Protective Effect of Resveratrol against IL-1β-Induced Inflammatory Response on Human Osteoarthritic Chondrocytes Partly via the TLR4/MyD88/NF-κB Signaling Pathway: An “in Vitro Study”

Resveratrol is a natural polyphenolic compound that prevents inflammation in chondrocytes and animal models of osteoarthritis (OA) via yet to be defined mechanisms. The purpose of this study was to determine whether the protective effect of resveratrol on IL-1β-induced human articular chondrocytes was associated with the TLR4/MyD88/NF-κB signaling pathway by incubating human articular chondrocytes (harvested from osteoarthritis patients) with IL-1β before treatment with resveratrol. Cell viability was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and TNFα levels in culture supernatants were measured by ELISA(Enzymelinked immunosorbent assay). The levels of TLR4 and its downstream signaling targets (MyD88 and TRAF6) and IL-1β were assessed by measuring the levels of mRNA and protein expression by real-time RT-PCR and western blot analysis, respectively, in addition to assessing NF-κB activation. In addition, TLR4 siRNA was used to block TLR4 expression in chondrocytes further demonstrating that resveratrol prevented IL-1β-mediated inflammation by TLR4 inhibition. We found that resveratrol prevented IL-1β-induced reduction in cell viability. Stimulation of chondrocytes with IL-1β caused a significant up-regulation of TLR4 and its downstream targets MyD88 and TRAF6 resulting in NF-κB activation associated with the synthesis of IL-1β and TNFα. These IL-1β-induced inflammatory responses were all effectively reversed by resveratrol. Furthermore, activation of NF-κB in chondrocytes treated with TLR4 siRNA was significantly attenuated, but not abolished, and exposure to resveratrol further reduced NF-κB translocation. These data suggested that resveratrol prevented IL-1β-induced inflammation in human articular chondrocytes at least in part by inhibiting the TLR4/MyD88/NF-κB signaling pathway suggesting that resveratrol has the potential to be used as a nutritional supplement to counteract OA symptoms.     

Pharmacological Effects of Polyphenol Phytochemicals on the Intestinal Inflammation via Targeting TLR4/NF-κB Signaling Pathway

TLR4/NF-κB is a key inflammatory signaling transduction pathway, closely involved in cell differentiation, proliferation, apoptosis, and pro-inflammatory response. Toll like receptor 4 (TLR4), the first mammalian TLR to be characterized, is the innate immune receptor that plays a key role in inflammatory signal transductions. Nuclear factor kappa B (NF-κB), the TLR4 downstream, is the key to accounting for the expression of multiple genes involved in inflammatory responses, such as pro-inflammatory cytokines. Inflammatory bowel disease (IBD) in humans is a chronic inflammatory disease with high incidence and prevalence worldwide. Targeting the TLR4/NF-κB signaling pathway might be an effective strategy to alleviate intestinal inflammation. Polyphenol phytochemicals have shown noticeable alleviative effects by acting on the TLR4/NF-κB signaling pathway in intestinal inflammation. This review summarizes the pharmacological effects of more than 20 kinds of polyphenols on intestinal inflammation via targeting the TLR4/NF-κB signaling pathway. We expected that polyphenol phytochemicals targeting the TLR4/NF-κB signaling pathway might be an effective approach to treat IBD in future clinical research applications.     

TLR4 Deficiency Affects the Microbiome and Reduces Intestinal Dysfunctions and Inflammation in Chronic Alcohol-Fed Mice

Chronic alcohol abuse causes an inflammatory response in the intestinal tract with damage to the integrity of the mucosa and epithelium, as well as dysbiosis in the gut microbiome. However, the role of gut bacteria in ethanol effects and how these microorganisms interact with the immune system are not well understood. The aim of the present study was to evaluate if TLR4 alters the ethanol-induced intestinal inflammatory response, and whether the response of this receptor affects the gut microbiota profile. We analyzed the 16S rRNA sequence of the fecal samples from wild-type (WT) and TLR4-knockout (TLR4-KO) mice with and without ethanol intake for 3 months. The results demonstrated that chronic ethanol consumption reduces microbiota diversity and causes dysbiosis in WT mice. Likewise, ethanol upregulates several inflammatory genes (IL-1β, iNOS, TNF-α) and miRNAs (miR-155-5p, miR-146a-5p) and alters structural and permeability genes (INTL1, CDH1, CFTR) in the colon of WT mice. Our results further demonstrated that TLR4-KO mice exhibit a different microbiota that can protect against the ethanol-induced activation of the immune system and colon integrity dysfunctions. In short, our results reveal that TLR4 is a key factor for determining the gut microbiota, which can participate in dysbiosis and the inflammatory response induced by alcohol consumption.     

Thalidomide Alleviates Pulmonary Fibrosis Induced by Silica in Mice by Inhibiting ER Stress and the TLR4-NF-κB Pathway

Silicosis is the most prevalent occupational disease in China. It is a form of pulmonary fibrosis caused by the inhalation of silicon particles. As there is no cure for the potentially lethal and progressive condition, the treatment of silicotic fibrosis is an important and difficult problem to address. Thalidomide, a drug with anti-inflammatory and immunoregulatory properties, has been reported to have lung-protective effects. The purpose of this study was to observe the therapeutic effect of thalidomide on silicotic mice and to determine the protective mechanism. By using silicotic mice models and MH-S cells, we found the expression of endoplasmic reticulum stress (ER stress) and Toll-like receptor 4 (TLR4)-nuclear factor kappa-B (NF-κB) pathway as well as inflammation-related factors were upregulated in the macrophages of silicotic mice. The same indexes were detected in silica-stimulated MH-S cells, and the results were consistent with those in vivo. That is, silica activated ER stress and the TLR4-NF-κB pathway as well as the inflammatory response in vitro. Treating both silicotic mice and silica-stimulated MH-S cells with thalidomide inhibited ER stress and the TLR4-NF-κB pathway as well as the inflammatory response. The present study demonstrates thalidomide as a potential therapeutic agent against silicosis.     

Ophiopogon Polysaccharide Liposome Regulated the Immune Activity of Kupffer Cell through miR-4796

The purpose of this article is to study the effects and mechanism of miR-4796 in the process of ophiopogon polysaccharide liposome (OPL) regulation of the immune activity of Kupffer cells (KCs). In this study, KCs were used as cell models, and were treated with OPL in different concentrations after being transfected with miR-4796 mimic or miR-4796 inhibitor. Firstly, the secretion of NO and iNOS, phagocytic activity, the expression of surface molecules CD14 and MHC II, apoptosis and ROS secretion were measured by Griess, flow cytometry, fluorescence staining and ELISA. Then, real-time PCR and Western blot were used to measure the expression of TLR4, IKKβ, MyD88 and NF-κB in the TLR4-NF-κB signaling pathway. The results showed that after transfection with miR-4796 mimic, the secretion of NO and iNOS, cell migration, cell phagocytosis and expression levels of CD14 and MHC II in the OPL group were significantly higher than those in the miR-4796 mimic control group (p < 0.05; p < 0.01). In addition, the mRNA and protein expression levels of TLR4, MyD88 and NF-κB were significantly higher than those in miR-4796 mimic control group (p < 0.05; p < 0.01). After transfection with miR-4796 inhibitor, the secretion of NO and iNOS, cell migration, cell phagocytosis, expression of CD14 and MHCII in OPL group were significantly higher than those in the miR-4796 inhibitor control group (p < 0.05; p < 0.01). These results indicated that OPL could regulate the immune activity of KCs by regulating miR-4796 and activating the TLR4-NF-κB signaling pathway.     

A Novel 1,8-Naphthyridine-2-Carboxamide Derivative Attenuates Inflammatory Responses and Cell Migration in LPS-Treated BV2 Cells via the Suppression of ROS Generation and TLR4/Myd88/NF-κB Signaling Pathway

Novel 1,8-naphthyridine-2-carboxamide derivatives with various substituents (HSR2101-HSR2113) were synthesized and evaluated for their effects on the production of pro-inflammatory mediators and cell migration in lipopolysaccharide (LPS)-treated BV2 microglial cells. Among the tested compounds, HSR2104 exhibited the most potent inhibitory effects on the LPS-stimulated production of inflammatory mediators, including nitric oxide (NO), tumor necrosis factor-α, and interleukin-6. Therefore, this compound was chosen for further investigation. We found that HSR2104 attenuated levels of inducible NO synthase and cyclooxygenase 2 in LPS-treated BV2 cells. In addition, it markedly suppressed LPS-induced cell migration as well as the generation of intracellular reactive oxygen species (ROS). Moreover, HSR2104 abated the LPS-triggered nuclear translocation of nuclear factor-κB (NF-κB) through inhibition of inhibitor kappa Bα phosphorylation. Furthermore, it reduced the expressions of Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) in LPS-treated BV2 cells. Similar results were observed with TAK242, a specific inhibitor of TLR4, suggesting that TLR4 is an upstream regulator of NF-κB signaling in BV2 cells. Collectively, our findings demonstrate that HSR2104 exhibits anti-inflammatory and anti-migratory activities in LPS-treated BV2 cells via the suppression of ROS and TLR4/MyD88/NF-κB signaling pathway. Based on our observations, HSR2104 may have a beneficial impact on inflammatory responses and microglial cell migration involved in the pathogenesis of various neurodegenerative disorders.     

Overview of the Importance of Biotics in Gut Barrier Integrity

Increased gut permeability is suggested to be involved in the pathogenesis of a growing number of disorders. The altered intestinal barrier and the subsequent translocation of bacteria or bacterial products into the internal milieu of the human body induce the inflammatory state. Gut microbiota maintains intestinal epithelium integrity. Since dysbiosis contributes to increased gut permeability, the interventions that change the gut microbiota and correct dysbiosis are suggested to also restore intestinal barrier function. In this review, the current knowledge on the role of biotics (probiotics, prebiotics, synbiotics and postbiotics) in maintaining the intestinal barrier function is summarized. The potential outcome of the results from in vitro and animal studies is presented, and the need for further well-designed randomized clinical trials is highlighted. Moreover, we indicate the need to understand the mechanisms by which biotics regulate the function of the intestinal barrier. This review is concluded with the future direction and requirement of studies involving biotics and gut barrier.     

Modulation of Gut Microbiota Combined with Upregulation of Intestinal Tight Junction Explains Anti-Inflammatory Effect of Corylin on Colitis-Associated Cancer in Mice

Inflammatory bowel disease (IBD) involves chronic inflammation, loss of epithelial integrity, and gastrointestinal microbiota dysbiosis, resulting in the development of a colon cancer known as colitis-associated colorectal cancer (CAC). In this study, we evaluated the effects of corylin in a mouse model of dextran sodium sulfate (DSS)-induced colitis. The results showed corylin could improved the survival rate and colon length, maintained body weight, and ameliorated the inflammatory response in the colon. Then, we further identified the possible antitumor effects after 30-day treatment of corylin on an azoxymethane (AOM)/DSS-induced CAC mouse model. Biomarkers associated with inflammation, the colon tissue barrier, macrophage polarization (CD11c, CCR7, CD163, and CD206), and microbiota dysbiosis were monitored in the AOM/DSS group versus corylin groups. Corylin downregulated pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1β, and IL-6) mRNA expression and inflammatory signaling-associated markers (TLR4, MyD88, AP-1, CD11b, and F4/80). In addition, a colon barrier experiment revealed that epithelial cell proliferation of the mucus layer (Lgr5, Cyclin D1, and Olfm4) was downregulated and tight junction proteins (claudin-1 and ZO-1) were upregulated. Furthermore, the Firmicutes/Bacteroidetes ratio changed with corylin intervention, and the microbial diversity and community richness of the AOM/DSS mice were improved by corylin. The comparative analysis of gut microbiota revealed that Bacteroidetes, Patescibacteria, Candidatus Saccharimonas, Erysipelatoclostridium, and Enterorhabdus were significantly increased but Firmicutes, Turicibacter, Romboutsia, and Blautia decreased after corylin treatment. Altogether, corylin administration showed cancer-ameliorating effects by reducing the risk of colitis-associated colon cancer via regulation of inflammation, carcinogenesis, and compositional change of gut microbiota. Therefore, corylin could be a novel, potential health-protective, natural agent against CAC.     

Melatonin-Mediated Colonic Microbiota Metabolite Butyrate Prevents Acute Sleep Deprivation-Induced Colitis in Mice

Radical cure colitis is a severe public health threat worldwide. Our previous studies have confirmed that melatonin can effectively improve gut microbiota disorder and mucosal injury caused by sleep deprivation (SD). The present study further explored the mechanism whereby exogenous melatonin prevented SD-induced colitis. 16S rRNA high-throughput sequencing and metabolomics analysis were used to explore the correlation between SD-induced colitis and intestinal microbiota and metabolite composition in mice. Fecal microbiota transplantation (FMT) and melatonin or butyrate supplementation tests verified the core role of gut microbiota in melatonin-alleviating SD-induced colitis. Further, in vitro tests studied the modulatory mechanism of metabolite butyrate. The results demonstrated that SD leads to reductions in plasma melatonin levels and colonic Card9 expression and consequent occurrence of colitis and gut microbiota disorder, especially the downregulation of Faecalibacterium and butyrate levels. The FMT from SD-mice to normal mice could restore SD-like colitis, while butyrate supplementation to SD-mice inhibited the occurrence of colitis, but with no change in the plasma melatonin level in both treatments. However, melatonin supplementation reversed all inductions in SD-mice. In intestinal epithelial cells, the inflammatory ameliorative effect of butyrate was blocked with pretreatments of HDAC3 agonist and HIF-1α antagonist but was mimicked by GSK-3β and p-P65 antagonists. Therefore, the administration of MLT may be a better therapy for SD-induced colitis relative to butyrate. A feasible mechanism would involve that melatonin up-regulated the Faecalibacterium population and production of its metabolite butyrate and MCT1 expression and inhibited HDAC3 in the colon, which would allow p-GSK-3β/β-catenin/HIF-1α activation and NF-κB/NLRP3 suppression to up-regulate Card9 expression and suppress inflammation response.     

Lactoferrin Ameliorates Dry Eye Disease Potentially through Enhancement of Short-Chain Fatty Acid Production by Gut Microbiota in Mice

Lactoferrin is a glycoprotein found at high concentrations within exocrine secretions, including tears. Low levels of lactoferrin have been implicated in the loss of tear secretion and ageing. Furthermore, lactoferrin possesses a range of functionalities, including anti-inflammatory properties and the ability to modulate the gut microbiota. Expanding evidence demonstrates a crucial role of the gut microbiota in immune regulation and development. The specific composition of bacterial species of the gut has a profound influence on local and systemic inflammation, leading to a protective capacity against a number of inflammatory diseases, potentially by the induction of regulatory immune cells. In this study, we demonstrated that oral administration of lactoferrin maintains tear secretion in a restraint and desiccating stress induced mouse model of dry eye disease. Furthermore, we revealed that lactoferrin induces the reduction of inflammatory cytokines, modulates gut microbiota, and induces short-chain fatty acid production. Whereas, the antibiotic vancomycin abrogates the effects of lactoferrin on dry eye disease and significantly reduces short-chain fatty acid concentrations. Therefore, this protective effect of LF against a mice model of DED may be explained by our observations of an altered gut microbiota and an enhanced production of immunomodulatory short-chain fatty acids.     

Resveratrol Attenuates Acute Inflammatory Injury in Experimental Subarachnoid Hemorrhage in Rats via Inhibition of TLR4 Pathway

Toll-like receptor 4 (TLR4) has been proven to play a critical role in neuroinflammation and to represent an important therapeutic target following subarachnoid hemorrhage (SAH). Resveratrol (RSV), a natural occurring polyphenolic compound, has a powerful anti-inflammatory property. However, the underlying molecular mechanisms of RSV in protecting against early brain injury (EBI) after SAH remain obscure. The purpose of this study was to investigate the effects of RSV on the TLR4-related inflammatory signaling pathway and EBI in rats after SAH. A prechiasmatic cistern SAH model was used in our experiment. The expressions of TLR4, high-mobility group box 1 (HMGB1), myeloid differentiation factor 88 (MyD88), and nuclear factor-κB (NF-κB) were evaluated by Western blot and immunohistochemistry. The expressions of Iba-1 and pro-inflammatory cytokines in brain cortex were determined by Western blot, immunofluorescence staining, or enzyme-linked immunosorbent assay. Neural apoptosis, brain edema, and neurological function were further evaluated to investigate the development of EBI. We found that post-SAH treatment with RSV could markedly inhibit the expressions of TLR4, HMGB1, MyD88, and NF-κB. Meanwhile, RSV significantly reduced microglia activation, as well as inflammatory cytokines leading to the amelioration of neural apoptosis, brain edema, and neurological behavior impairment at 24 h after SAH. However, RSV treatment failed to alleviate brain edema and neurological deficits at 72 h after SAH. These results indicated that RSV treatment could alleviate EBI after SAH, at least in part, via inhibition of TLR4-mediated inflammatory signaling pathway.     

Anti-Inflammatory Effect of Auranofin on Palmitic Acid and LPS-Induced Inflammatory Response by Modulating TLR4 and NOX4-Mediated NF-κB Signaling Pathway in RAW264.7 Macrophages

Chronic inflammation, which is promoted by the production and secretion of inflammatory mediators and cytokines in activated macrophages, is responsible for the development of many diseases. Auranofin is a Food and Drug Administration-approved gold-based compound for the treatment of rheumatoid arthritis, and evidence suggests that auranofin could be a potential therapeutic agent for inflammation. In this study, to demonstrate the inhibitory effect of auranofin on chronic inflammation, a saturated fatty acid, palmitic acid (PA), and a low concentration of lipopolysaccharide (LPS) were used to activate RAW264.7 macrophages. The results show that PA amplified LPS signals to produce nitric oxide (NO) and various cytokines. However, auranofin significantly inhibited the levels of NO, monocyte chemoattractant protein-1, and pro-inflammatory cytokines, such as interleukin (IL)-1β, tumor necrosis factor-α, and IL-6, which had been increased by co-treatment with PA and LPS. Moreover, the expression of inducible NO synthase, IL-1β, and IL-6 mRNA and protein levels increased by PA and LPS were reduced by auranofin. In particular, the upregulation of NADPH oxidase (NOX) 4 and the translocation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) induced by PA and LPS were suppressed by auranofin. The binding between the toll-like receptor (TLR) 4 and auranofin was also predicted, and the release of NO and cytokines was reduced more by simultaneous treatment with auranofin and TLR4 inhibitor than by auranofin alone. In conclusion, all these findings suggested that auranofin had anti-inflammatory effects in PA and LPS-induced macrophages by interacting with TLR4 and downregulating the NOX4-mediated NF-κB signaling pathway.     

Candida Administration in Bilateral Nephrectomy Mice Elevates Serum (1→3)-β-D-glucan That Enhances Systemic Inflammation Through Energy Augmentation in Macrophages

Systemic inflammation, from gut translocation of organismal molecules, might worsen uremic complications in acute kidney injury (AKI). The monitoring of gut permeability integrity and/or organismal molecules in AKI might be clinically beneficial. Due to the less prominence of Candida albicans in human intestine compared with mouse gut, C. albicans were orally administered in bilateral nephrectomy (BiN) mice. Gut dysbiosis, using microbiome analysis, and gut permeability defect (gut leakage), which was determined by fluorescein isothiocyanate-dextran and intestinal tight-junction immunofluorescent staining, in mice with BiN-Candida was more severe than BiN without Candida. Additionally, profound gut leakage in BiN-Candida also resulted in gut translocation of lipopolysaccharide (LPS) and (1→3)-β-D-glucan (BG), the organismal components from gut contents, that induced more severe systemic inflammation than BiN without Candida. The co-presentation of LPS and BG in mouse serum enhanced inflammatory responses. As such, LPS with Whole Glucan Particle (WGP, a representative BG) induced more severe macrophage responses than LPS alone as determined by supernatant cytokines and gene expression of downstream signals (NFκB, Malt-1 and Syk). Meanwhile, WGP alone did not induced the responses. In parallel, WGP (with or without LPS), but not LPS alone, accelerated macrophage ATP production (extracellular flux analysis) through the upregulation of genes in mitochondria and glycolysis pathway (using RNA sequencing analysis), without the induction of cell activities. These data indicated a WGP pre-conditioning effect on cell energy augmentation. In conclusion, Candida in BiN mice accelerated gut translocation of BG that augmented cell energy status and enhanced pro-inflammatory macrophage responses. Hence, gut fungi and BG were associated with the enhanced systemic inflammation in acute uremia.     

Urolithin A Inactivation of TLR3/TRIF Signaling to Block the NF-κB/STAT1 Axis Reduces Inflammation and Enhances Antioxidant Defense in Poly(I:C)-Induced RAW264.7 Cells

Urolithin A is an active compound of gut-microbiota-derived metabolites of polyphenol ellagic acid that has anti-aging, antioxidative, and anti-inflammatory effects. However, the effects of urolithin A on polyinosinic acid-polycytidylic acid (poly(I:C))-induced inflammation remain unclear. Poly(I:C) is a double-stranded RNA (dsRNA) similar to a virus and is recognized by Toll-like receptor-3 (TLR3), inducing an inflammatory response in immune cells, such as macrophages. Inflammation is a natural defense process of the innate immune system. Therefore, we used poly(I:C)-induced RAW264.7 cells and attenuated the inflammation induced by urolithin A. First, our data suggested that 1–30 μM urolithin A does not reduce RAW264.7 cell viability, whereas 1 μM urolithin A is sufficient for antioxidation and the decreased production of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and C-C chemokine ligand 5. The inflammation-related proteins cyclooxygenase-2 and inducible nitric oxide synthase were also downregulated by urolithin A. Next, 1 μM urolithin A inhibited the levels of interferon (INF)-α and INF-β. Urolithin A was applied to investigate the blockade of the TLR3 signaling pathway in poly(I:C)-induced RAW264.7 cells. Moreover, the TLR3 signaling pathway, subsequent inflammatory-related pathways, and antioxidation pathways showed changes in nuclear factor-κB (NF-κB) signaling and blocked ERK/mitogen-activated protein kinase (MAPK) signaling. Urolithin A enhanced catalase (CAT) and superoxide dismutase (SOD) activities, but decreased malondialdehyde (MDA) levels in poly(I:C)-induced RAW264.7 cells. Thus, our results suggest that urolithin A inhibits TLR3-activated inflammatory and oxidative-associated pathways in macrophages, and that this inhibition is induced by poly(I:C). Therefore, urolithin A may have antiviral effects and could be used to treat viral-infection-related diseases.     

Artemisinin Attenuates Amyloid-Induced Brain Inflammation and Memory Impairments by Modulating TLR4/NF-κB Signaling

The abnormal immune response is an early change in the pathogenesis of Alzheimer’s disease (AD). Microglial activation is a crucial regulator of the immune response, which contributes to progressive neuronal injury by releasing neurotoxic products. Therefore, finding effective drugs to regulate microglial homeostasis and neuroinflammation has become a new AD treatment strategy. Artemisinin has potent anti-inflammatory and immune activities. However, it is unclear whether Artemisinin contributes to the regulation of microglial activation, thereby improving AD pathology. This study found that Artemisinin significantly reduced amyloid beta-peptide 1–42 (Aβ_1–42)-induced increases in nitric oxide and reactive oxygen species and inflammatory factors in BV2 cells. In addition, Artemisinin inhibited the migration of microglia and prevented the expansion of the inflammatory cascade. The mechanical studies showed Artemisinin inhibited neuroinflammation and exerted neuroprotective effects by regulating the Toll-like receptor 4 (TLR4)/Nuclear factor-kappa B (NF-κB) signaling pathway. Similar results were obtained in AD model mice, in which Artemisinin administration attenuated Aβ_1–42-induced neuroinflammation and neuronal injury, reversing spatial learning and memory deficits. The anti-inflammatory effect of Artemisinin is also accompanied by the activation of the TLR4/NF-κB signaling pathway in the animal model. Our results indicate that Artemisinin attenuated Aβ_1–42-induced neuroinflammation and neuronal injury by stimulating the TLR4/NF-κB signaling pathway. These findings suggest that Artemisinin is a potential therapeutic agent for AD.     

Caffeine Inhibits NLRP3 Inflammasome Activation by Downregulating TLR4/MAPK/NF-κB Signaling Pathway in an Experimental NASH Model

Caffeine elicits protective effects against liver diseases, such as NASH; however, its mechanism of action involving the pyrin domain-containing-3 (NLRP3) inflammasome signaling pathway remains to be elucidated. This study aimed to evaluate the effect of caffeine on the NLRP3 inflammasome signaling pathway in a rat model of NASH. NASH was induced by feeding rats a high-fat, -sucrose, and -cholesterol diet (HFSCD) for 15 weeks along with a weekly low dose (400 mg/kg, i.p.) of CCl₄. Caffeine was administered at 50 mg/kg p.o. The effects of HFSCD+CCl₄ and caffeine on the liver were evaluated using biochemical, ultrastructural, histological, and molecular biological approaches. The HFSCD+CCl₄-treated rats showed fat accumulation in the liver, elevated levels of inflammatory mediators, NLRP3 inflammasome activation, antioxidant dysregulation, and liver fibrosis. Caffeine reduced necrosis, cholestasis, oxidative stress, and fibrosis. Caffeine exhibited anti-inflammatory effects by attenuating NLRP3 inflammasome activation. Moreover, caffeine prevented increases in toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) protein levels and mitigated the phosphorylation of mitogen-activated protein kinase (MAPK). Importantly, caffeine prevented the activation of hepatic stellate cells. This study is the first to report that caffeine ameliorates NASH by inhibiting NLRP3 inflammasome activation through the suppression of the TLR4/MAPK/NF-κB signaling pathway.