46] (Figure 4d). Oxaliplatin stimulates NF-κB through the JAK/STAT pathway and causes the production of proinflammatory factors such as CXCL12. Furthermore, this leads to increased MC-P1 (CCL2) transcription in the DRG and its receptor (CCL2R) on macrophages. Activation of CCL2R induces proinflammatory cytokine release, increasing the innate immune response [47]. Cisplatin also uses NF-κB to mediate its proinflammatory effects through the production of nitric oxide and prostaglandins. These factors contribute to inflammation, which leads to membrane depolarization and an increased chance of an AP firing [48].

Increased GABA Release
The release of more NT by the mechanisms described above increases interneuron activation and GABA release. GABA acts on GABAA receptors on DRG neurons, causing an efflux of Clthat contributes to the dorsal root reflex, by which the axonal membrane is depolarized in the reverse direction [54]. The reverse ion flow enhances the depolarization of the nociceptor membrane, causing increased release of substance P and cGRP and the consequent release of proinflammatory mediators in a positive feedback loop (see Section 3). In a cisplatin-induced neuropathic pain model, the flavonoid 6-methoxy-flavone increased Clinflux through GABAA channels [18] and thus decreased presynaptic membrane depolarization and the dorsal root reflex. Smaller amounts of NT were released, decreasing the frequency of action potentials in the postsynaptic neuron and alleviating static and dynamic allodynia [18].

Enhanced Activation of Satellite Glial Cells
Oxaliplatin and taxol contribute to CIPN by activating and increasing satellite glial cells' coupling (SGC). SGC coupling and increases in GFAP due to ROS production are hypothesized to activate SGCs. Oxaliplatin induces the release of the pro-inflammatory cytokines IL-6 and TNF-α, promoting neuronal hyperexcitability and upregulating the purinergic receptor P2X, increasing the sensitivity to ATP, whose concentration is elevated due to increased action potential firing. Moreover, P2X upregulation increases intra-SGC Ca 2+ flow and causes intracellular Ca 2+ waves (ICW) that travel through gap junctions between SGCs and neurons. ICW transmission between SGCs surrounding different neurons causes both SGC and neuronal hyperexcitability [55]. Increased SGC coupling is explained by the oxaliplatin-induced upregulation of Cx-43, a crucial connexon between SGCs. Oxaliplatin also downregulates the inward rectifier channel K ir 4.1, reducing the resting membrane potential and disrupting extracellular K + concentrations. Both of these effects cause the depolarization of SGC-surrounded neurons and thus increase the likelihood of action potential firing (Figure 4h).

Increased Oxidative Stress
Oxaliplatin increases the production of nitric oxide and superoxide, which react to form peroxynitrite. Peroxynitrite is highly reactive and gives rise to nitrogen dioxide and hydroxyl radicals that interact directly with lipids and tyrosine, leading to lipid peroxidation and protein nitrosylation in the DRG ultimately neuronal damage and death through ROS elevation and GSH depletion. Cisplatin accumulation also causes lipid peroxidation and increases inducible nitric oxide synthase (iNOS) activity, and thereby induces oxidative stress and neurotoxicity (Figure 4f). Similarly, paclitaxel induces oxidative stress by increasing DPPH and nitric oxide levels in the DRG [48]. In contrast, thalidomide may cause axonal sensory neuropathy through the depletion of nerve growth factor (NGF). NGF regulates neuronal growth, maintenance, and survival; its depletion is associated with peripheral neuropathy [45].
No research on the potential effects of flavonoids on SGCs was found ( Figure 4i); thus, future studies should investigate this.

General Effects of Anticancer Drugs and Flavonoids
Cisplatin and carboplatin increase proinflammatory cytokine release by astrocytes and microglial cells [7,64], contributing to peripheral sensitization. Oxaliplatin similarly enhances cytokine release and peripheral sensitization by increasing astrocyte coupling [65]. Paclitaxel and bortezomib increase synaptic glutamate concentrations [66,67], increasing the likelihood of action potential generation in the postsynaptic neuron (Figure 5a). Increased postsynaptic action potential frequency intensifies neuropathic pain. Quercetin reverses the paclitaxel-induced decrease in thermal hyperalgesia and mechanical allodynia thresholds [41], while icariin alleviates paclitaxel-induced mechanical allodynia and spinal neuroinflammation [42].

Effects of Anticancer Drugs
Cisplatin and carboplatin are ligands for TLR4 [7,64], and cisplatin upregulates the TREM-2 ligand [68] (Table 5). Subsequent activation of the TLR4 and TREM-2 pathways eventually results in NF-κB activation, culminating in proinflammatory cytokine release by astrocytes and microglial cells [69]. Chemokines released by DRG neurons, such as CX3CL1, also cause NF-κB activation and proinflammatory cytokine release [7,70] ( Figure 5b). Proinflammatory cytokines cause peripheral sensitization via the processes mentioned in Section 2; IL-1β acts on IL-1R on astrocytes, stimulating NF-κB activation and hence the release of more proinflammatory cytokines [71]. These cytokines also stimulate the fusion of presynaptic glutamate vesicles with the DRG neuron membrane [67]. Table 5. Studies investigating the mechanisms by which anticancer drugs exert their effects.

Mode of Administration/Concentration Animal Reference
Strong TREM2/DAP12 signaling continuously activated microglial cells, which resulted in neuropathic pain.

Cisplatin induced
Intraperitoneal/Accumulated dose of 23 mg/kg delivered in 2 rounds daily for 5 days with a 5 day break between rounds.

Bortezomib induced (in vitro and in vivo)
Male Sprague Dawley rats, S1pr1 knockout and knockdown mice [67] Downregulation of GLAST and GLT-1 on astrocyte membranes Paclitaxel induced  Paclitaxel inhibits GLAST and GLT-1 (glutamate uptake channels) located on astrocyte membranes [66]. Bortezomib increases sphingolipid metabolism, resulting in increased synthesis of S1P receptors [67]. Alternatively, S1P is upregulated by increases in peripheral TNF-α, IL-1β, and IL-6, commonly seen in neuropathic pain states. Increased S1P is correlated with increased presynaptic membrane fusion of glutamate vesicles [67]. These changes increase synaptic glutamate levels, upregulating NMDA and AMPA on the postsynaptic membrane. They contribute to neuropathic pain by increasing the postsynaptic neuron's depolarization and consequently increasing the chance of generating an action potential.
Oxaliplatin upregulates the CX43 gap junctional protein, increasing coupling between astrocytes [65]. This increases astrocyte activation and proinflammatory cytokine release. Furthermore, histamine released by mast cells acts on HIR; the subsequent cascade results in arachidonic acid production. Arachidonic acid activates 12 HPETE, which in turn sensitizes TRPV1 (Figure 5d) [41]. More Ca 2+ enters the presynaptic neuron, causing more vesicle fusion and NT release, thereby increasing the likelihood of generating a postsynaptic action potential, increasing neuropathic pain intensity.

Flavonoids Counter the Effects of Anticancer Drugs Quercetin and Icariin
The flavonoid quercetin inhibits mast cell degranulation and PKC epsilon movement to the membrane, thus decreasing the activation of TRPV1 (Figure 5e) [41]. This reduces Ca 2+ entry and thus the fusion of vesicles with the membrane, decreasing the likelihood of a postsynaptic action potential. Icariin inhibits NF-κB in the spinal cord dorsal horn (Figure 5e) (Table 6); thus, fewer proinflammatory cytokines are released, reducing the fusion of vesicles with the membrane. Overall, the frequency of action potentials in the postsynaptic neuron decreases, decreasing neuropathy intensity [42] (Figure 5c). Suppressed GFAP and astrocyte production of TNF-α, IL-1b, and IL-6.

Upregulation of the S1P Pathway
Bioactive sphingolipid metabolites are potent signaling molecules involved in bortezomib-induced CIPN. Bortezomib affects the S1P signaling pathway by increasing ceramide and its biosynthetic precursors such as S1P (Figure 6b). As astrocytes express S1PR1 (at higher levels than glial cells), they mediate bortezomib-induced CIPN; S1P triggers them to become reactive (Table 7). This form of astrocyte activation is associated with increases in GFAP, TNF, and IL-1β. S1PR1-induced inflammation establishes a feed-forward mechanism that dysregulates sphingolipid production, as TNF and IL-1β cause the activation of enzymes in the ceramide and S1P pathways. S1PR1 also increases glutamate release, which sustains neuropathic pain [67].

Upregulation of the TLR4 Pathway
Cisplatin activates TLR4 receptors via the MYD88/ NF-κB pathway, leading to the release of inflammatory cytokines like TNF and consequent mechanical allodynia. Activating transcription factor 3 (ATF3), whose concentration increases during inflammation, augments TLR4 signaling to NF-κB. This pathway is also seen in microglia [64].

Increased Expression of Cx-43
Besides upregulating GFAP (as with paclitaxel), oxaliplatin upregulates Cx-43, a component of the gap junctions between astrocytes. Cx-43 mediates the exchange of ions, metabolites, glia-transmitters, and the propagation of Ca 2+ waves between astrocytes. It increases astrocyte coupling and activation and consequently plays a role in oxaliplatininduced hypersensitivity [63].

Downregulation of Glutamate Uptake Receptors and Increased GFAP
Paclitaxel activates astrocytes and not microglial cells. As glutamate is a major excitatory NT, regulation of its uptake is essential. Paclitaxel downregulates the glial glutamate receptors GLAST and GLT-1 on astrocytes. Consequently, paclitaxel induces nociceptive behaviors and hypersensitivity to peripheral thermal and mechanical stimuli due to glutamate's impaired clearance in the inter-synaptic space [56]. Paclitaxel also increases the expression of GFAP, further supporting the astrocyte activation hypothesis in parallel with hypersensitivity [42].

Increased CX3CL1 Expression
CX3CL1 is a chemokine that stimulates spinal microglia by acting in its G-protein coupled receptor CX3CL1R to release pain mediators. Vincristine upregulates Iba-1, CX3CR1, and p-p38, which eventually activate NF-κB and CREB. NF-κB and CREB activate the microglia, which release proinflammatory cytokines (TNF-α and IL-1β) (Figure 6d). Soluble CX3CL1 activates the microglia-specific receptor CX3CR1, causing the phosphorylation of p38 MAPK, thereby promoting proinflammatory cytokines' secretion. Targeting of the Notch signaling pathway inhibits this CX3CL1/p38 signaling pathway through a mechanism not entirely elucidated [72]. No specific effects of flavonoids on spinal microglial cells are known to date ( Figure 6e); thus, this is an area of investigation of future research.

The Flavonoids Icariin and Astragli Radix Counter the Effects of Anticancer Drugs
Icariin counteracts paclitaxel-induced GFAP expression and thus represses astrocyte activation. Icariin also inhibits the production of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 in the spinal cord [42] (Table 8) (Figure 6c). In addition to isolated flavonoids, Astragali radix is an adaptogenic herbal product that improves chemotherapy patients' quality of life. Astragai radix is rich in various phytochemicals, including isoflavonoids, while isoflavones are more concentrated in hydroalcoholic extracts of Astragali radix when compared with aqueous. Besides, 50% hydroalcoholic extracts of Astragali radix reduced ATF-3 nuclear immunoreactivity in L4-L5 DRG, oxaliplatin-induced molecular and morphometric alterations in peripheral nerve and dorsal root ganglia, and the activation of microglia and astrocytes in a Sprague-Dawley rat model of oxaliplatin-induced neurotoxicity [73]. Reduction of Oxaliplatin-induced molecular and morphometric alterations in peripheral nerve and dorsal-root ganglia. Decrease in the activation of microglia and astrocytes [73]

General Effects of Anticancer Drugs and Flavonoids
Anticancer drugs cause mitochondrial and neuronal damage [76,77] and upregulate iNOS [56]. Flavonoids restore ATP levels and mitochondrial protective enzymes, reverse neuronal damage, and decrease ROS production [78][79][80][81] (Figure 7a). Thus, they reduce spontaneous pain, ongoing pain, and mechanical hypersensitivity. Mitochondrial damage and sensitization of primary afferent channels cause ectopic discharges, which contribute to spontaneous pain, ongoing pain, and mechanical hypersensitivity; (c) Flavonoids alleviate neuronal damage caused by anticancer drugs, reducing spontaneous pain, mechanical hypersensitivity, and the likelihood of apoptosis.

Neuronal Damage
Furthermore, the activity of the Na + /K + exchanger will decrease, leading to increased ectopic activity [88]. Ectopic activity leads to spontaneous pain, ongoing pain, and mechanical hypersensitivity [76]. Mitochondrial DNA adducts damage the electron transport chain's proteins, increasing oxidative stress and H 2 O 2 release [76]. H 2 O 2 causes ROS formation and demyelinates the neuron; ROS causes p. 53 and Bax release, leading to neuronal apoptosis [77]. ROS also increases the release of proinflammatory cytokines, which cause peripheral sensitization, further contributing to ectopic activity [89]. Damage to electron transport chain proteins causes the downregulation of protective enzymes such as SOD, GAPDH, GSH, and GPX, leading to excessive ROS production [76].

Enhanced iNOS Expression
Oxaliplatin increases LPS-induced iNOS expression [56]. Ca 2+ , together with iNOS, leads to the production of NOS, which increases NO. NO combines with oxygen free radicals to form ONO -2, enhancing DNA adducts production [56].

Flavonoids Counter the Effects of Anticancer Drugs
The flavonoids morin and GSPE restore GSH levels, and morin restores ATP levels ( Figure 7c) [78,79]. Increased ATP levels restore the activity of the Na + /K + exchanger, decreasing ectopic activity and thus neuropathic pain, while elevated GSH levels reduce H 2 O 2 and ROS production. These effects reduce the chance of apoptosis and also decrease ectopic activity. Thus, spontaneous pain, ongoing pain, and mechanical hypersensitivity will all decrease. Genistein restores mitochondrial GPX levels while isoorientin ameliorates axonal swelling and prevents demyelination [80,81]. Quercetin, rutin, and genistein reduce LPS-induced iNOS expression [56,81] and thereby reduce ONO 2 production and DNA adduct formation. Besides, the natural flavonoid silibinin prevents oxidative damage and exerts antineuropathic effects in a rat model of painful oxaliplatin-induced neuropathy [90] ( Table 10).

Flavonoids: Promise, Applications, and Side Effects
CIPN is a multifactorial disease with various pharmacological mechanisms. Effective treatments should influence the mechanisms that contribute most to the symptoms. Damage to DRG neuronal cell bodies or axons contributes most strongly to CIPN symptom development. Likewise, flavonoids with the most success in animal models affect these areas by reducing peripheral sensitization of DRG neurons, modulating synaptic transmission at the spinal dorsal horn, and reducing mitochondrial damage in DRG neurons, among other mechanisms. However, how these mechanisms interact with and influence each other to cause symptoms still requires extensive investigation.
Moreover, pain results from interactions between central and peripheral mechanisms [93,94]. More information about central-peripheral interaction and central nervous system mechanisms of pain transmission (neuromodulators, neuroplasticity, central sensitization, and NTs) is needed to understand the pathophysiology of CIPN [50] entirely. Also, there are many clinical phenotypes of CIPN, and each requires a specific standardized treatment approach. For example, oxaliplatin and paclitaxel induce neuropathy through different mechanisms [93]. Adding a different complexity level, combinations of different anticancer drugs are used in treatment regimens [4,95].
It is essential to consider the concentrations of flavonoids that are useful in the treatment of CIPN. Flavonoids are present in relatively low concentrations in fruits and vegetables; these sources also contain a mixture of secondary plant metabolites such as vitamin C, folate, potassium, and fiber [96]. These secondary metabolites have known health benefits that cannot be replaced by a single compound (e.g., flavonoids) given as a dietary supplement [96]. Suppliers of flavonoid supplements recommend daily doses many times higher than those found in a flavonoid-rich diet. For example, quercetin is offered as a supplement with daily doses of 1 g or more [96], while its daily dietary intake is estimated to be between 10-100 mg [96].
Other issues to be considered in evaluating flavonoids as dietary supplements include drug interactions, trace element chelation, and thyroid status [97]. In vitro experiments indicate that purified flavonoids and flavonoid-rich extracts chelate iron, posing a risk for iron deficiency individuals. Flavonoids also interact with copper, manganese, and vitamin C [97,98]. They may exhibit antithyroid and goitrogenic activity. For example, quercetin and isoflavones inhibit iodothyronine deiodinase activity. High-dose isoflavones inhibit thyroid hormone biosynthesis, have estrogenic effects, and are goitrogenic [99,100]. Flavonoids may interfere with the absorption, tissue distribution, metabolism, and excretion of classical xenobiotics due to similar metabolic pathways. Notably, flavonoids interfere with all phase II enzymes, affecting the organism's ability to detoxify endogenous and exogenous xenobiotics. For example, quercetin and kaempferol increase either the transcription or activity of the enzyme UDP-glucosyltransferase A1 [101].
Another vital factor to consider while reviewing in vitro studies is the effect of flavonoid distribution on local concentrations and drug interactions. For instance, quercetin and kaempferol inhibit CYP3A4 and, consequently, the metabolism of the Ca 2+ channel blockers nifedipine and felodipine in human liver microsomes at concentrations >10 µmol/L [102]. On the other hand, quercetin did not inhibit CYP3A4 metabolism of the statin simvastatin in pigs. A possible explanation for this is a lower hepatic concentration than observed in vitro [103]. Flavonoids (with C5 hydroxy and methoxy groups [104,105]) inhibit ABC transposers. The inhibition has positive consequences for poorly absorbed drugs but may result in drug toxicities for low therapeutic index drugs [106].
Given these side effects, it has been concluded by some that whole fruits and vegetables are more beneficial to health than any single plant constituent [107]. On the flip side, overexpression of ABC transporters is one of the major mechanisms of multidrug resistance encountered during chemotherapy treatments. Cancer cells overexpress the ABC transporter, which pumps out anticancer drugs before they can have a significant effect. Thus, flavonoids can reduce drug resistance and thereby enhance the efficacy of chemotherapy. Moreover, several studies suggest that flavonoids sensitize cancer cells to chemotherapy [108,109]. Quercetin especially has promise, combined with vincristine, to increase breast cancer treatment efficacy [110]. Therefore, the potential of flavonoids as adjuncts in chemotherapy creates an additional incentive to investigate further their potential in counteracting CIPN, especially in studies involving humans.
Flavonoids have potential as therapeutic agents for preventing CIPN; however, many questions remain unanswered due to the lack of flavonoid studies with human subjects; e.g., what serum concentration must be achieved to get a significant therapeutic effect? Can this concentration be achieved by supplementing the diet with flavonoid-rich foods, or are intravenous injections a must? According to Mongiovi et al., increasing citrus fruit intake poses its own problems in patients undergoing chemotherapy-their results showed a positive association between citrus fruit intake (rich in flavonoids) and neuropathic pain symptoms [111], which may indicate potential detrimental interactions between flavonoids and other substances in citrus fruits. However, other studies evaluating the effects of diet on CIPN did not show this association [112], and thus more studies are required to further elucidate the effects of citrus fruits on CIPN symptoms. Furthermore, due to flavonoids' potential side effects, an extensive cost-benefit analysis in humans is needed to determine whether a flavonoid-based treatment should be explored further.
Translating results obtained from mouse models to humans presents challenges of its own. Sex, differences in social structure, and variations in genotype and neuroanatomy all influence pain pathways and pain perception [3,113]. Due to the differences in symptoms experienced by humans and mice [113], the effects of flavonoids on humans are expected to be highly variable. Another complicating factor is that, unlike patients with CIPN, most mice in these studies do not have cancer. Furthermore, murine chemotherapy delivery methods may not match clinical ones; additionally, the sex of the animals does not match clinical demographics-studies with animals use mostly male mice, but there are many female cancer patients as well [3]. Also, in animal models, only acute (experienced within the first six months after treatment), not chronic (occurring about two years following treatment), neuropathic pain is studied [3]. Thus, the elucidated mechanisms are related to the acute phase only, and treatment with flavonoids may not have the same effect on chronic neuropathic pain [3]. The mechanisms of chronic neuropathic pain warrant more research in animal models, especially in the context of flavonoids. The symptoms of chronic neuropathic pain vary from those of the acute phase. While the acute phase is marked by dysesthesia, paraesthesia, and hyperesthesia, chronic neuropathy is mainly associated with sensory ataxia, insomnia, anxiety, depression, cognitive and functional deficits, and fall risk [10,11,114]. So far, only one study has assessed the effects of flavonoids on symptoms specific to chronic neuropathy: Chtourou et al. investigated cisplatin exposure to acetylcholinesterase, ATPase, and oxidative stress biomarkers and the potential association this may have on behavioral performance in aged rats. The protective mechanisms of the flavonoid naringin were also studied. While cisplatin decreased enzymatic and nonenzymatic antioxidant activity in the hippocampus and raised levels of ROS, NO, MDA, and PCO, naringin reversed these effects and alleviated cisplatin-induced cognitive deficits (as seen by improved performance on the behavioral test administered) [115]. Flavonoids thus reverse anticancer drug-induced cognitive decline in chronic CIPN; in this light, their effects on the central nervous system merit further investigation. Also, flavonoids may hold promise in treating depression linked to chronic CIPN, since they are effective as antidepressants due to their antioxidant activity [116]. Furthermore, recent clinical results indicate that pediatric patients receiving azole antifungal treatment along with one-hour infusions of vincristine develop less severe peripheral neuropathy than patients receiving only vincristine. Similar studies should be conducted with flavonoids, in which isolated or mixed flavonoids are delivered during chemotherapy treatment to decrease the onset and severity of symptoms [117].
In short, increasing evidence indicates that flavonoids may alleviate the symptoms of both acute and chronic CIPN, increase the efficacy of chemotherapy, and reduce the cognitive dysfunction that results from it; thus, their side effects, effects in humans, and mechanisms of action are priorities for further investigation.

Conclusions
Flavonoids hold great promise in the management of CIPN. Investigating the mechanisms through which flavonoids act furthers the understanding of peripheral neuropathy and offers new methods to overcome it. The burden of CIPN and the promise of flavonoids encourages future research into their actions in humans, as well as their therapeutic index and side effects.  Acknowledgments: All figures were produced using BioRender.

Conflicts of Interest:
The authors declare no conflict of interest.