Search Results (541)

Chronic inflammation constitutes a well-established driver of colorectal carcinogenesis, yet the molecular circuitry linking inflammatory receptor signalling to tumour cell survival remains incompletely delineated. Here we demonstrate that the HMG-CoA reductase inhibitors atorvastatin and rosuvastatin modulate inflammatory survival pathways in colorectal cancer cells in a manner consistent with targeted interference with the protease-activated receptor 2 (PAR-2)–extracellular signal-regulated kinase (ERK)–tumour necrosis factor-α (TNF-α) signalling axis. Using lipopolysaccharide-stimulated HT-29 and Caco-2 cells as complementary models of inflammatory colorectal malignancy, we show that both statins selectively attenuate PAR-2 expression at the protein and transcript levels while leaving structurally related PAR-1 unaffected. This pattern of receptor modulation is accompanied by suppression of total ERK1/2 expression, ERK1/2 phosphorylation, and the transcriptional target DUSP6, together with attenuation of TNF-α secretion. Importantly, these signaling shifts are associated with dual apoptotic programs; the extrinsic pathway, reflected by transcriptional upregulation and proteolytic activation of caspase-8; and the intrinsic mitochondrial pathway, evidenced by reciprocal modulation of Bcl-2 family proteins favoring Bax over Bcl-2. Both pathways converge upon activation of executioner caspase-3 and an increase in Annexin V-defined apoptotic fractions, indicating re-engagement of programmed cell death under inflammatory stress. Notably, rosuvastatin consistently demonstrates superior potency across signaling endpoints, achieving comparable biological effects at lower concentrations than atorvastatin. Collectively, these data indicate that clinically deployed statins target the PAR-2–ERK axis and are associated with re-activation of apoptotic pathways in inflammatory colorectal cancer models, while leaving open the possibility that additional statin-responsive networks contribute to their pro-apoptotic effects. This mechanistic framework provides biological plausibility for epidemiologic observations linking statin use with reduced colorectal cancer risk and improved outcomes, and supports further translational evaluation of PAR-2-directed statin strategies in colorectal malignancy. Full article

Clostridial neurotoxins, botulinum neurotoxins (BoNTs), and tetanus neurotoxin (TeNT) are potent toxins responsible for severe diseases, botulism and tetanus, respectively. BoNTs associate with non-toxic proteins (non-toxic non-hemagglutinin, hemagglutinins, and OrfXs), which protect BoNTs against acidic pH and protease degradation and facilitate BoNT passage through the intestinal barrier. TeNT enters motor neurons and undergoes a retrograde axonal transport until the target inhibitory interneurons in the central nervous system. BoNTs and TeNT recognize specific cell surface receptors which consist of complex sets of protein(s)-glycan-gangliosides and determine specific cell entry pathways. Recent data on structural and functional investigations of BoNT and TeNT receptors bring a better understanding of toxin trafficking in the host and entry into target neuronal cells, which is useful for the development of updated strategies of prevention and treatment of the corresponding diseases. Since clostridial neurotoxins, notably BoNTs, are important therapeutic tools, detailed knowledge of their activity opens the way of the development of engineered molecules for specific clinical applications. Full article

Upon RNA virus infection, viral RNA is sensed by the RIG-I-like receptors (RLRs), which signal through the adaptor protein VISA/MAVS to induce an innate antiviral response. How the VISA-mediated innate antiviral response is regulated and whether it can be targeted for drug development against diseases caused by RNA virus infection needs to be further investigated. Here we report that physalin F, a natural secosteroid isolated from Physalis angulata L., inhibits innate immune response to RNA virus. Mechanistically, physalin F binds to and promotes the activation of the mitochondrial m-AAA protease AFG3L2, which subsequently mediates the degradation of VISA. Knockdown of AFG3L2 promotes RLR-mediated innate antiviral signaling, whereas physalin F inhibits innate immune response to RNA virus both in cells and mice. Our study discovers physalin F as an inhibitor of VISA-mediated innate antiviral response as well as a candidate compound for the treatment of related diseases. More importantly, our findings suggest that AFG3L2 constitutively mediates degradation of VISA under physiological conditions, which represents a novel negative regulatory mechanism of RLR-mediated innate antiviral response. Full article

Background: PAR 1 (protease-activated receptor 1) and endothelin 1 are biomarkers that could be of significance in kidney diseases. Methods: We measured the plasma levels of PAR1 and endothelin 1 in patients with membranous nephropathy (n = 19), focal and segmental glomerulosclerosis (FSGS) (n = 30), systemic lupus erythematosus (SLE) (n = 22), IgA nephropathy (n = 16), mesangial proliferative (non-IgA) glomerulonephritis (n = 7), chronic kidney disease (CKD) (n = 27), and hemodialysis (n = 26), as well as a healthy control group (n = 22). Then, for two years, we tracked the patients’ clinical progress (creatinine, total protein, and albumin levels) and compared the outcomes with their initial PAR 1 and endothelin 1 levels. Moreover, we checked the correlations between PAR 1 and endothelin 1 and the results of anti-PAR1 and anti-ETAR (endothelin A receptor) evaluations. Results: Membranous nephropathy, FSGS, IgA nephropathy, CKD, and hemodialysis patients had higher PAR 1 levels than the control group. PAR 1 correlated with total protein, albumin in SLE, total protein in IgA nephropathy, and creatinine in CKD. Endothelin 1 correlated with albumin in membranous nephropathy, total protein, albumin, creatinine in FSGS, total protein in IgA nephropathy, total protein, and albumin in CKD. PAR 1 correlated with anti-PAR 1 in FSGS. Anti-ETAR correlated with anti-PAR 1 in membranous nephropathy, FSGS, and IgA nephropathy. Conclusions: PAR 1 levels are elevated in some kidney diseases compared to the healthy population. Both PAR 1 and endothelin 1 are supposed to be related to the clinical course of specific kidney diseases. Full article

Cigarette smoking is the leading preventable cause of chronic diseases (e.g., COPD, cardiovascular disease, cancer), largely driven by persistent immune-inflammatory mechanisms. This review synthesizes the molecular and cellular cascades linking cigarette smoke (CS) exposure to chronic pathology. CS constituents, particularly ROS/RNS, induce rapid oxidative stress that overwhelms antioxidant defenses and generates damage-associated molecular patterns (DAMPs). These DAMPs activate pattern recognition receptors (PRRs) and the NLRP3 inflammasome, initiating NF-κB signaling and the release of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6). CS exposure causes profound innate immune dysregulation, including airway epithelial barrier disruption, hyperactivated neutrophils, and dysfunctional alveolar macrophages (AMs) that release destructive proteases (e.g., MMP-12) and acquire foam-cell–like characteristics. Furthermore, CS drives adaptive immunity toward a Th1/Th17-dominant phenotype while suppressing regulatory T-cell (Treg) function, thereby promoting autoimmunity and chronic tissue injury. Critically, CS induces epigenetic reprogramming (e.g., DNA methylation, miRNA dysregulation), locking immune cells into a persistent pro-inflammatory state. This convergence of oxidative stress, innate and adaptive immune dysregulation, and epigenetic alterations underlies the systemic low-grade inflammation that fuels smoking-related chronic diseases, highlighting key targets for novel therapeutic interventions. Full article

This study systematically evaluated insect resistance in transgenic poplar lines carrying three distinct Bacillus thuringiensis (Bt) gene vector architectures: a single-gene pb vector (Cry1Ac), a reverse-oriented double-gene n19 vector (Cry1Ac-Cry3A), and a forward-oriented double-gene n5 vector (Cry3A-Cry1Ac). The transgenic lines were accordingly designated as pb8/pb9, n19a/n19b, and DB7/DB16, respectively. Molecular analyses confirmed stable Bt gene integration, with the expression of Cry3A being consistently higher than that of Cry1Ac expression. Bioassays showed that dual-gene lines conferred broader insect resistance to pests than that of single-gene lines against both lepidopteran (Hyphantria cunea) and coleopteran (Plagiodera versicolora, Anoplophora glabripennis) pests. In contrast, the single-gene line pb9 exhibited specialized, high efficacy against H. cunea, achieving 100% mortality. Transcriptomic analysis of P. versicolora larvae fed the double-gene high-resistance n19a line and low-resistance DB16 line revealed multi-level molecular responses to Bt stress, including up-regulation of toxin-activating proteases, altered receptor expression, and suppression of growth-related genes. These changes were associated with significant developmental delay (8.33–20.83% reduction in the molting index). Our findings characterize the insect resistance and molecular profiles of the six transgenic poplar lines, as follows: multi-gene lines (n19a/n19b and DB7/DB16) confer broad-spectrum pest resistance, whereas single-gene lines (pb8/pb9) exhibit targeted efficacy. These results support the utility of these lines for pest-specific poplar breeding programs. Full article

Proteases represent a diverse family of enzymes that catalyze the hydrolysis of peptide bonds, modulating numerous biological processes. Among their substrates, CD74—also known as the invariant chain—has received increased research attention due to its multifunctional roles in both innate and adaptive immunity. This review provides an overview of current knowledge on protease-mediated interactions with CD74. The protein was originally identified as a chaperone for major histocompatibility complex class II (MHC-II) molecules. Proteolytic cleavage of CD74, most notably by cathepsin S, is essential for the release of MHC-II and the initiation of antigen presentation. However, CD74 has since emerged as a central regulator of processes extending well beyond antigen presentation. More recent findings reveal that CD74, acting as a receptor of macrophage migration inhibitory factor, also participates in signaling pathways in non-immune cells, independent of its classical chaperone function. Proteolytic processing of CD74 can trigger signaling cascades that modulate gene expression, underscoring its multifunctionality. Dysregulation of CD74 cleavage and its interaction with proteases has been linked to diverse pathological conditions, including cancer and autoimmune diseases, where aberrant protease activity disrupts CD74 function and promotes disease progression. Full article

Background: Pruritus is burdensome across dermatoses. Beyond Staphylococcus, broader components of the cutaneous microbiome—bacteria, fungi, and viruses—and their products shape itch via barrier disruption, immune polarization, and direct neurosensory activation. Methods: We conducted a narrative review of human and translational studies. PubMed/MEDLINE, Scopus, and Web of Science were searched from inception to 27 August 2025 using terms for itch, skin microbiome, bacteriotherapy, proteases, PAR, TRP channels, IL-31, Malassezia, and AHR ligands. English and Italian records were screened; randomized trials, systematic reviews, and mechanistic studies were prioritized; and unsupported single case reports were excluded. Results: Beyond Staphylococcus aureus, microbial drivers include secreted proteases activating PAR-2/4; pore-forming peptides and toxins engaging MRGPRs and sensitizing TRPV1/TRPA1; and metabolites, especially tryptophan-derived AHR ligands, that recalibrate barrier and neuro-immune circuits. Commensal taxa can restore epidermal lipids, tight junctions, and antimicrobial peptides. Early studies of topical live biotherapeutics—Roseomonas mucosa and Staphylococcus hominis A9—report reductions in disease severity and itch. Fungal communities, particularly Malassezia, contribute via lipases and bioactive metabolites with context-dependent effects. Across studies, heterogeneous itch metrics, small samples, and short follow-up limit certainty. Conclusions: The cutaneous microbiome actively contributes to itch and is targetable. Future studies should prioritize standardized itch endpoints, responder stratification, and robust safety for live biotherapeutics. Full article

The controlled pro-inflammatory immune response is critical for fighting against external and endogenous threats, such as microbes/pathogens, allergens, xenobiotics, various antigens, and dying host cells and their mediators (DNA, RNA, and nuclear proteins) released into the circulation and cytosol (PAMPs, MAMPs, and DAMPs). Several pattern recognition receptors (PRRs) and their downstream adaptor molecules, expressed by innate and adaptive immune cells, are critical in generating the inflammatory immune response by recognizing PAMPs, MAMPs, and DAMPs. However, their dysregulation may predispose the host to develop inflammation-associated organ damage, neurodegeneration, autoimmunity, cancer, and even death due to the absence of the inflammation resolution phase. The cytosolic calcium (Ca²⁺) level regulates the survival, proliferation, and immunological functions of immune cells. Cysteine-rich proteases, specifically calpains, are Ca²⁺-dependent proteases that become activated during inflammatory conditions, playing a critical role in the inflammatory process and associated organ damage. Therefore, this article discusses the expression and function of calpain-1 and calpain-2 (ubiquitous calpains) in various innate (epithelial, endothelial, dendritic, mast, and NK cells, as well as macrophages) and adaptive (T and B cells) immune cells, affecting inflammation and immune regulation. As inflammatory diseases are on the rise due to several factors, such as environment, lifestyle, and an aging population, we must not just investigate but strive for a deeper understanding of the inflammation and immunoregulation under the calpain system (calpain-1 and calpain-2 and their endogenous negative regulator calpastatin) lens, which is ubiquitous and senses cytosolic Ca²⁺ changes to impact immune response. Full article

Chronic inflammation constitutes a significant characteristic of sustained infections caused by viral and fungal pathogens, with a strong correlation to the development of cancer, autoimmune disorders, and tissue fibrosis. Viral proteins such as HIV-1 Tat, HBV X (HBx), HPV E6/E7, and EBV LMP1 modulate the host’s immune signaling pathways, primarily through the activation of the NF-κB signaling cascade and the disruption of cytokine equilibrium. These molecular interactions result in a pro-inflammatory microenvironment that facilitates viral persistence, immune evasion, and the process of oncogenesis. Structural investigations have elucidated the mechanisms by which these viral proteins interact with host signaling complexes, thereby highlighting their potential as viable therapeutic targets. Similarly, fungal proteins, including secreted aspartyl proteases (Saps), ribotoxin Asp f1, and chitin-binding proteins, incite chronic inflammation by activating pattern recognition receptors and triggering inflammasome activation. Despite the limited structural information of these fungal proteins, emerging models and bioinformatic analyses identified conserved motifs that are crucial for host interactions. Biologic therapies, encompassing antiviral and antifungal peptides as well as monoclonal antibodies, are currently under development to disrupt these protein-host interactions and modulate inflammatory responses. This review provides structural and functional insight into viral and fungal inflammatory proteins and evaluates the potential of biologics as targeted therapeutic interventions for chronic inflammation associated with infections. We discuss the ongoing clinical trials involving neutralizing antibodies targeting HIV, peptide vaccines aimed at HPV and other promising molecules. Finally, we discuss the current limitations of biologics and possible solutions to translate these promising therapeutics into clinical practice. Full article

Plasma-activated water (PAW) is an emerging disinfectant; however, its potential as a quorum sensing inhibitor (QSI) for biofilm control remains underexplored, and its action mechanisms have not been elucidated. This study investigated the effects of PAW on biofilm formation and spoilage factors secretion in Pseudomonas fluorescens under sub-inhibitory conditions. PAW generated by treating water for 60 s (PAW-60) reduced biofilm biomass by up to 1.29 log CFU/mL after 12 h incubation. It also completely inhibited protease production (100%) and decreased siderophore production by 31.87%. N-butyryl-homoserine lactone (C₄-HSL) was identified as the dominant signaling molecule, with its production decreasing by 34.34–84.07% following PAW treatments. Meanwhile, C₄-HSL activity was significantly suppressed by 42.58–65.38%. An FTIR analysis revealed the formation of a new C=O group, indicating oxidative degradation of acyl homoserine lactones (AHLs). Exogenous C₄-HSL progressively restored the biofilm biomass, spoilage factors production, and QS-related gene expression levels, with no significant difference observed compared with the control at 0.05 µg/mL (p < 0.05). The results suggest that the inhibitory effects of PAW are primarily due to the disruption of AHLs transduction in the QS pathway. Molecular docking showed that the long-lived reactive species in PAW could bind to AHLs’ synthetic protein (FadD1) and receptor protein (LuxR) via hydrogen bonding. PAW-60 reduced the spoilage activity of P. fluorescens inoculated into fish muscle juice and extended its shelf life from 8 to 10 days during storage at 4 °C. A strong positive correlation was observed between AHLs accumulation and the spoilage process. These findings demonstrate that PAW mitigates biofilm formation and food spoilage by blocking signaling transduction, which involves suppression of AHLs production, oxidative degradation of AHLs molecules, and disruption of AHLs recognition. Full article

Ankyrin-repeat and SOCS-box protein 9 (ASB9) is a member of the ASB family of proteins, which act as a substrate recognition component of E3 ubiquitin ligases and regulate various reproductive processes. ASB9 was previously identified as being induced in bovine granulosa cells (GCs) by LH/hCG, and its binding partners, including protease-activated receptor 1 (PAR1), were reported. The aim of this study was to decipher ASB9’s mechanisms of action in GCs and determine whether ASB9 induction by LH/hCG is necessary for the regulation of PAR1 and the signaling pathways involved in GC function and activity. Cultured GCs were treated with different doses of FSH, LH, and thrombin. RT-qPCR analyses revealed that thrombin increased PAR1 expression, while FSH had no effect on PAR1. Treatment with LH significantly downregulated PAR1, even in the presence of thrombin, possibly via ASB9. The phosphorylation profile of MAPK3/1 in thrombin-treated GCs suggests PAR1-mediated control. ASB9 induction appeared to have a negative effect on the MAPK pathway, although thrombin treatment briefly (within an hour) blocked the negative effect of ASB9 on PAR1. Proliferation assays showed that ASB9 negatively regulated the GC number while increasing apoptosis. These data provide evidence of ASB9’s mode of action and its potent functional effects on PAR1 regulation, GC proliferation, and, potentially, the ovulatory process in bovine species. Full article

Background/Objectives: Global warming and concomitant extreme weather events have markedly increased the incidence of heat stroke. Heat stroke (HS) poses a substantial threat to cerebral health by triggering neuroinflammation and accelerating neurodegenerative processes. The activation of the Nod-like receptor protein 3 (NLRP3) inflammasome for interleukin-1β (IL-1β) secretion has been implicated as a critical mechanism underlying HS-related fatalities. However, the potential role of specific dietary factors to counteract heat stroke-induced neurotoxicity remains largely underexplored. We previously reported that eicosapentaenoic acid (EPA) and urolithin A (UroA), a gut metabolite of ellagic acid, effectively suppress NLRP3 inflammasome activation against metabolic or pathogenic insults. This study aimed to assess the impact of eicosapentaenoic acid (EPA), urolithin A (UroA), and their combination on mitigating heatstroke-mediated NLRP3 inflammasome activation in microglial cells. Methods: In vitro heatstroke conditions were replicated by subjecting murine BV2 microglial cells to a high temperature (41 °C) under hypoxic conditions. To achieve nutrient loading, BV2 cells were preincubated with either EPA (50 µM) or UroA (10 µM). NLRP3 inflammasome activation was evaluated by proinflammatory gene expression, caspase-1 cleavage in cells, and IL-1β secretion to the medium. The caspase-1 activation was determined using a luciferase-based inflammasome and protease activity reporter (iGLuc) assay. Results: Exposure to high temperatures under hypoxia successfully mimicked HS conditions and promoted NLRP3 inflammasome activation in BV2 cells. Both EPA and UroA substantially attenuated the heat stroke-induced priming of proinflammatory genes. More importantly, EPA and UroA demonstrated a synergistic effect in mitigating HS-induced active caspase-1 production, leading to a dramatic decrease in IL-1β secretion. This synergistic effect between EPA and UroA was further confirmed by the iGLuc reporter assay. Conclusions: Dietary enrichment with EPA and UroA precursors may constitute an efficacious strategy for mitigating heat stroke-mediated neuroinflammation and neurodegenerative diseases. Full article

Background: Chronic inflammation drives colorectal cancer (CRC) progression, with PAR-2, a G-protein coupled receptor, linking extracellular inflammatory signals to tumor-promoting pathways via ERK1/2 phosphorylation, calcium mobilization, TNF-α upregulation, and apoptosis suppression. While curcumin has notable anti-inflammatory and anti-cancer properties, its effects on PAR-2 signaling in inflammation-driven CRC remain underexplored. Objective: This study investigates how curcumin modulates PAR-2 expression and downstream oncogenic signaling in inflammation-driven CRC cells and explores its potential direct interaction with PAR-2 at the structural level. Methods: HT 29 and Caco-2 CRC cell lines were exposed to lipopolysaccharide (LPS) to induce an inflammatory phenotype, followed by treatment with curcumin at 50 µM and 100 µM. PAR-2 and PAR-1 expression, along with downstream markers including ERK1/2, p-ERK, TNF-α, caspase-8, cleaved caspase-8, caspase-3, Bcl 2, and Bax, were analyzed by Western blot and quantitative PCR. Calcium mobilization was assessed using Fluo-4 dye-based fluorescence imaging. Apoptosis was quantified using MTT viability assays, AO/EtBr dual staining, and Annexin V/PI flow cytometry. In parallel, AlphaFold-predicted structural models of PAR-2 were used to perform molecular docking with curcumin using CB-Dock2, to identify potential binding pockets and assess binding energetics. Results: Curcumin selectively downregulated PAR-2—but not PAR-1—at both transcript and protein levels in a dose-dependent manner. This downregulation was accompanied by suppression of ERK phosphorylation and calcium signaling, inhibition of TNF-α secretion, and reversal of the anti-apoptotic signaling axis (Bcl 2 downregulation and Bax and caspase-3/-8 upregulation). Functional assays confirmed enhanced apoptosis in curcumin-treated cells. Computational docking revealed a high-affinity binding interaction between curcumin and the transmembrane domain of PAR-2, supporting the hypothesis of direct G-Protein-Coupled Receptor (GPCR) modulation. Conclusions: Our findings reveal that curcumin targets the PAR-2/ERK/TNF-α axis and reactivates apoptotic pathways in inflammation-driven CRC, establishing it as a potent, mechanistically validated candidate for therapeutic repurposing in CRC. Full article

Atopic dermatitis is a chronic inflammatory skin disorder characterized by persistent inflammation and severe pruritus. Current anti-inflammatory agents carry risks of long-term adverse effects, while antihistamines provide limited relief of pruritus. Protease-activated receptor 2 (PAR2) has emerged as a critical mediator of both inflammation and pruritus, representing a promising therapeutic target. In this study, we investigated the therapeutic potential of punicalagin (PCG), a potent PAR2 antagonist, in atopic dermatitis. PCG fully and potently inhibited trypsin-induced PAR2 activation in HaCaT cells with an IC₅₀ of 1.30 µM, exhibiting over 40-fold greater selectivity over PAR1. PCG significantly inhibited PAR2-induced phosphorylation of ERK1/2 and NF-κB in both HaCaT and human dermal fibroblast cells and reduced IL-8 secretion in HaCaT cells. In addition, PCG did not significantly affect other pruritus-related GPCRs including H1R, H4R, TGR5, 5HT2A, 5HT2B, and MRGPRX2 at 30 µM. Notably, PCG strongly blocked PAR2-AP-induced scratching in mice. In addition, PCG improved skin lesions, reduced dermatitis severity scores, and alleviated scratching behavior in a DNFB-induced atopic dermatitis model. These effects were associated with reduced epidermal thickness, decreased serum TSLP levels, and inhibition of PAR2-dependent calcium signaling in dorsal root ganglion neurons. These findings demonstrate that PCG is a selective PAR2 antagonist that effectively alleviates both inflammatory and pruritic symptoms of atopic dermatitis, suggesting its potential as a novel therapeutic agent. Full article