Rutin Attenuates Liver Fibrosis by the IRG1-Itaconate-Nrf2 Axis: Modulation of Oxidative Stress and NLRP3 Inflammasome Activation

Rutin Attenuates Liver Fibrosis by the IRG1-Itaconate-Nrf2 Axis: Modulation of Oxidative Stress and NLRP3 Inflammasome Activation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Rutin Attenuates Liver Fibrosis by the IRG1-Itaconate-Nrf2 Axis: Modulation of Oxidative Stress and NLRP3 Inflammasome Activation Ningman Jiang, Jiao Zhang, Ge Kuang, Guohao Liu, Jingyuan Wan, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8939517/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 4 You are reading this latest preprint version Abstract Liver fibrosis, a common outcome of chronic liver injury, is characterized by excessive inflammation and oxidative stress. Rutin, a bioactive flavonoid with known antioxidant and anti-inflammatory properties, has not been thoroughly investigated for its potential anti-fibrotic mechanisms. This study aimed to elucidate the role of Rutin in liver fibrosis and its underlying molecular pathways. A carbon tetrachloride (CCl₄)-induced murine liver fibrosis model was employed. Liver injury, fibrotic deposition, inflammatory response, and oxidative stress were evaluated through histopathological examination, Western blotting, quantitative real-time PCR, and RNA sequence. The involvement of immune-responsive gene 1 (IRG1) was investigated using IRG1-knockout mice, while molecular docking and cellular thermal shift assay (CETSA) were performed to assess Rutin-IRG1 binding. The results showed that Rutin treatment significantly attenuated CCl₄-induced hepatic injury and collagen accumulation, accompanied by reduced markers of fibrosis. Mechanistically, Rutin activated the IRG1-itaconate axis, leading to a notable decrease in reactive oxygen species and pro-inflammatory cytokine release by Nrf2 activation and NLRP3 inflammasome containment. Molecular analyses confirmed direct binding of Rutin to IRG1, stabilizing its structure and enhancing its functional activity. The protective effects of Rutin were abolished in IRG1-deficient mice, underscoring the essential role of IRG1 in its anti-fibrotic action. In conclusion, Rutin ameliorates liver fibrosis by mitigating oxidative stress and suppressing NLRP3 inflammasome activation through targeting the IRG1/itaconate pathway, revealing a novel immunometabolic mechanism for its hepatoprotective effect. Rutin liver fibrosis IRG1 itaconate oxidative stress NLRP3 inflammasome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Liver fibrosis occurs primarily in chronic liver disease with chronic hepatocyte damage, iterative liver inflammation, excessive oxidative stress and persistent activation of hepatic fibrogenesis. These chronic liver diseases are caused by a variety of etiologies including viral liver disease, alcoholic liver disease, non-alcoholic fatty liver disease and autoimmune liver diseases [1] . Liver fibrosis is curable and reversible, but it is irreversible when progress to cirrhosis or even hepatocellular carcinoma [2, 3]. Currently, liver transplantation is the most effective treatment for liver fibrosis. However, because of the scarcity of organ donors, its clinical application is not popular. Herein, finding promising agents to alleviate liver dysfunction and decrease complications is necessary for liver fibrosis management. It has been widely accepted that oxidative stress and inflammation play critical roles in liver fibrosis [4]. When continuously stimulated by a variety of pathogenic factors, the balance of oxidative stress in the liver is broken, and the liver releases a large number of oxygen free radicals and reactive oxygen species (ROS), which induce hepatocyte injury and death. Dying hepatocytes release damage-associated molecular patterns (DAMPs) that are recognized by pattern recognition receptor (PRR), further activate innate immune signals including Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) and inflammasome NOD-like receptor pyrin domain-containing 3 (NLRP3), resulting in the production and release of numerous inflammatory signals including cytokines, chemokines and neutrophil-mediated ROS [5-7]. This, in turn, promotes apoptosis and necrosis of hepatocytes, forming a feedforward loop of oxidative stress-inflammation-cell death [7] . Meanwhile, ROS and inflammatory signals such as transforming growth factor beta 1 (TGF-β1), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) activate hepatic stellate cell (HSC), resulting in excessive collagen deposition in the extracellular matrix (ECM), leading to liver fibrosis [8-11] . Endogenous complex anti-inflammatory and antioxidant defense systems also exist in the liver, which are regulated by a series of pathways to ensure that the response to inflammation and ROS is sufficient to meet the body's needs [12]. Itaconate is a regulatory metabolite in the tricarboxylic acid (TCA) cycle, and it also acts as an immunomodulator. It is synthesized by cis-aconitic acid, a TCA cycle intermediate, and catalyzed by cis-aconitic acid decarboxylase encoded by the immune response gene 1 protein (IRG1) [13]. Recently, itaconate has received considerable attention due to its critical role in negatively regulating inflammatory responses and cytokine production as an anti-inflammatory metabolite [14, 15]. Nuclear factor E2-related factor 2 (Nrf2), a ubiquitous transcription factor, is the master antioxidant regulator for cells to cope with oxidative stress. The fundamental and induced expression of a string of antioxidant response element-dependent genes including heme oxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO1), are driven by Nrf2 to regulate the physiological and pathophysiological consequence of oxidant exposure [16, 17]. Previous studies had shown a link between the IRG1/itaconate pathway and Nrf2, and a recent study using IRG1 -/- mice demonstrated that IRG1 suppresses injury and inflammation during hepatic ischemia-reperfusion (I/R), and that the mechanism of the itaconate derivative 4-octyl itaconate (4-OI) protected against hepatic I/R injury requires Nrf2 [18]. Rutin, known as quercetin-3-O-Rutinoside and vitamin P, is a polyphenolic natural flavonoid originated from many medicinal plants and vegetables[19]. Its chemical structure was identified as (2-(3,4-dihydroxyphenyl)-4,5-dihydroxy-3-[3,4,5trihydroxy-6-[(3,4,5-trihydroxy-6methyl-oxan-2-yl) oxymethyl] oxan-2-yl] oxy -chromen-7-one (Fig. 1A) [20]. It is reported that Rutin shows a wide range of pharmacological/biological activities including antioxidant, anti-inflammatory, hepatoprotective and neuroprotective effects [21-25]. Previous studies had shown Rutin had hepatoprotection on liver injury induced by different models, including bile duct ligation and high-fat diet [23, 25]. However, there is little research on the therapeutic effect of Rutin on hepatic fibrosis, and the potential molecular mechanism remains undefined. In this study, we explored that whether Rutin had protective effects in CCl 4 -induced liver fibrosis model and whether these protective effects were related to its anti-inflammatory, antioxidant properties and IRG1-itaconate pathway. Materials and Methods Animals and Experimental Design IRG1 knockout (KO, IRG1 −/− ) mice (Cyagen, S-KO-02679) and their C57BL/6N cognate background control mice were bred and raised under specific pathogen-free conditions. All mice were 8-12 weeks old male mice weighing 20-30 g, housed in standard laboratory animal cages and maintained with standard management conditions of a 12 h light/ dark cycle. All experimental procedures were approved by the ethics committee of Chongqing Medical University, and conducted in accordance with the ARRIVE guidelines and the national research council’s guide for the care and use of laboratory animals. To explore the effect of Rutin dose on its biological effects, all mice were randomly divided into six groups. (1) Vehicle control, (2) Rutin, (3) CCl 4 , (4) CCl 4 +Rutin (15 mg/kg), (5) CCl 4 +Rutin (30 mg/kg), (6) CCl 4 +Rutin (60 mg/kg). To investigate the protective mechanism of Rutin against liver fibrosis, WT mice and IRG1 −/− mice were used in CCl 4 group or CCl 4 +Rutin (60 mg/kg) group. Rutin (suspended in 0.1% CMC-Na) was intragastrically (i.g.) administered to corresponding group of mice for 10 consecutive days. Vehicle groups of mice received corresponding volume of CMC-Na solution. Hepatic fibrosis was induced by intraperitoneal (i.p.) injections of CCl 4 (0.5 ml/kg, dissolved in olive oil, 1:19) three times a week for 8 weeks. Same volume of olive oil was injected to the normal control group. All mice were sacrificed 48 h after final CCl 4 injection. All serum samples and liver samples were collected for further examination. Biochemical Assay Blood samples were collected through retro-orbital bleeding. The serum samples were obtained by centrifugation at 3600 rpm for 10 min at 4°C. Serum enzyme activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by spectrophotometry using corresponding detection kits (Nan Jing Jan Cheng Biochemical Institute, Nanjing, China) according to the instruction. Histologic Analysis Some excised liver specimens were fixed with 4% paraformaldehyde solution and then embedded in paraffin. Paraffin sections were stained with hematoxylin and eosin (H&E), and then were scored using the Suzuki methodology to assess tissue inflammation and necrosis. Sirius red staining and Masson staining were performed in paraffin sections according to standard protocol. Detection of Tissue ROS Some liver samples were snap frozen in liquid nitrogen and kept at -80°C. To determine the hepatic ROS levels, the frozen liver samples were sectioned transversely. The 10 μm thick of frozen liver section was treated with 10 μmol/L fluorescent dye dihydroethidium (DHE) and incubated at 37°C for 30 min in the dark. Then the nuclei were stained with DAPI and the fluorescence images were obtained by fluorescence microscope (Nikon, Tokyo, Japan). Measurement of Peroxidation The peroxidation products of various biomacromolecules in liver tissue were detected, including the lipid peroxidation product 4-HNE, the protein peroxidation products Protein Carbonyl and the nucleic acid peroxidation products 8-OHG, 8-OHdG and 8-hydroxyguanine. The production of 4-HNE was demonstrated by immunohistochemistry, and the contents of protein carbonyl and 8-OHG, 8-OHdG, 8-hydroxyguanine were measured by ELISA. The specific methods are in 2.9 and 2.10. Examination for Antioxidant Enzymes Commercially available assay kits for the examination of hepatic superoxide dismutase (SOD) and catalase (CAT) activities were obtained from Nan Jing Jan Cheng Biochemical Institute (Nanjing, China). The activity of SOD was determined by colorimetry. The principle, briefly, is that superoxide anions (O 2 • − ) are produced by xanthine and xanthine oxidase reaction systems, which reduce WST-1 to orange or purple WST-1 Formazan dye. The reactions of CAT decomposing H 2 O 2 are quickly terminated by adding ammonium molybdate and the remaining H 2 O 2 reacts with ammonium molybdate to produce a pale-yellow complex. The activity of CAT can be calculated by measuring its change at 405 nm. Hydroxyproline (Hyp) Assay The level of Hepatic Hyp was examined by using the corresponding kit (Nan Jing Jan Cheng Biochemical Institute, Nanjing, China) according to the supplier's protocol. The oxidation product of hydroxyproline reacts with dimethylaminobenzaldehyde to form a purple complex, and the level of Hyp can be calculated by colorimetry. Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) and RNA Sequencing The total RNA of liver tissues was extracted using Trizol reagent. Then, NanoDrop One microvolume spectrophotometer was used to quantify total RNA. Equal amount of RNA was reversibly transcribed into complementary DNA. PCR reactions were used to amplify the target gene. Its procedure, briefly, consisted of starting at 94°C for 5 min, followed by 32-40 cycles of amplification (denaturation at 94°C for 1 min, annealing at 58°C for 30 s, and extension at 72°C for 30 s) with a final primer extra-extension at 72°C for 7 min. The PCR products were separated on 2% agarose gel and stained with ethidium bromide. The intensity of each target gene mRNA band was quantified by molecular imaging system and β-actin was used as internal control. The sequences of the primers are listed in Table 1 . Tissue Preparation and Western Blot Analysis Liver tissue removed from -80°C, triturated and lysed in RIPA lysis buffer containing protease inhibitors. Total protein concentrations were measured with BCA kit and normalized with buffer. Then, proteins were electrophoretically separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes. After blocking with 5% nonfat dry milk for 1 h, the membranes were incubated overnight with the indicated antibodies (IRG1, NQO1, HO-1, NLRP3, ASC, pro-caspase1, TLR4, IκB, p65) in 4°C. Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h, then visualized by chemiluminescence (Bio-Rad). Immunohistochemical and Immunofluorescence Staining Paraffin sections were used for immunohistochemical staining. They were deparaffinized, hydrated and incubated with H 2 O 2 to block endogenous peroxidase. After retrieved by microwave and blocked with 5% BSA, the liver sections were incubated with primary antibody overnight at 4°C. Sections were then co-incubated with biotinylated secondary antibodies for 1 h, followed by avidin-biotin-peroxidase complexes. The immunoreactive signal is generated by color deposition with diaminobenzidine as a substrate. For immunofluorescence staining, the frozen sections were washed in TBS, and then blocked in 5% BSA for 1 h. Following overnight incubation with primary antibodies, the corresponding fluorescent-labeled secondary antibodies were applied. DAPI dye was used to stain the nuclei. After mounting with anti-quenching fluorescent mounting medium, images were obtained using a fluorescence microscope (Leica, USA). Enzyme-Linked ImmunoSorbent Assay (ELISA) The levels of cytokines, chemokines and peroxidation products in liver cell supernatants were measured using ELISA Bender medsystems kits according to the manufacturer's instructions. Measurements of Itaconate Samples were injected by Thermo Fisher Vanquish ultra-high-performance liquid chromatography (HPLC) and separated over a reversed-phase Thermo Fisher Hypercarb porous graphitic column maintaining at 52°C. The mobile phase contains solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). Itaconate concentrations were quantified using liquid chromatography-mass spectrometry analysis. Deuterated (D4)-taurine and (D3)-lactate (Sigma-Aldrich) were used as an internal standard. Molecular Docking and Dynamics Simulations The molecular docking study was performed to explore the binding mode and affinity between Rutin and the IRG1 protein. The three-dimensional crystal structure of human IRG1 was retrieved from the Protein Data Bank. Docking was carried out with AutoDock4 software. The IRG1 protein was defined as the rigid receptor, while all rotatable bonds in Rutin were set as flexible. Ten independent docking runs were performed, and the resulting poses were ranked according to their binding affinity (kcal/mol). The pose with the lowest binding energy and highest cluster occupancy was selected for further analysis. Visualization and interaction analysis (hydrogen bonds, hydrophobic contacts) were conducted using Pymol2 software . To evaluate the stability and dynamic behavior of the Rutin-IRG1 complex, molecular dynamics (MD) simulations were performed using the GROMACS package. The complex obtained from molecular docking was solvated in a cubic water box. Production MD simulation was then carried out for 100 ns under periodic boundary conditions. The algorithm constrained bond lengths, and the Particle Mesh Ewald method was used for long-range electrostatic interactions. Trajectories were analyzed for root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent-accessible surface area (SASA), and hydrogen bond occupancy. The binding free energy was calculated. Cellular Thermal Shift Assay (CETSA) The Cellular Thermal Shift Assay was employed to validate the direct binding between Rutin and the intracellular target IRG1 in a cellular context. HepG2 cells were seeded in 6-well plates and allowed to adhere overnight. Cells were then treated with either Rutin (10 µM) or an equivalent volume of vehicle control (DMSO). Following treatment, cells were harvested, washed with PBS, and resuspended. The cell suspension was aliquoted into PCR tubes and heated at a gradient of temperatures (ranging from 37°C to 67°C) for 3 minutes in a thermal cycler, followed by incubation at room temperature for an additional 3 minutes. Cells were then subjected to three freeze-thaw cycles using liquid nitrogen to lyse the cells. The soluble protein fraction was separated from cell debris by centrifugation at 12,000 × g for 15 minutes at 4°C. The supernatant was collected, and the protein concentration was determined. Equal amounts of protein from each temperature point were resolved by SDS-PAGE, followed by Western blotting analysis using a specific anti-IRG1 antibody. The band intensity of soluble IRG1 remaining at each temperature was quantified using ImageJ software. Statistical Analyses These data were expressed as mean ± SD. Statistical analysis was carried out using one-way or two-way ANOVA followed by a Bonferoni comparison or Dunnett's multiple comparison test to determine the differences among groups. P < 0.05 was considered as statistically significant. Results Rutin Attenuates CCl₄-Induced Liver Injury and Fibrosis in Mice Rutin exhibited significant hepatoprotective and antifibrotic effects in a mouse model of CCl₄-induced liver injury. Its characteristic flavonoid molecular structure underlies its notable bioactivity ( Fig. 1A ). Biochemical assays showed that CCl₄ administration markedly elevated AST and ALT levels, indicating hepatocellular injury, whereas Rutin treatment significantly reduced these enzyme levels ( Fig. 1B, 1C ). Histopathological examination using H&E staining revealed that CCl₄ disrupted hepatic lobular architecture, caused hepatocellular swelling, collagen deposition, and substantial inflammatory infiltration, all of which were alleviated in a dose-dependent manner by Rutin ( Fig. 1D ). In the context of liver fibrosis, Masson’s trichrome and Sirius Red staining demonstrated excessive collagen accumulation in CCl₄-treated livers, which was significantly reduced following Rutin administration ( Fig. 1E, 1F ). Consistently, hepatic hydroxyproline content was elevated in the CCl₄ group and markedly decreased in the Rutin-treated groups ( Fig. 1G ). Immunohistochemical analyses further revealed that CCl₄ induced strong α-smooth muscle actin (α-SMA) and type I collagen alpha 1 (Col1a1) expression in the periportal region, reflecting HSC activation and fibrogenesis, both of which were significantly attenuated by Rutin ( Fig. 1H, 1I ). RT-qPCR analysis confirmed that CCl₄ markedly upregulated the mRNA expression of fibrosis-related genes, including Col1a1, CTGF, TGF-β, and TIMP-1, while Rutin treatment significantly suppressed these transcriptional changes ( Fig. 1J–1M ). Together, these results show that Rutin attenuates CCl₄-induced liver injury and fibrosis, as evidenced by reduced serum transaminase levels, improved histological architecture, decreased collagen deposition, and suppression of fibrotic marker expression. Rutin Attenuates CCl₄-Induced Hepatic Inflammation and Oxidative Stress in Mice To comprehensively elucidate the anti-inflammatory and antioxidant effects of Rutin in a CCl₄-induced mouse model of liver injury, we conducted a series of analyses at multiple levels, including gene expression, immune cell infiltration, oxidative damage markers, and antioxidant enzyme activity. RT-qPCR analysis demonstrated that Rutin treatment significantly downregulated the hepatic mRNA expression of pro-inflammatory cytokines IL-6 and TNF-α, as well as chemokines CCL2 and CXCL1 ( Fig. 2A–2D ), indicating its potent anti-inflammatory capacity. Immunofluorescence staining further revealed that CCl₄ exposure markedly increased the infiltration of F4/80-positive macrophages and CD11b-positive neutrophils in liver tissue, which was notably alleviated by Rutin administration ( Fig. 2E, 2F ), suggesting an inhibitory effect on inflammatory cell recruitment. Regarding oxidative stress, immunohistochemical staining showed a substantial elevation of the lipid peroxidation marker 4-hydroxynonenal (4-HNE) following CCl₄ treatment, which was significantly reduced upon Rutin intervention ( Fig. 2G ). ELISA assays confirmed that CCl₄ induced a marked increase in hepatic levels of oxidative damage markers, including the protein peroxidation product protein carbonyl and nucleic acid oxidation products 8-OHG, 8-OHdG, and 8-hydroxyguanine, all of which were significantly attenuated by Rutin ( Fig. 2H, 2I ). Furthermore, enzymatic activity assays showed that CCl₄ markedly suppressed the hepatic activities of key antioxidant enzymes SOD and CAT, while Rutin treatment effectively restored their activity levels ( Fig. 2J, 2K ). These results indicate that Rutin reduces hepatic inflammation and oxidative stress in CCl₄-treated mice, as evidenced by decreased pro-inflammatory cytokine expression, reduced immune cell infiltration, lower oxidative damage markers, and restored antioxidant enzyme activity. Rutin Activates the Nrf2 Pathway and Inhibits the NLRP3–NF-κB Axis in the Liver of CCl₄-Treated Mice Rutin exerts significant regulatory effects on oxidative stress and inflammatory signaling pathways in the liver. Specifically, Rutin treatment markedly upregulated the expression of Nrf2 and its downstream antioxidant enzymes NQO1 and HO-1 in the liver of CCl₄-treated mice ( Fig. 3A ), which was further confirmed by semi-quantitative Western blot analysis showing consistent increases in the protein levels of Nrf2, NQO1, and HO-1 ( Fig. 3B–3D ), suggesting that Rutin enhances hepatic antioxidant defense via activation of the Nrf2 pathway. Conversely, the expression of NLRP3 and its downstream signaling molecules ASC and pro-caspase-1, which were significantly induced by CCl₄, were markedly reduced following Rutin administration ( Fig. 3E ). This inhibitory effect was further supported by Western blot analysis, which demonstrated consistent downregulation of NLRP3, ASC, caspase-1, and IL-1β protein levels ( Fig. 3F–3I ). Additionally, Western blot analysis revealed that CCl₄ markedly activated the NF-κB signaling pathway, as evidenced by elevated expression of TLR4, p-IκB, and p-p65, whereas Rutin effectively suppressed these changes ( Fig. 3J ) and the semi-quantitative analysis of NLRP3, ASC, caspase-1, and IL-1β protein levels confirmed this trend ( Fig. 3K–3N ). Furthermore, HPLC analysis demonstrated that Rutin significantly increased the hepatic content of itaconate in CCl₄-treated mice ( Fig. 3O ), indicating a potential involvement of endogenous immunometabolic regulation in its anti-inflammatory and antioxidant effects. These results show that Rutin upregulates the hepatic Nrf2 pathway and its downstream antioxidant enzymes, while suppressing the NLRP3 inflammasome and NF-κB signaling activation induced by CCl₄. Additionally, Rutin increases hepatic itaconate levels, suggesting involvement of endogenous metabolic regulation in these responses. Molecular interactions between Rutin and IRG1 This study investigates the interactions between Rutin and IRG1 through molecular docking and molecular dynamics (MD) simulations. Molecular docking results show that Rutin forms hydrogen bonds with several amino acids of IRG1, including ASN-425, ILE-347, ASP-285, ARG-273, and ASP-417, enhancing binding affinity. Additionally, Rutin interacts with ARG-273 through cation-π conjugation and strengthens the binding further through hydrophobic interactions with ARG-273, VAL-349, LYS-421, and ALA-424 ( Fig. 4A ). MD simulation reveals that the RMSD of the complex quickly reaches equilibrium within 0–10 ns (around 1.2 Å), then gradually increases to 2.0 Å between 40–60 ns, rising sharply to 3.5 Å at 60 ns, and stabilizing at approximately 3.0 Å, indicating the system reaches a new equilibrium state ( Fig. 4 B ). RMSF analysis shows that the protein’s flexibility decreases, with values generally below 2 Å, indicating rigidity in the protein core, which supports stable binding and enzymatic activity ( Fig. 4C ). RoG analysis shows slight conformational relaxation between 0–20 ns, with RoG increasing from 22.2 Å to 22.5 Å, and stabilizing between 22.6–22.7 Å after a significant increase at 60 ns, indicating a new equilibrium state ( Fig. 4D ). MM-GBSA calculations show a binding energy of -16.38±4.79 kcal/mol, indicating strong binding affinity, primarily driven by electrostatic and van der Waals interactions ( Fig. 4E ). Energy decomposition identifies key amino acids contributing to binding, such as GLU 297, ALA 296, and VAL 295 ( Fig. 4F ). Hydrogen bond analysis shows an initial increase to 10 bonds, followed by a drop to zero between 20–60 ns, and a recovery to 2–6 bonds between 60–100 ns, suggesting conformational shifts and a new interaction equilibrium ( Fig. 4G ). and CETSA analysis showed that Rutin significantly enhances IRG1 thermal stability ( Fig. 4H ). These findings indicate that Rutin binds IRG1 through multiple stable interactions and induces conformational stabilization of the protein. This structural basis supports a potential role for Rutin in modulating IRG1 function at the molecular level. IRG1 Deficiency Abolishes Rutin-Mediated Activation of Nrf2 and Inhibition of NF-κB Signaling To further investigate whether the antifibrotic effect of Rutin is dependent on the IRG1 pathway, we established an IRG1⁻/⁻ mouse model of CCl₄-induced liver fibrosis. Western blot analysis revealed that Rutin markedly upregulated the protein expression of Nrf2 and its downstream antioxidant enzymes NQO1 and HO-1 in liver tissue; however, this upregulation was significantly attenuated in IRG1-deficient mice ( Fig. 5A ), and semi-quantitative analysis showed consistent trends for Nrf2, NQO1, and HO-1 levels ( Fig. 5B–5D ), suggesting that IRG1 plays a crucial role in mediating Rutin-induced activation of the Nrf2 pathway. Similarly, in WT mice, Rutin significantly inhibited the CCl₄-induced expression of NLRP3 inflammasome-related proteins, including NLRP3, ASC, Caspase-1, and IL-1β, whereas this inhibitory effect was markedly diminished in IRG1⁻/⁻ mice ( Fig. 5E ), which was further supported by Western blot semi-quantitative analysis ( Fig. 5F–5I ), indicating a potential involvement of IRG1 in the negative regulation of inflammasome activation by Rutin. In addition, Western blot results showed that CCl₄ administration significantly activated the NF-κB signaling pathway, as evidenced by elevated protein levels of TLR4, p-IκB, and p-p65 in the liver, and these increases were effectively suppressed by Rutin in WT mice but not in IRG1⁻/⁻ mice ( Fig. 5J ); consistent trends were observed in the semi-quantitative analysis of TLR4, p-IκB, IκB, p-p65, and p65 expression levels ( Fig. 5K–5N ), further supporting the role of IRG1 in Rutin-mediated inhibition of NF-κB activation. Notably, HPLC analysis demonstrated that Rutin significantly increased hepatic itaconate content in CCl₄-treated WT mice, while this elevation was abolished in IRG1⁻/⁻ mice ( Fig. 5O ), further supporting the hypothesis that Rutin exerts its anti-inflammatory and antioxidant effects, at least in part, through the IRG1/itaconate metabolic pathway. These findings indicate that the presence of IRG1 is essential for Rutin to exert its regulatory effects on the Nrf2 antioxidant response and the NLRP3–NF-κB inflammatory axis, highlighting the importance of the IRG1/itaconate pathway in the hepatoprotective mechanism of Rutin. IRG1 Deficiency Attenuates the Anti-inflammatory and Antioxidant Effects of Rutin in CCl₄-Induced Liver Injury In the context of IRG1 gene knockout, we further evaluated hepatic inflammation and antioxidant responses to determine whether the anti-inflammatory and antioxidant effects of Rutin are mediated through the IRG1 pathway. RT-qPCR analysis revealed that Rutin significantly downregulated the mRNA expression of pro-inflammatory cytokines IL-6 and TNF-α, as well as chemokines CCL2 and CXCL1 in the liver of CCl₄-treated mice, indicating a potent anti-inflammatory effect. However, this inhibitory effect was markedly attenuated in IRG1⁻/⁻ mice ( Fig. 6A–6D ). Consistently, immunofluorescence staining showed that CCl₄ exposure significantly increased the hepatic infiltration of F4/80-positive macrophages and CD11b-positive neutrophils, which was notably reduced by Rutin treatment in wild-type mice but not in IRG1-deficient mice ( Fig. 6E, 6F ). Regarding oxidative stress, immunohistochemical analysis demonstrated that hepatic levels of the lipid peroxidation marker 4-HNE were significantly elevated following CCl₄ exposure, whereas Rutin administration markedly reduced 4-HNE accumulation; this reduction was impaired in IRG1⁻/⁻ mice ( Fig. 6G ). ELISA further confirmed that the levels of hepatic oxidative damage markers, including the protein peroxidation product protein carbonyl and nucleic acid oxidation products 8-OHG, 8-OHdG, and 8-hydroxyguanine, were significantly increased in CCl₄-treated mice and effectively attenuated by Rutin, whereas these protective effects were substantially diminished in IRG1⁻/⁻ mice ( Fig. 6H, 6I ). Additionally, enzymatic activity assays revealed that CCl₄ significantly suppressed the hepatic activities of the key antioxidant enzymes SOD and CAT, which were restored by Rutin treatment in wild-type mice, but this restoration was abolished in IRG1-deficient mice ( Fig. 6J, 6K ). These findings indicate that IRG1 plays a critical regulatory role in the anti-inflammatory and antioxidant effects of Rutin, and that the absence of IRG1 significantly compromises the hepatoprotective efficacy of Rutin, highlighting the IRG1/itaconate pathway as a potential mechanistic basis for its therapeutic actions. IRG1 Deficiency Impairs the Hepatoprotective and Antifibrotic Effects of Rutin in CCl₄-Induced Liver Injury In the context of IRG1 gene deficiency, we further evaluated hepatic injury and fibrosis to determine whether the protective effects of Rutin against tissue damage and fibrosis are associated with the IRG1 pathway. Biochemical analysis revealed that CCl₄ administration significantly elevated serum levels of AST and ALT, indicating hepatocellular injury, whereas Rutin treatment markedly reduced these enzyme levels. However, this hepatoprotective effect was substantially attenuated in IRG1⁻/⁻ mice ( Fig. 7A, 7B ). Histopathological examination using H&E staining demonstrated that CCl₄ disrupted hepatic lobular architecture, causing hepatocyte swelling, collagen deposition, and extensive inflammatory infiltration, all of which were alleviated by Rutin, while this improvement was significantly impaired in IRG1⁻/⁻ mice ( Fig. 7C ). In the context of liver fibrosis, Masson's trichrome and Sirius Red staining revealed excessive collagen accumulation in the liver following CCl₄ exposure, which was markedly reduced by Rutin treatment in wild-type mice but not in IRG1-deficient mice ( Fig. 7D, 7E ). Consistently, hepatic hydroxyproline content, which was elevated after CCl₄ treatment, was significantly reduced by Rutin, whereas this effect was blunted in IRG1⁻/⁻ mice ( Fig. 7F ). Immunohistochemical analysis further showed that CCl₄ induced strong perivascular expression of α-SMA and Col1a1, reflecting hepatic stellate cell activation and fibrogenesis, both of which were significantly attenuated by Rutin in wild-type mice but not in IRG1-deficient mice ( Fig. 7G, 7H ). Moreover, RT-qPCR analysis confirmed that CCl₄ markedly upregulated the mRNA expression of fibrosis-related genes, including Col1a1, CTGF, TGF-β, and TIMP-1, and these transcriptional changes were significantly suppressed by Rutin, while this suppressive effect was abolished in IRG1⁻/⁻ mice ( Fig. 7I-7L ). These findings indicate that IRG1 plays a critical role in mediating the protective effects of Rutin against CCl₄-induced hepatic injury and fibrosis, highlighting the IRG1/itaconate pathway as a potential mechanistic basis for its antifibrotic activity. Discussion CCl 4 has been widely used to induce liver injury and fibrosis in mice [26]. CCl 4 is one of halogenated alkanes that activated by cytochrome (CYP)2E1 to form a highly reactive trichloromethyl radical and promote excessive generation of ROS. The trichloromethyl radical and its peroxy radical CCl 3 OO •− are involved in lipid peroxidation processes and several free radical reactions that contribute to the activation of Kupffer cells and the induction of an inflammatory response [27, 28]. Kupffer cells activated by CCl 4 release TNF-α, TGF-β, and IL-1, IL-6, and IL-10. HSCs activated by CCl 4 and inflammatory mediators over-produce type-I collagen, and thus promote liver fibrosis. In brief, CCl 4 -induced fibrosis is closely related to oxidative stress and inflammation. In our study, Rutin was found to inhibit CCl 4 -induced liver inflammation and oxidative stress. Rutin might be a potent agent to treat liver fibrosis owing to its antioxidant and anti-inflammatory biological activities. Oxidative stress is closely associated with the progress of CCl 4 -induced liver injury and fibrosis. CCl 4 -induced increase in hepatic ROS level. Several studies had shown that ROS played an important role in the regulation of HSCs activation and fibrotic gene expression [29]. After activated by ROS, HSCs proliferate and excessively release collagen into the ECM propagating liver fibrosis. The excessive ROS can also activate the lipid peroxidation of hepatocytes to cause the increase generation of lipid peroxides including 4-HNE, which easily modify proteins and inhibit critical enzyme activities. Antioxidant enzymes, especially SOD and CAT, can reduce the level of superoxide (O 2 − ) and hydrogen peroxide (H 2 O 2 ), which are the most-produced ROS [30]. Nrf2 is one of master regulators to cope with oxidative stress. In the oxidatively stimulated state, Nrf2 remains stable and enters the nucleus, where it binds to antioxidant response elements, resulting in transcriptional activation of genes encoding a series of antioxidant enzymes, including HO-1 and NQO1, to restore redox homeostasis in the body [31-35]. For these reasons, we observed the helpful effect of Rutin on CCl 4 -induced liver injury and fibrosis by detecting hepatic ROS level, production of lipid peroxidation products, antioxidant enzyme activities and Nrf2 pathway. The results showed that Rutin could significantly reduce the hepatic ROS level, decrease the production of 4-HNE, and increase the activities of SOD and CAT, and activation of Nrf2 pathway, suggesting that the protective effect of Rutin on liver injury and fibrosis might be associated with its antioxidant activity. Inflammation is another critical pathological mechanism promoting CCl 4 -induced liver injury and fibrosis. DAMPs released from damaged or dying hepatocytes are recognized by PRRs on Kupffer cells and further activate Kupffer cells [36] . Activated kupffer cells produce cytokines and chemokines including IL-1β, IL-6, TNF-α, CCL2 and CXCL1, participate in the inflammatory response and induce the activation of HSCs [11]. IL-1β is a main pro-inflammatory cytokine produced during NLRP3 inflammasome activation. TNF-α is considered to be the main endogenous harmful mediator during liver injury, which has the ability to directly damage tissues and trigger inflammatory cascades [37]. IL-6 is another pro-inflammatory cytokine that antagonizes TNF-α-induced tissue damage. In this study, Rutin decreased the mRNA and protein levels of IL-1β, IL-6, TNF-α in CCl 4 -treated mice. The release of chemokines such as CCL2 and CXCL1 recruit macrophages and neutrophils to participate in the initial stage of inflammatory response in the process of liver fibrosis. In our results, we observed Rutin sharply decreased the mRNA and protein levels of CCL2 and CXCL1 in CCl 4 -treated mice. In addition, we found aggregation of macrophages and neutrophils in the liver of mice following CCl 4 administration, whereas Rutin treatment significantly reduced this activity. Previous studies have demonstrated that inhibition of NLRP3 inflammasome ameliorates CCl 4 -induced liver fibrosis [38-41]. ASC and pro-caspase 1 are key components of NLRP3 inflammasome [42, 43]. In the present study, the decline of NLRP3, ASC and pro-caspase 1 protein levels was observed in CCl 4 mice after Rutin treatment. TLR4 is a critical PRR involved in the triggering of CCl 4 -induced cascades of inflammatory responses. NF-κB is a classic inflammatory signaling pathway that promotes the occurrence of inflammatory responses, of which IkB and p65 are important components of this pathway [11]. It has been reported that the knockout of TLR4 can alleviate inflammatory response to attenuate CCl 4 -induced hydroxyproline and α-SMA [44]. In the present study, Rutin was observed to decrease the level of TLR4 and the phosphorylation of IκB and p65 in the liver of CCl 4 -treated mice. These results suggest that Rutin inhibits liver inflammation by inhibiting the activation of NF-κB and inflammasome NLRP3 signaling pathways after CCl 4 exposure. To sum up, Rutin inhibits hepatic oxidative stress by promoting Nrf2 signaling, and inhibits hepatic inflammation by inhibiting the activation of NF-κB and inflammasome NLRP3 signaling pathways, so we speculate whether there is an intermediate molecule affected by Rutin, which is involved in the regulation of the above signaling pathways. Itaconate, an endogenous biological metabolite catalyzed by IRG1 and accumulated by metabolic disorders after inflammatory activation of macrophages, is also acts as an immunomodulator. Previous study had shown a link between the IRG1/itaconate pathway and Nrf2, when itaconate was shown to directly alkylate cysteine residues in Kelch-like ECH-associated protein 1 (Keap1), leading to an increase in Nrf2 in macrophages during endotoxemia [45]. Endogenous itaconate and its derivative 4-OI have been presented to alkylate cysteine residues on plenty of proteins, including Keap1, and this form of cysteine alkylation, called 2, 3-dicarboxypropylation or itaconation, has been exhibited to be a crucial component involved in the anti-inflammatory properties of itaconate [46-48]. In a recent study, 4-OI inhibited the inflammatory response by inhibiting NF-κB signal pathway in RAW264.7 cells[49]. A previous study has reported that itaconate induced dicarboxypropylation of NLRP3 cysteine C548 and inhibited inflammasome activation, providing evidence that itaconate is a specific endogenous inhibitor of NLRP3 inflammasome activation [50]. We hypothesized that that Rutin further promotes Nrf2 pathway and inhibits NF-κB and inflammasome NLRP3 signaling pathways by regulating IRG1-itaconate axis. Therefore, we repeated the above experiments with IRG1 −/− mice and found that the protective effect of Rutin on liver injury and fibrosis was weakened. It indicated that the protective effects of Rutin on CCl 4 -induced liver injury and fibrosis was related to the IRG1-itaconate axis. Conclusion The results of this experiment support the hepatic beneficial effects of Rutin, and Rutin supplementation may reduce drug-induced liver injury and fibrosis in mice. We have demonstrated that Rutin has a certain inhibitory effect on CCl 4 -induced liver injury, inflammatory response, oxidative stress and liver fibrosis in mice, possibly by further upregulating Nrf2 activity through IRG1-itaconate pathway and inhibiting inflammatory related pathways NLRP3 and NF-κB. Abbreviations ROS, reactive oxygen species; DAMPs, damage-associated molecular patterns; PRR, pattern recognition receptor; NF-κB, Nuclear Factor kappa-light-chain-enhancer of activated B cells; NLRP3, NOD-like receptor pyrin domain-containing 3; TGF-β1, transforming growth factor beta 1; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; ECM, extracellular matrix; TCA, tricarboxylic acid; IRG1, immune response gene 1 protein; Nrf2, Nuclear factor E2-related factor 2; HO-1, heme oxygenase-1; NQO1, quinone oxidoreductase 1; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DHE, Dihydroethidium; SOD, superoxide dismutase; CAT, Catalase; HPLC, high-performance liquid chromatography; HSC, hepatic stellate cell; O 2− , superoxide; H 2 O 2 , hydrogen peroxide. Declarations Acknowledgements The study was supported by Chongqing Natural Science Foundation (CSTB2022NSCQ-MSX0061) Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethics Approval Animal studies were ethically approved by the Chongqing Medical University Ethics Committee (Certification Number: 20220305). Data availability Data will be made available upon request. References Parola M, Pinzani M. Liver fibrosis: Pathophysiology, pathogenetic targets and clinical issues. Molecular aspects of medicine 2019; 65:37-55. Sun M, Kisseleva T. Reversibility of liver fibrosis. Clin Res Hepatol Gastroenterol 2015; 39 Suppl 1:S60-3. Asrani SK, Devarbhavi H, Eaton J, Kamath PS. Burden of liver diseases in the world. Journal of hepatology 2019; 70:151-171. Koyama Y, Brenner DA. Liver inflammation and fibrosis. 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Caspase-1-induced pyroptotic cell death. Immunol Rev 2011; 243:206-14. Bala S, Csak T, Saha B, Zatsiorsky J, Kodys K, Catalano D, et al. The pro-inflammatory effects of miR-155 promote liver fibrosis and alcohol-induced steatohepatitis. J Hepatol 2016; 64:1378-87. Mills EL, Ryan DG, Prag HA, Dikovskaya D, Menon D, Zaslona Z, et al. Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1. Nature 2018; 556:113-117. Liao ST, Han C, Xu DQ, Fu XW, Wang JS, Kong LY. 4-Octyl itaconate inhibits aerobic glycolysis by targeting GAPDH to exert anti-inflammatory effects. Nat Commun 2019; 10:5091. Qin W, Zhang Y, Tang H, Liu D, Chen Y, Liu Y, et al. Chemoproteomic Profiling of Itaconation by Bioorthogonal Probes in Inflammatory Macrophages. J Am Chem Soc 2020; 142:10894-10898. Qin W, Qin K, Zhang Y, Jia W, Chen Y, Cheng B, et al. S-glycosylation-based cysteine profiling reveals regulation of glycolysis by itaconate. Nat Chem Biol 2019; 15:983-991. Yang W, Wang Y, Zhang P, Sun X, Chen X, Yu J, et al. Immune-responsive gene 1 protects against liver injury caused by concanavalin A via the activation Nrf2/HO-1 pathway and inhibition of ROS activation pathways. Free Radic Biol Med 2022; 182:108-118. Hooftman A, Angiari S, Hester S, Corcoran SE, Runtsch MC, Ling C, et al. The Immunomodulatory Metabolite Itaconate Modifies NLRP3 and Inhibits Inflammasome Activation. Cell Metab 2020; 32:468-478.e7. Tables Table 1. The primer sequences used for real-time PCR Gene Forward Primers (5’–3’) Reverse Primers (5’–3’) GAPDH IL-1β IL-6 TNF-α CCL2 Col1a1 AGG TCG GTG TGA ACG GAT TTG GCA ACT GTT CCT GAA CTC AAC T TAG TCC TTC CTA CCC CAA TTT CC CCC TCA CAC TCA GAT CAT CTT CT TTA AAA ACC TGG ATC GGA ACC AA GCT CCT CTT AGG GGC CAC T TGT AGA CCA TGT AGT TGA GGT CA ATC TTT TGG GGT CCG TCA ACT TTG GTC CTT AGC CAC TCC TTC GCT ACG ACG TGG GCT ACA G GCA TTA GCT TCA GAT TTA CGG GT CCA CGT CTC ACC ATT GGG G CTGF TCT CCA CCC GAG TTA CCA ATG AAT GTT TTC CTC CAG GTC AGC TGF-b TTG CTT CAG CTC CAC AGA GA TGG TTG TAG AGG GCA AGG AC TIMP-1 TGG TTG TAG AGG GCA AGG AC TGG TTG TAG AGG GCA AGG AC Additional Declarations No competing interests reported. Supplementary Files GA.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 12 Mar, 2026 Editor assigned by journal 28 Feb, 2026 Submission checks completed at journal 28 Feb, 2026 First submitted to journal 22 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8939517","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":605238942,"identity":"3f1c5cda-0534-4876-b7d7-437e12bb612e","order_by":0,"name":"Ningman Jiang","email":"","orcid":"","institution":"The First Affiliated Hospital of Chongqing Medical University,","correspondingAuthor":false,"prefix":"","firstName":"Ningman","middleName":"","lastName":"Jiang","suffix":""},{"id":605238943,"identity":"1b369b5e-ba8f-4479-81ec-b4ebb24f21b9","order_by":1,"name":"Jiao Zhang","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiao","middleName":"","lastName":"Zhang","suffix":""},{"id":605238944,"identity":"208af1f8-67fb-4a3c-aeb4-6be110e8cfaf","order_by":2,"name":"Ge Kuang","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ge","middleName":"","lastName":"Kuang","suffix":""},{"id":605238945,"identity":"7f481146-a297-4547-a307-24030aa45111","order_by":3,"name":"Guohao Liu","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Guohao","middleName":"","lastName":"Liu","suffix":""},{"id":605238946,"identity":"d5c22cde-5125-4b28-86fb-92075a5f9c48","order_by":4,"name":"Jingyuan Wan","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jingyuan","middleName":"","lastName":"Wan","suffix":""},{"id":605238947,"identity":"b0146f73-c378-43d6-8b52-89d0e9b8e0a0","order_by":5,"name":"Bin wang","email":"","orcid":"","institution":"The First Affiliated Hospital of Chongqing Medical University,","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"wang","suffix":""},{"id":605238948,"identity":"68aacb59-de11-4f0e-83db-a6c1d88ad0bc","order_by":6,"name":"Zizuo Zhao","email":"data:image/png;base64,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","orcid":"","institution":"The First Affiliated Hospital of Chongqing Medical University,","correspondingAuthor":true,"prefix":"","firstName":"Zizuo","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2026-02-22 13:53:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8939517/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8939517/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105323452,"identity":"0757c9e0-81e0-4993-8891-c1da77f7049f","added_by":"auto","created_at":"2026-03-24 17:55:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2457059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRutin attenuates CCl₄-induced liver injury and fibrosis in mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Chemical structure of Rutin. (B) Serum ALT levels. (C) Serum AST levels. (D) H\u0026amp;E staining. (E) Masson staining. (F) Sirius Red staining. (G) Hepatic hydroxyproline content. (H) α-SMA (IHC). (I–M) mRNA levels of Col1a1, CTGF, TGF-β, and TIMP-1 (RT-qPCR). Data were expressed as mean ± SD; n = 6. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8939517/v1/620de626e9020f7be67e0a41.png"},{"id":105323444,"identity":"8375fd1c-155c-4064-b132-6d96694a290b","added_by":"auto","created_at":"2026-03-24 17:55:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1084587,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRutin reduces hepatic inflammation and oxidative stress in CCl₄-treated mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A–D) mRNA levels of IL-6, TNF-α, CCL2, and CXCL1 (RT-qPCR). E. Hepatic neutrophils (IHF). F. Hepatic macrophages (IHF). (G) 4-HNE expression (IHC). (H) Protein carbonyl levels (ELISA). (I) Hepatic levels of the nucleic acid oxidation product 8-OH-dG (ELISA). (J) SOD activity. (K) CAT activity. Data were expressed as mean ± SD; n = 6. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8939517/v1/2c1d77e096ece04679478678.png"},{"id":105323453,"identity":"4c25eab1-55f7-4590-a26e-06b5a0824ed4","added_by":"auto","created_at":"2026-03-24 17:55:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":497286,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRutin activates Nrf2 and inhibits the NLRP3-NF-κB pathway in CCl₄-treated mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Nrf2, NQO1, and HO-1 protein levels (WB). (B–D) Quantification of Nrf2, NQO1, and HO-1. (E) NLRP3, ASC, and pro-caspase-1 protein levels (WB). (F–I) Quantification of NLRP3, ASC, caspase-1, and IL-1β. (J) TLR4, IκB, and p65 protein levels (WB). (K–M) Quantification of TLR4, IκB, and p65. (N) Hepatic itaconate levels (HPLC). Data were expressed as mean ± SD; n = 3. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8939517/v1/af3122d28c23d792b8201d04.png"},{"id":105323445,"identity":"5cfdf7f5-22ab-4398-998c-e94ec6db9234","added_by":"auto","created_at":"2026-03-24 17:55:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":655435,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular interaction between Rutin and IRG1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Molecular docking analysis. (B) RMSD of the Rutin-IRG1 complex. (C) RMSF of IRG1 residues. (D) Radius of gyration (RoG). (E) Binding energy (MM-GBSA). (F) Key residues contributing to binding. (G) Hydrogen bond dynamics. (H) CETSA showing IRG1 thermal stability.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8939517/v1/f10bdf1635dbc9cec18974a4.png"},{"id":105323449,"identity":"caa9733b-400a-4327-9c10-13a31e87b8da","added_by":"auto","created_at":"2026-03-24 17:55:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":577419,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIRG1 is required for Rutin-mediated activation of Nrf2 and inhibition of the NLRP3–NF-κB pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Nrf2, NQO1, and HO-1 protein levels (WB). (B–D) Quantification of Nrf2, NQO1, and HO-1. (E) NLRP3, ASC, and pro-caspase-1 protein levels (WB). (F–I) Quantification of NLRP3, ASC, pro-caspase-1, and IL-1β. (J) TLR4, IκB, and p65 protein levels (WB). (K–M) Quantification of TLR4, IκB, and p65. (N) Hepatic itaconate levels (HPLC). Data were expressed as mean ± SD; n = 3. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8939517/v1/fcf76e826caea7cbd57d3731.png"},{"id":105323447,"identity":"73865493-8c2c-457b-a64f-c8ca3fcc1c9d","added_by":"auto","created_at":"2026-03-24 17:55:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1239074,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIRG1 is required for the anti-inflammatory and antioxidant effects of Rutin.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, B) Hepatic mRNA levels of IL-6 and TNF-α (RT-qPCR). (C, D) Hepatic mRNA levels of CCL2 and CXCL1 (RT-qPCR). (E) Hepatic neutrophils (IHF). (F) Hepatic macrophages (IHF). (G) Hepatic 4-HNE levels (IHC). (H) Hepatic protein carbonyl levels (ELISA). (I) Hepatic 8-OH-dG levels (ELISA). (J) Hepatic SOD activity.\u003c/p\u003e\n\u003cp\u003e(K) Hepatic CAT activity. Data were expressed as mean ± SD; n = 6. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8939517/v1/77d51aa95d05cf1b82614a4d.png"},{"id":105323448,"identity":"c0c7bd17-2f31-42c6-bfae-2bd9dcc179c4","added_by":"auto","created_at":"2026-03-24 17:55:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2723779,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIRG1 deficiency impairs the hepatoprotective and antifibrotic effects of Rutin.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Serum ALT levels. (B) Serum AST levels. (C) H\u0026amp;E staining of liver tissue. (D) Masson staining. (E) Sirius Red staining. (F) Hepatic hydroxyproline content. (G) α-SMA expression (IHC). (H–K) mRNA levels of Col1a1, CTGF, TGF-β, and TIMP-1 (RT-qPCR). (L) Mechanistic summary diagram. Data were expressed as mean ± SD; n = 6. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8939517/v1/a2a64be85690141bd747cbad.png"},{"id":105565213,"identity":"f829c8f5-7050-4198-ad70-813fb6042eb1","added_by":"auto","created_at":"2026-03-27 12:52:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9931487,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8939517/v1/98d3db68-307c-411c-b618-8bc1c8db47bd.pdf"},{"id":105323450,"identity":"30f56883-2465-406c-baed-f12d8ddd4ba4","added_by":"auto","created_at":"2026-03-24 17:55:37","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":235640,"visible":true,"origin":"","legend":"","description":"","filename":"GA.docx","url":"https://assets-eu.researchsquare.com/files/rs-8939517/v1/1bbbea71993dbc428c4f23e8.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Rutin Attenuates Liver Fibrosis by the IRG1-Itaconate-Nrf2 Axis: Modulation of Oxidative Stress and NLRP3 Inflammasome Activation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLiver fibrosis occurs primarily in chronic liver disease\u0026nbsp;\u003cstrong\u003ewith\u0026nbsp;\u003c/strong\u003echronic hepatocyte damage, iterative liver inflammation, excessive oxidative stress and persistent activation of hepatic fibrogenesis. These chronic liver diseases are caused by a variety of etiologies including viral liver disease, alcoholic liver disease, non-alcoholic fatty liver disease and autoimmune liver diseases [1] . Liver fibrosis is curable and reversible, but it is irreversible when progress to cirrhosis or even hepatocellular carcinoma [2, 3]. Currently, liver transplantation is the most effective treatment for liver fibrosis. However, because of the scarcity of organ donors, its clinical application is not popular. Herein, finding promising agents to alleviate liver dysfunction and decrease complications is necessary for liver fibrosis management.\u003c/p\u003e\n\u003cp\u003eIt has been widely accepted that oxidative stress and inflammation play critical roles in liver fibrosis [4]. When continuously stimulated by a variety of pathogenic factors, the balance of oxidative stress in the liver is broken, and the liver releases a large number of oxygen free radicals and reactive oxygen species (ROS), which induce hepatocyte injury and death. Dying hepatocytes release damage-associated molecular patterns (DAMPs) that are recognized by pattern recognition receptor (PRR), further activate innate immune signals including Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) and inflammasome NOD-like receptor pyrin domain-containing 3 (NLRP3), resulting in the production and release of numerous inflammatory signals including cytokines, chemokines and neutrophil-mediated ROS [5-7]. This, in turn, promotes apoptosis and necrosis of hepatocytes, forming a feedforward loop of oxidative stress-inflammation-cell death [7] . Meanwhile, ROS and inflammatory signals such as transforming growth factor beta 1 (TGF-β1), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) activate hepatic stellate cell (HSC), resulting in excessive collagen deposition in the extracellular matrix (ECM), leading to liver fibrosis [8-11] .\u003c/p\u003e\n\u003cp\u003eEndogenous complex anti-inflammatory and antioxidant defense systems also exist in the liver, which are regulated by a series of pathways to ensure that the response to inflammation and ROS is sufficient to meet the body's needs [12]. Itaconate is a regulatory metabolite in the tricarboxylic acid (TCA) cycle, and it also acts as an immunomodulator. It is synthesized by cis-aconitic acid, a TCA cycle intermediate, and catalyzed by cis-aconitic acid decarboxylase encoded by the immune response gene 1 protein (IRG1) [13]. Recently, itaconate has received considerable attention due to its critical role in negatively regulating inflammatory responses and cytokine production as an anti-inflammatory metabolite [14, 15]. Nuclear factor E2-related factor 2 (Nrf2), a ubiquitous transcription factor, is the master antioxidant regulator for cells to cope with oxidative stress. The fundamental and induced expression of a string of antioxidant response element-dependent genes including heme oxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO1), are driven by Nrf2 to regulate the physiological and pathophysiological consequence of oxidant exposure [16, 17]. Previous studies had shown a link between the IRG1/itaconate pathway and Nrf2, and a recent study using IRG1\u003csup\u003e-/-\u003c/sup\u003emice demonstrated that IRG1 suppresses injury and inflammation during hepatic ischemia-reperfusion (I/R), and that the mechanism of the itaconate derivative 4-octyl itaconate (4-OI) protected against hepatic I/R injury requires Nrf2 [18].\u003c/p\u003e\n\u003cp\u003eRutin, known as quercetin-3-O-Rutinoside and vitamin P, is a polyphenolic natural flavonoid originated from many medicinal plants and vegetables[19]. Its chemical structure was identified as (2-(3,4-dihydroxyphenyl)-4,5-dihydroxy-3-[3,4,5trihydroxy-6-[(3,4,5-trihydroxy-6methyl-oxan-2-yl) oxymethyl] oxan-2-yl] oxy -chromen-7-one (Fig. 1A) [20]. It is reported that Rutin shows a wide range of pharmacological/biological activities including antioxidant, anti-inflammatory, hepatoprotective and neuroprotective effects [21-25]. Previous studies had shown Rutin had hepatoprotection on liver injury induced by different models, including bile duct ligation and high-fat diet [23, 25]. However, there is little research on the therapeutic effect of Rutin on hepatic fibrosis, and the potential molecular mechanism remains undefined.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, we explored that whether Rutin had protective effects in CCl\u003csub\u003e4\u003c/sub\u003e-induced liver fibrosis model and whether these protective effects were related to its anti-inflammatory, antioxidant properties and IRG1-itaconate pathway.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals and Experimental Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIRG1 knockout (KO, IRG1\u003csup\u003e−/−\u003c/sup\u003e) mice (Cyagen, S-KO-02679) and their C57BL/6N cognate background control mice were bred and raised under specific pathogen-free conditions. All mice were 8-12 weeks old male mice weighing 20-30 g, housed in standard laboratory animal cages and maintained with standard management conditions of a 12 h light/ dark cycle. All experimental procedures were approved by the ethics committee of Chongqing Medical University, and conducted in accordance with the ARRIVE guidelines and the national research council’s guide for the care and use of laboratory animals.\u003c/p\u003e\n\u003cp\u003eTo explore the effect of Rutin dose on its biological effects, all mice were randomly divided into six groups. (1) Vehicle control, (2) Rutin, (3) CCl\u003csub\u003e4\u003c/sub\u003e, (4) CCl\u003csub\u003e4\u003c/sub\u003e+Rutin (15 mg/kg), (5) CCl\u003csub\u003e4\u003c/sub\u003e+Rutin (30 mg/kg), (6) CCl\u003csub\u003e4\u003c/sub\u003e+Rutin (60 mg/kg). To investigate the protective mechanism of Rutin against liver fibrosis, WT mice and IRG1\u003csup\u003e−/−\u003c/sup\u003e mice were used in CCl\u003csub\u003e4\u0026nbsp;\u003c/sub\u003egroup or CCl\u003csub\u003e4\u003c/sub\u003e+Rutin (60 mg/kg) group. Rutin (suspended in 0.1% CMC-Na) was intragastrically (i.g.) administered to corresponding group of mice for 10 consecutive days. Vehicle groups of mice received corresponding volume of CMC-Na solution. Hepatic fibrosis was induced by intraperitoneal (i.p.) injections of CCl\u003csub\u003e4\u003c/sub\u003e (0.5 ml/kg, dissolved in olive oil, 1:19) three times a week for 8 weeks. Same volume of olive oil was injected to the normal control group. All mice were sacrificed 48 h after final CCl\u003csub\u003e4\u0026nbsp;\u003c/sub\u003einjection. All serum samples and liver samples were collected for further examination.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiochemical Assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood samples were collected through retro-orbital bleeding. The serum samples were obtained by centrifugation at 3600 rpm for 10 min at 4°C. Serum enzyme activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by spectrophotometry using corresponding detection kits (Nan Jing Jan Cheng Biochemical Institute, Nanjing, China) according to the instruction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistologic Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSome excised liver specimens were fixed with 4% paraformaldehyde solution and then embedded in paraffin. Paraffin sections were stained with hematoxylin and eosin (H\u0026amp;E), and then were scored using the Suzuki methodology to assess tissue inflammation and necrosis. Sirius red staining and Masson staining were performed in paraffin sections according to standard protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of Tissue ROS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSome liver samples were snap frozen in liquid nitrogen and kept at -80°C. To determine the hepatic ROS levels, the frozen liver samples were sectioned transversely. The 10 μm thick of frozen liver section was treated with 10 μmol/L fluorescent dye dihydroethidium (DHE) and incubated at 37°C for 30 min in the dark. Then the nuclei were stained with DAPI and the fluorescence images were obtained by fluorescence microscope (Nikon, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of Peroxidation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe peroxidation products of various biomacromolecules in liver tissue were detected, including the lipid peroxidation product 4-HNE, the protein peroxidation products Protein Carbonyl and the nucleic acid peroxidation products 8-OHG, 8-OHdG and 8-hydroxyguanine. The production of 4-HNE was demonstrated by immunohistochemistry, and the contents of protein carbonyl and 8-OHG, 8-OHdG, 8-hydroxyguanine were measured by ELISA. The specific methods are in 2.9 and 2.10.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExamination for Antioxidant Enzymes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCommercially available assay kits for the examination of hepatic superoxide dismutase (SOD) and catalase (CAT) activities were obtained from Nan Jing Jan Cheng Biochemical Institute (Nanjing, China). The activity of SOD was determined by colorimetry. The principle, briefly, is that superoxide anions (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e• −\u003c/sup\u003e) are produced by xanthine and xanthine oxidase reaction systems, which reduce WST-1 to orange or purple WST-1 Formazan dye. The reactions of CAT decomposing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e are quickly terminated by adding ammonium molybdate and the remaining H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e reacts with ammonium molybdate to produce a pale-yellow complex. The activity of CAT can be calculated by measuring its change at 405 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHydroxyproline (Hyp) Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe level of Hepatic Hyp was examined by using the corresponding kit (Nan Jing Jan Cheng Biochemical Institute, Nanjing, China) according to the supplier's protocol. The oxidation product of hydroxyproline reacts with dimethylaminobenzaldehyde to form a purple complex, and the level of Hyp can be calculated by colorimetry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) and RNA Sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe total RNA of liver tissues was extracted using Trizol reagent. Then, NanoDrop One microvolume spectrophotometer was used to quantify total RNA. Equal amount of RNA was reversibly transcribed into complementary DNA. PCR reactions were used to amplify the target gene. Its procedure, briefly, consisted of starting at 94°C for 5 min, followed by 32-40 cycles of amplification (denaturation at 94°C for 1 min, annealing at 58°C for 30 s, and extension at 72°C for 30 s) with a final primer extra-extension at 72°C for 7 min. The PCR products were separated on 2% agarose gel and stained with ethidium bromide. The intensity of each target gene mRNA band was quantified by molecular imaging system and β-actin was used as internal control. The sequences of the primers are listed in \u003cstrong\u003eTable 1\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue Preparation and Western Blot Analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLiver tissue removed from -80°C, triturated and lysed in RIPA lysis buffer containing protease inhibitors. Total protein concentrations were measured with BCA kit and normalized with buffer. Then, proteins were electrophoretically separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes. After blocking with 5% nonfat dry milk for 1 h, the membranes were incubated overnight with the indicated antibodies (IRG1, NQO1, HO-1, NLRP3, ASC, pro-caspase1, TLR4, IκB, p65) in 4°C. Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h, then visualized by chemiluminescence (Bio-Rad).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical and Immunofluorescence Staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParaffin sections were used for immunohistochemical staining. They were deparaffinized, hydrated and incubated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to block endogenous peroxidase. After retrieved by microwave and blocked with 5% BSA, the liver sections were incubated with primary antibody overnight at 4°C. Sections were then co-incubated with biotinylated secondary antibodies for 1 h, followed by avidin-biotin-peroxidase complexes. The immunoreactive signal is generated by color deposition with diaminobenzidine as a substrate.\u003c/p\u003e\n\u003cp\u003eFor immunofluorescence staining, the frozen sections were washed in TBS, and then blocked in 5% BSA for 1 h. Following overnight incubation with primary antibodies, the corresponding fluorescent-labeled secondary antibodies were applied. DAPI dye was used to stain the nuclei. After mounting with anti-quenching fluorescent mounting medium, images were obtained using a fluorescence microscope (Leica, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme-Linked ImmunoSorbent Assay (ELISA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe levels of cytokines, chemokines and peroxidation products in liver cell supernatants were measured using ELISA Bender medsystems kits according to the manufacturer's instructions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurements of Itaconate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSamples were injected by Thermo Fisher Vanquish ultra-high-performance liquid chromatography (HPLC) and separated over a reversed-phase Thermo Fisher Hypercarb porous graphitic column maintaining at 52°C. The mobile phase contains solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). Itaconate concentrations were quantified using liquid chromatography-mass spectrometry analysis. Deuterated (D4)-taurine and (D3)-lactate (Sigma-Aldrich) were used as an internal standard.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular Docking and Dynamics Simulations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe molecular docking study was performed to explore the binding mode and affinity between Rutin and the IRG1 protein. The three-dimensional crystal structure of human IRG1 was retrieved from the Protein Data Bank. Docking was carried out with \u003cem\u003eAutoDock4\u0026nbsp;\u003c/em\u003esoftware. The IRG1 protein was defined as the rigid receptor, while all rotatable bonds in Rutin were set as flexible. Ten independent docking runs were performed, and the resulting poses were ranked according to their binding affinity (kcal/mol). The pose with the lowest binding energy and highest cluster occupancy was selected for further analysis. Visualization and interaction analysis (hydrogen bonds, hydrophobic contacts) were conducted using \u003cem\u003ePymol2 software\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eTo evaluate the stability and dynamic behavior of the Rutin-IRG1 complex, molecular dynamics (MD) simulations were performed using the GROMACS package. The complex obtained from molecular docking was solvated in a cubic water box. Production MD simulation was then carried out for 100 ns under periodic boundary conditions. The algorithm constrained bond lengths, and the Particle Mesh Ewald method was used for long-range electrostatic interactions. Trajectories were analyzed for root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent-accessible surface area (SASA), and hydrogen bond occupancy. The binding free energy was calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCellular Thermal Shift Assay (CETSA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Cellular Thermal Shift Assay was employed to validate the direct binding between Rutin and the intracellular target IRG1 in a cellular context. HepG2 cells were seeded in 6-well plates and allowed to adhere overnight. Cells were then treated with either Rutin (10 µM) or an equivalent volume of vehicle control (DMSO). Following treatment, cells were harvested, washed with PBS, and resuspended. The cell suspension was aliquoted into PCR tubes and heated at a gradient of temperatures (ranging from 37°C to 67°C) for 3 minutes in a thermal cycler, followed by incubation at room temperature for an additional 3 minutes. Cells were then subjected to three freeze-thaw cycles using liquid nitrogen to lyse the cells. The soluble protein fraction was separated from cell debris by centrifugation at 12,000 × g for 15 minutes at 4°C. The supernatant was collected, and the protein concentration was determined. Equal amounts of protein from each temperature point were resolved by SDS-PAGE, followed by Western blotting analysis using a specific anti-IRG1 antibody. The band intensity of soluble IRG1 remaining at each temperature was quantified using ImageJ software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analyses\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese data were expressed as mean ± SD. Statistical analysis was carried out using one-way or two-way ANOVA followed by a Bonferoni comparison or Dunnett's multiple comparison test to determine the differences among groups. \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 was considered as statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eRutin Attenuates CCl₄-Induced Liver Injury and Fibrosis in Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRutin exhibited significant hepatoprotective and antifibrotic effects in a mouse model of CCl₄-induced liver injury. Its characteristic flavonoid molecular structure underlies its notable bioactivity (\u003cstrong\u003eFig. 1A\u003c/strong\u003e). Biochemical assays showed that CCl₄ administration markedly elevated AST and ALT levels, indicating hepatocellular injury, whereas Rutin treatment significantly reduced these enzyme levels (\u003cstrong\u003eFig. 1B, 1C\u003c/strong\u003e). Histopathological examination using H\u0026amp;E staining revealed that CCl₄ disrupted hepatic lobular architecture, caused hepatocellular swelling, collagen deposition, and substantial inflammatory infiltration, all of which were alleviated in a dose-dependent manner by Rutin (\u003cstrong\u003eFig. 1D\u003c/strong\u003e). In the context of liver fibrosis, Masson’s trichrome and Sirius Red staining demonstrated excessive collagen accumulation in CCl₄-treated livers, which was significantly reduced following Rutin administration (\u003cstrong\u003eFig. 1E, 1F\u003c/strong\u003e). Consistently, hepatic hydroxyproline content was elevated in the CCl₄ group and markedly decreased in the Rutin-treated groups (\u003cstrong\u003eFig. 1G\u003c/strong\u003e). Immunohistochemical analyses further revealed that CCl₄ induced strong α-smooth muscle actin (α-SMA) and type I collagen alpha 1 (Col1a1) expression in the periportal region, reflecting HSC activation and fibrogenesis, both of which were significantly attenuated by Rutin (\u003cstrong\u003eFig. 1H, 1I\u003c/strong\u003e). RT-qPCR analysis confirmed that CCl₄ markedly upregulated the mRNA expression of fibrosis-related genes, including Col1a1, CTGF, TGF-β, and TIMP-1, while Rutin treatment significantly suppressed these transcriptional changes (\u003cstrong\u003eFig. 1J–1M\u003c/strong\u003e). Together, these results show that Rutin attenuates CCl₄-induced liver injury and fibrosis, as evidenced by reduced serum transaminase levels, improved histological architecture, decreased collagen deposition, and suppression of fibrotic marker expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRutin Attenuates CCl₄-Induced Hepatic Inflammation and Oxidative Stress in Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo comprehensively elucidate the anti-inflammatory and antioxidant effects of Rutin in a CCl₄-induced mouse model of liver injury, we conducted a series of analyses at multiple levels, including gene expression, immune cell infiltration, oxidative damage markers, and antioxidant enzyme activity. RT-qPCR analysis demonstrated that Rutin treatment significantly downregulated the hepatic mRNA expression of pro-inflammatory cytokines IL-6 and TNF-α, as well as chemokines CCL2 and CXCL1 (\u003cstrong\u003eFig. 2A–2D\u003c/strong\u003e), indicating its potent anti-inflammatory capacity. Immunofluorescence staining further revealed that CCl₄ exposure markedly increased the infiltration of F4/80-positive macrophages and CD11b-positive neutrophils in liver tissue, which was notably alleviated by Rutin administration (\u003cstrong\u003eFig. 2E, 2F\u003c/strong\u003e), suggesting an inhibitory effect on inflammatory cell recruitment. Regarding oxidative stress, immunohistochemical staining showed a substantial elevation of the lipid peroxidation marker 4-hydroxynonenal (4-HNE) following CCl₄ treatment, which was significantly reduced upon Rutin intervention (\u003cstrong\u003eFig. 2G\u003c/strong\u003e). ELISA assays confirmed that CCl₄ induced a marked increase in hepatic levels of oxidative damage markers, including the protein peroxidation product protein carbonyl and nucleic acid oxidation products 8-OHG, 8-OHdG, and 8-hydroxyguanine, all of which were significantly attenuated by Rutin (\u003cstrong\u003eFig. 2H, 2I\u003c/strong\u003e). Furthermore, enzymatic activity assays showed that CCl₄ markedly suppressed the hepatic activities of key antioxidant enzymes SOD and CAT, while Rutin treatment effectively restored their activity levels (\u003cstrong\u003eFig. 2J, 2K\u003c/strong\u003e). These results indicate that Rutin reduces hepatic inflammation and oxidative stress in CCl₄-treated mice, as evidenced by decreased pro-inflammatory cytokine expression, reduced immune cell infiltration, lower oxidative damage markers, and restored antioxidant enzyme activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRutin Activates the Nrf2 Pathway and Inhibits the NLRP3–NF-κB Axis in the Liver of CCl₄-Treated Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRutin exerts significant regulatory effects on oxidative stress and inflammatory signaling pathways in the liver. Specifically, Rutin treatment markedly upregulated the expression of Nrf2 and its downstream antioxidant enzymes NQO1 and HO-1 in the liver of CCl₄-treated mice (\u003cstrong\u003eFig. 3A\u003c/strong\u003e), which was further confirmed by semi-quantitative Western blot analysis showing consistent increases in the protein levels of Nrf2, NQO1, and HO-1 (\u003cstrong\u003eFig. 3B–3D\u003c/strong\u003e), suggesting that Rutin enhances hepatic antioxidant defense via activation of the Nrf2 pathway. Conversely, the expression of NLRP3 and its downstream signaling molecules ASC and pro-caspase-1, which were significantly induced by CCl₄, were markedly reduced following Rutin administration (\u003cstrong\u003eFig. 3E\u003c/strong\u003e). This inhibitory effect was further supported by Western blot analysis, which demonstrated consistent downregulation of NLRP3, ASC, caspase-1, and IL-1β protein levels (\u003cstrong\u003eFig. 3F–3I\u003c/strong\u003e). Additionally, Western blot analysis revealed that CCl₄ markedly activated the NF-κB signaling pathway, as evidenced by elevated expression of TLR4, p-IκB, and p-p65, whereas Rutin effectively suppressed these changes (\u003cstrong\u003eFig. 3J\u003c/strong\u003e) and the semi-quantitative analysis of NLRP3, ASC, caspase-1, and IL-1β protein levels confirmed this trend (\u003cstrong\u003eFig. 3K–3N\u003c/strong\u003e). Furthermore, HPLC analysis demonstrated that Rutin significantly increased the hepatic content of itaconate in CCl₄-treated mice (\u003cstrong\u003eFig. 3O\u003c/strong\u003e), indicating a potential involvement of endogenous immunometabolic regulation in its anti-inflammatory and antioxidant effects. These results show that Rutin upregulates the hepatic Nrf2 pathway and its downstream antioxidant enzymes, while suppressing the NLRP3 inflammasome and NF-κB signaling activation induced by CCl₄. Additionally, Rutin increases hepatic itaconate levels, suggesting involvement of endogenous metabolic regulation in these responses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular interactions between Rutin and IRG1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study investigates the interactions between Rutin and IRG1 through molecular docking and molecular dynamics (MD) simulations. Molecular docking results show that Rutin forms hydrogen bonds with several amino acids of IRG1, including ASN-425, ILE-347, ASP-285, ARG-273, and ASP-417, enhancing binding affinity. Additionally, Rutin interacts with ARG-273 through cation-π conjugation and strengthens the binding further through hydrophobic interactions with ARG-273, VAL-349, LYS-421, and ALA-424 (\u003cstrong\u003eFig. 4A\u003c/strong\u003e). MD simulation reveals that the RMSD of the complex quickly reaches equilibrium within 0–10 ns (around 1.2 Å), then gradually increases to 2.0 Å between 40–60 ns, rising sharply to 3.5 Å at 60 ns, and stabilizing at approximately 3.0 Å, indicating the system reaches a new equilibrium state (\u003cstrong\u003eFig. 4\u003c/strong\u003e\u003cstrong\u003eB\u003c/strong\u003e). RMSF analysis shows that the protein’s flexibility decreases, with values generally below 2 Å, indicating rigidity in the protein core, which supports stable binding and enzymatic activity (\u003cstrong\u003eFig. 4C\u003c/strong\u003e). RoG analysis shows slight conformational relaxation between 0–20 ns, with RoG increasing from 22.2 Å to 22.5 Å, and stabilizing between 22.6–22.7 Å after a significant increase at 60 ns, indicating a new equilibrium state (\u003cstrong\u003eFig. 4D\u003c/strong\u003e). MM-GBSA calculations show a binding energy of -16.38±4.79 kcal/mol, indicating strong binding affinity, primarily driven by electrostatic and van der Waals interactions (\u003cstrong\u003eFig. 4E\u003c/strong\u003e). Energy decomposition identifies key amino acids contributing to binding, such as GLU 297, ALA 296, and VAL 295 (\u003cstrong\u003eFig. 4F\u003c/strong\u003e). Hydrogen bond analysis shows an initial increase to 10 bonds, followed by a drop to zero between 20–60 ns, and a recovery to 2–6 bonds between 60–100 ns, suggesting conformational shifts and a new interaction equilibrium (\u003cstrong\u003eFig. 4G\u003c/strong\u003e). and CETSA analysis showed that Rutin significantly enhances IRG1 thermal stability (\u003cstrong\u003eFig. 4H\u003c/strong\u003e). These findings indicate that Rutin binds IRG1 through multiple stable interactions and induces conformational stabilization of the protein. This structural basis supports a potential role for Rutin in modulating IRG1 function at the molecular level.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIRG1 Deficiency Abolishes Rutin-Mediated Activation of Nrf2 and Inhibition of NF-κB Signaling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate whether the antifibrotic effect of Rutin is dependent on the IRG1 pathway, we established an IRG1⁻/⁻ mouse model of CCl₄-induced liver fibrosis. Western blot analysis revealed that Rutin markedly upregulated the protein expression of Nrf2 and its downstream antioxidant enzymes NQO1 and HO-1 in liver tissue; however, this upregulation was significantly attenuated in IRG1-deficient mice (\u003cstrong\u003eFig. 5A\u003c/strong\u003e), and semi-quantitative analysis showed consistent trends for Nrf2, NQO1, and HO-1 levels (\u003cstrong\u003eFig. 5B–5D\u003c/strong\u003e), suggesting that IRG1 plays a crucial role in mediating Rutin-induced activation of the Nrf2 pathway. Similarly, in WT mice, Rutin significantly inhibited the CCl₄-induced expression of NLRP3 inflammasome-related proteins, including NLRP3, ASC, Caspase-1, and IL-1β, whereas this inhibitory effect was markedly diminished in IRG1⁻/⁻ mice (\u003cstrong\u003eFig. 5E\u003c/strong\u003e), which was further supported by Western blot semi-quantitative analysis (\u003cstrong\u003eFig. 5F–5I\u003c/strong\u003e), indicating a potential involvement of IRG1 in the negative regulation of inflammasome activation by Rutin. In addition, Western blot results showed that CCl₄ administration significantly activated the NF-κB signaling pathway, as evidenced by elevated protein levels of TLR4, p-IκB, and p-p65 in the liver, and these increases were effectively suppressed by Rutin in WT mice but not in IRG1⁻/⁻ mice (\u003cstrong\u003eFig. 5J\u003c/strong\u003e); consistent trends were observed in the semi-quantitative analysis of TLR4, p-IκB, IκB, p-p65, and p65 expression levels (\u003cstrong\u003eFig. 5K–5N\u003c/strong\u003e), further supporting the role of IRG1 in Rutin-mediated inhibition of NF-κB activation. Notably, HPLC analysis demonstrated that Rutin significantly increased hepatic itaconate content in CCl₄-treated WT mice, while this elevation was abolished in IRG1⁻/⁻ mice (\u003cstrong\u003eFig. 5O\u003c/strong\u003e), further supporting the hypothesis that Rutin exerts its anti-inflammatory and antioxidant effects, at least in part, through the IRG1/itaconate metabolic pathway. These findings indicate that the presence of IRG1 is essential for Rutin to exert its regulatory effects on the Nrf2 antioxidant response and the NLRP3–NF-κB inflammatory axis, highlighting the importance of the IRG1/itaconate pathway in the hepatoprotective mechanism of Rutin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIRG1 Deficiency Attenuates the Anti-inflammatory and Antioxidant Effects of Rutin in CCl₄-Induced Liver Injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the context of IRG1 gene knockout, we further evaluated hepatic inflammation and antioxidant responses to determine whether the anti-inflammatory and antioxidant effects of Rutin are mediated through the IRG1 pathway. RT-qPCR analysis revealed that Rutin significantly downregulated the mRNA expression of pro-inflammatory cytokines IL-6 and TNF-α, as well as chemokines CCL2 and CXCL1 in the liver of CCl₄-treated mice, indicating a potent anti-inflammatory effect. However, this inhibitory effect was markedly attenuated in IRG1⁻/⁻ mice (\u003cstrong\u003eFig. 6A–6D\u003c/strong\u003e). Consistently, immunofluorescence staining showed that CCl₄ exposure significantly increased the hepatic infiltration of F4/80-positive macrophages and CD11b-positive neutrophils, which was notably reduced by Rutin treatment in wild-type mice but not in IRG1-deficient mice (\u003cstrong\u003eFig. 6E, 6F\u003c/strong\u003e). Regarding oxidative stress, immunohistochemical analysis demonstrated that hepatic levels of the lipid peroxidation marker 4-HNE were significantly elevated following CCl₄ exposure, whereas Rutin administration markedly reduced 4-HNE accumulation; this reduction was impaired in IRG1⁻/⁻ mice (\u003cstrong\u003eFig. 6G\u003c/strong\u003e). ELISA further confirmed that the levels of hepatic oxidative damage markers, including the protein peroxidation product protein carbonyl and nucleic acid oxidation products 8-OHG, 8-OHdG, and 8-hydroxyguanine, were significantly increased in CCl₄-treated mice and effectively attenuated by Rutin, whereas these protective effects were substantially diminished in IRG1⁻/⁻ mice (\u003cstrong\u003eFig. 6H, 6I\u003c/strong\u003e). Additionally, enzymatic activity assays revealed that CCl₄ significantly suppressed the hepatic activities of the key antioxidant enzymes SOD and CAT, which were restored by Rutin treatment in wild-type mice, but this restoration was abolished in IRG1-deficient mice (\u003cstrong\u003eFig. 6J, 6K\u003c/strong\u003e). These findings indicate that IRG1 plays a critical regulatory role in the anti-inflammatory and antioxidant effects of Rutin, and that the absence of IRG1 significantly compromises the hepatoprotective efficacy of Rutin, highlighting the IRG1/itaconate pathway as a potential mechanistic basis for its therapeutic actions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIRG1 Deficiency Impairs the Hepatoprotective and Antifibrotic Effects of Rutin in CCl₄-Induced Liver Injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the context of IRG1 gene deficiency, we further evaluated hepatic injury and fibrosis to determine whether the protective effects of Rutin against tissue damage and fibrosis are associated with the IRG1 pathway. Biochemical analysis revealed that CCl₄ administration significantly elevated serum levels of AST and ALT, indicating hepatocellular injury, whereas Rutin treatment markedly reduced these enzyme levels. However, this hepatoprotective effect was substantially attenuated in IRG1⁻/⁻ mice (\u003cstrong\u003eFig. 7A, 7B\u003c/strong\u003e). Histopathological examination using H\u0026amp;E staining demonstrated that CCl₄ disrupted hepatic lobular architecture, causing hepatocyte swelling, collagen deposition, and extensive inflammatory infiltration, all of which were alleviated by Rutin, while this improvement was significantly impaired in IRG1⁻/⁻ mice (\u003cstrong\u003eFig. 7C\u003c/strong\u003e). In the context of liver fibrosis, Masson's trichrome and Sirius Red staining revealed excessive collagen accumulation in the liver following CCl₄ exposure, which was markedly reduced by Rutin treatment in wild-type mice but not in IRG1-deficient mice (\u003cstrong\u003eFig. 7D, 7E\u003c/strong\u003e). Consistently, hepatic hydroxyproline content, which was elevated after CCl₄ treatment, was significantly reduced by Rutin, whereas this effect was blunted in IRG1⁻/⁻ mice (\u003cstrong\u003eFig. 7F\u003c/strong\u003e). Immunohistochemical analysis further showed that CCl₄ induced strong perivascular expression of α-SMA and Col1a1, reflecting hepatic stellate cell activation and fibrogenesis, both of which were significantly attenuated by Rutin in wild-type mice but not in IRG1-deficient mice (\u003cstrong\u003eFig. 7G, 7H\u003c/strong\u003e). Moreover, RT-qPCR analysis confirmed that CCl₄ markedly upregulated the mRNA expression of fibrosis-related genes, including Col1a1, CTGF, TGF-β, and TIMP-1, and these transcriptional changes were significantly suppressed by Rutin, while this suppressive effect was abolished in IRG1⁻/⁻ mice (\u003cstrong\u003eFig. 7I-7L\u003c/strong\u003e). These findings indicate that IRG1 plays a critical role in mediating the protective effects of Rutin against CCl₄-induced hepatic injury and fibrosis, highlighting the IRG1/itaconate pathway as a potential mechanistic basis for its antifibrotic activity.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCCl\u003csub\u003e4\u003c/sub\u003e has been widely used to induce liver injury and fibrosis in mice [26]. CCl\u003csub\u003e4\u003c/sub\u003e is one of halogenated alkanes that activated by cytochrome (CYP)2E1 to form a highly reactive trichloromethyl radical and promote excessive generation of ROS. The trichloromethyl radical and its peroxy radical CCl\u003csub\u003e3\u003c/sub\u003eOO\u003csup\u003e•−\u0026nbsp;\u003c/sup\u003eare involved in lipid peroxidation processes and several free radical reactions that contribute to the activation of Kupffer cells and the induction of an inflammatory response [27, 28]. Kupffer cells activated by CCl\u003csub\u003e4\u003c/sub\u003e release TNF-α, TGF-β, and IL-1, IL-6, and IL-10. HSCs activated by CCl\u003csub\u003e4\u003c/sub\u003e and inflammatory mediators over-produce type-I collagen, and thus promote liver fibrosis. In brief, CCl\u003csub\u003e4\u003c/sub\u003e-induced fibrosis is closely related to oxidative stress and inflammation. In our study, Rutin was found to inhibit CCl\u003csub\u003e4\u003c/sub\u003e-induced liver inflammation and oxidative stress. Rutin might be a potent agent to treat liver fibrosis owing to its antioxidant and anti-inflammatory biological activities.\u003c/p\u003e\n\u003cp\u003eOxidative stress is closely associated with the progress of CCl\u003csub\u003e4\u003c/sub\u003e-induced liver injury and fibrosis. CCl\u003csub\u003e4\u003c/sub\u003e-induced increase in hepatic ROS level. Several studies had shown that ROS played an important role in the regulation of HSCs activation and fibrotic gene expression [29]. After activated by ROS, HSCs proliferate and excessively release collagen into the ECM\u0026nbsp;propagating\u0026nbsp;liver fibrosis. The excessive ROS can also activate the lipid peroxidation of hepatocytes to cause the increase generation of lipid peroxides including 4-HNE, which easily modify proteins and inhibit critical enzyme activities. Antioxidant enzymes, especially SOD and CAT, can reduce the level of superoxide (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e−\u003c/sup\u003e) and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), which are the most-produced ROS\u0026nbsp;[30]. Nrf2 is one of master regulators to cope with oxidative stress.\u0026nbsp;In the oxidatively stimulated state, Nrf2 remains stable and enters the nucleus, where it binds to antioxidant response elements, resulting in transcriptional activation of genes encoding a series of antioxidant enzymes, including HO-1 and NQO1, to restore redox homeostasis in the body\u0026nbsp;[31-35].\u0026nbsp;For these reasons, we\u0026nbsp;observed the\u0026nbsp;helpful\u0026nbsp;effect of\u0026nbsp;Rutin\u0026nbsp;on\u0026nbsp;CCl\u003csub\u003e4\u003c/sub\u003e-induced\u0026nbsp;liver injury\u0026nbsp;and fibrosis\u0026nbsp;by detecting\u0026nbsp;hepatic ROS level, production of lipid peroxidation products, antioxidant enzyme activities and Nrf2 pathway.\u0026nbsp;The results showed that\u0026nbsp;Rutin\u0026nbsp;could significantly reduce the\u0026nbsp;hepatic ROS level, decrease the production of 4-HNE,\u0026nbsp;and increase the activities\u0026nbsp;of SOD\u0026nbsp;and CAT, and activation of Nrf2 pathway, suggesting that the protective effect of\u0026nbsp;Rutin\u0026nbsp;on\u0026nbsp;liver injury\u0026nbsp;and fibrosis\u0026nbsp;might\u0026nbsp;be associated with its antioxidant activity.\u003c/p\u003e\n\u003cp\u003eInflammation is another critical pathological mechanism promoting CCl\u003csub\u003e4\u003c/sub\u003e-induced liver injury and fibrosis. DAMPs released from damaged or dying hepatocytes are recognized by PRRs on Kupffer cells and further activate Kupffer cells [36] . Activated kupffer cells produce cytokines and chemokines including IL-1β, IL-6, TNF-α, CCL2 and CXCL1, participate in the inflammatory response and induce the activation of HSCs [11]. IL-1β is a main pro-inflammatory cytokine produced during NLRP3 inflammasome activation. TNF-α is considered to be the main endogenous harmful mediator during liver injury, which has the ability to directly damage tissues and trigger inflammatory cascades [37]. IL-6 is another pro-inflammatory cytokine that antagonizes TNF-α-induced tissue damage. In this study, Rutin decreased the mRNA and protein levels of IL-1β, IL-6, TNF-α in CCl\u003csub\u003e4\u003c/sub\u003e-treated mice. The release of chemokines such as CCL2 and CXCL1 recruit macrophages and neutrophils to participate in the initial stage of inflammatory response in the process of liver fibrosis. In our results, we observed Rutin sharply decreased the mRNA and protein levels of CCL2 and CXCL1 in CCl\u003csub\u003e4\u003c/sub\u003e-treated mice. In addition, we found aggregation of macrophages and neutrophils in the liver of mice following CCl\u003csub\u003e4\u003c/sub\u003e administration, whereas Rutin treatment significantly reduced this activity. Previous studies have demonstrated that inhibition of NLRP3 inflammasome ameliorates CCl\u003csub\u003e4\u003c/sub\u003e-induced liver fibrosis [38-41]. ASC and pro-caspase 1 are key components of NLRP3 inflammasome [42, 43]. In the present study, the decline of NLRP3, ASC and pro-caspase 1 protein levels was observed in CCl\u003csub\u003e4\u0026nbsp;\u003c/sub\u003emice after Rutin treatment. TLR4 is a critical PRR involved in the triggering of CCl\u003csub\u003e4\u003c/sub\u003e-induced cascades of inflammatory responses. NF-κB is a classic inflammatory signaling pathway that promotes the occurrence of inflammatory responses, of which IkB and p65 are important components of this pathway [11]. It has been reported that the knockout of TLR4 can alleviate inflammatory response to attenuate CCl\u003csub\u003e4\u003c/sub\u003e-induced hydroxyproline and α-SMA [44]. In the present study, Rutin was observed to decrease the level of TLR4 and the phosphorylation of IκB and p65 in the liver of CCl\u003csub\u003e4\u003c/sub\u003e-treated mice. These results suggest that Rutin inhibits liver inflammation by inhibiting the activation of NF-κB and inflammasome NLRP3 signaling pathways after CCl\u003csub\u003e4\u003c/sub\u003e exposure.\u003c/p\u003e\n\u003cp\u003eTo sum up, Rutin inhibits hepatic oxidative stress by promoting Nrf2 signaling, and inhibits hepatic inflammation by inhibiting the activation of NF-κB and inflammasome NLRP3 signaling pathways, so we speculate whether there is an intermediate molecule affected by Rutin, which is involved in the regulation of the above signaling pathways.\u0026nbsp;Itaconate, an endogenous biological metabolite catalyzed by IRG1 and accumulated by metabolic disorders after inflammatory activation of macrophages, is also acts as an immunomodulator.\u0026nbsp;Previous study had shown a link between the IRG1/itaconate pathway and Nrf2, when itaconate was shown to directly alkylate cysteine residues in\u0026nbsp;Kelch-like ECH-associated protein 1 (Keap1), leading to an increase in Nrf2 in macrophages during endotoxemia\u0026nbsp;[45].\u0026nbsp;Endogenous itaconate and its derivative 4-OI have been presented to alkylate cysteine residues on plenty of proteins, including Keap1, and this form of cysteine alkylation, called 2, 3-dicarboxypropylation or itaconation, has been exhibited to be a crucial component involved in the anti-inflammatory properties of itaconate\u0026nbsp;[46-48]. In a recent study, 4-OI inhibited the inflammatory response by inhibiting\u0026nbsp;NF-κB\u0026nbsp;signal pathway in RAW264.7 cells[49]. A previous study has reported that itaconate induced dicarboxypropylation of NLRP3 cysteine C548 and inhibited inflammasome activation, providing evidence that itaconate is a specific endogenous inhibitor of NLRP3 inflammasome activation\u0026nbsp;[50]. We hypothesized that that Rutin further promotes Nrf2 pathway and\u0026nbsp;inhibits\u0026nbsp;NF-κB and inflammasome NLRP3 signaling pathways\u0026nbsp;by regulating IRG1-itaconate axis. Therefore, we repeated the above experiments with IRG1\u003csup\u003e−/−\u003c/sup\u003e mice and found that the protective effect of Rutin on liver injury and fibrosis was weakened. It indicated that the protective effects of Rutin on CCl\u003csub\u003e4\u003c/sub\u003e-induced liver injury and fibrosis was related to the IRG1-itaconate axis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe results of this experiment support the hepatic beneficial effects of Rutin, and Rutin supplementation may reduce drug-induced liver injury and fibrosis in mice. We have demonstrated that Rutin has a certain inhibitory effect on CCl\u003csub\u003e4\u003c/sub\u003e-induced liver injury, inflammatory response, oxidative stress and liver fibrosis in mice, possibly by further upregulating Nrf2 activity through IRG1-itaconate pathway and inhibiting inflammatory related pathways NLRP3 and NF-κB.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eROS, reactive oxygen species; DAMPs, damage-associated molecular patterns; PRR, pattern recognition receptor; NF-κB, Nuclear Factor kappa-light-chain-enhancer of activated B cells; NLRP3, NOD-like receptor pyrin domain-containing 3; TGF-β1, transforming growth factor beta 1; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; ECM, extracellular matrix; TCA, tricarboxylic acid; IRG1, immune response gene 1 protein; Nrf2, Nuclear factor E2-related factor 2; HO-1, heme oxygenase-1; NQO1, quinone oxidoreductase 1; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DHE, Dihydroethidium; SOD, superoxide dismutase; CAT, Catalase; HPLC, high-performance liquid chromatography; HSC, hepatic stellate cell; O\u003csup\u003e2−\u003c/sup\u003e, superoxide; H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, hydrogen peroxide.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was supported by Chongqing Natural Science Foundation (CSTB2022NSCQ-MSX0061)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003eEthics Approval Animal studies were ethically approved by the Chongqing Medical University Ethics Committee (Certification Number:\u003c/p\u003e\n\u003cp\u003e20220305).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eParola M, Pinzani M. 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Caspase-1-induced pyroptotic cell death. \u003cem\u003eImmunol Rev\u003c/em\u003e 2011; 243:206-14.\u003c/li\u003e\n\u003cli\u003eBala S, Csak T, Saha B, Zatsiorsky J, Kodys K, Catalano D, et al. The pro-inflammatory effects of miR-155 promote liver fibrosis and alcohol-induced steatohepatitis. \u003cem\u003eJ Hepatol\u003c/em\u003e 2016; 64:1378-87.\u003c/li\u003e\n\u003cli\u003eMills EL, Ryan DG, Prag HA, Dikovskaya D, Menon D, Zaslona Z, et al. Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1. \u003cem\u003eNature\u003c/em\u003e 2018; 556:113-117.\u003c/li\u003e\n\u003cli\u003eLiao ST, Han C, Xu DQ, Fu XW, Wang JS, Kong LY. 4-Octyl itaconate inhibits aerobic glycolysis by targeting GAPDH to exert anti-inflammatory effects. \u003cem\u003eNat Commun\u003c/em\u003e 2019; 10:5091.\u003c/li\u003e\n\u003cli\u003eQin W, Zhang Y, Tang H, Liu D, Chen Y, Liu Y, et al. 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The Immunomodulatory Metabolite Itaconate Modifies NLRP3 and Inhibits Inflammasome Activation. \u003cem\u003eCell Metab\u003c/em\u003e 2020; 32:468-478.e7.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. The primer sequences used for real-time PCR\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"576\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eGene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 255px;\"\u003e\n \u003cp\u003eForward Primers (5\u0026rsquo;\u0026ndash;3\u0026rsquo;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 255px;\"\u003e\n \u003cp\u003eReverse Primers (5\u0026rsquo;\u0026ndash;3\u0026rsquo;)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eGAPDH\u003c/p\u003e\n \u003cp\u003eIL-1\u0026beta;\u003c/p\u003e\n \u003cp\u003eIL-6\u003c/p\u003e\n \u003cp\u003eTNF-\u0026alpha;\u003c/p\u003e\n \u003cp\u003eCCL2\u003c/p\u003e\n \u003cp\u003eCol1a1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 255px;\"\u003e\n \u003cp\u003eAGG TCG GTG TGA ACG GAT TTG\u003c/p\u003e\n \u003cp\u003eGCA ACT GTT CCT GAA CTC AAC T\u003c/p\u003e\n \u003cp\u003eTAG TCC TTC CTA CCC CAA TTT CC\u003c/p\u003e\n \u003cp\u003eCCC TCA CAC TCA GAT CAT CTT CT\u003c/p\u003e\n \u003cp\u003eTTA AAA ACC TGG ATC GGA ACC AA\u003c/p\u003e\n \u003cp\u003eGCT CCT CTT AGG GGC CAC T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 255px;\"\u003e\n \u003cp\u003eTGT AGA CCA TGT AGT TGA GGT CA\u003c/p\u003e\n \u003cp\u003eATC TTT TGG GGT CCG TCA ACT\u003c/p\u003e\n \u003cp\u003eTTG GTC CTT AGC CAC TCC TTC\u003c/p\u003e\n \u003cp\u003eGCT ACG ACG TGG GCT ACA G\u003c/p\u003e\n \u003cp\u003eGCA TTA GCT TCA GAT TTA CGG GT\u003c/p\u003e\n \u003cp\u003eCCA CGT CTC ACC ATT GGG G\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eCTGF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 255px;\"\u003e\n \u003cp\u003eTCT CCA CCC GAG TTA CCA ATG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 255px;\"\u003e\n \u003cp\u003eAAT GTT TTC CTC CAG GTC AGC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eTGF-b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 255px;\"\u003e\n \u003cp\u003eTTG CTT CAG CTC CAC AGA GA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 255px;\"\u003e\n \u003cp\u003eTGG TTG TAG AGG GCA AGG AC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eTIMP-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 255px;\"\u003e\n \u003cp\u003eTGG TTG TAG AGG GCA AGG AC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 255px;\"\u003e\n \u003cp\u003eTGG TTG TAG AGG GCA AGG AC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"inflammation-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inre","sideBox":"Learn more about [Inflammation Research](http://link.springer.com/journal/11)","snPcode":"11","submissionUrl":"https://submission.nature.com/new-submission/11/3","title":"Inflammation Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Rutin, liver fibrosis, IRG1, itaconate, oxidative stress, NLRP3 inflammasome","lastPublishedDoi":"10.21203/rs.3.rs-8939517/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8939517/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLiver fibrosis, a common outcome of chronic liver injury, is characterized by excessive inflammation and oxidative stress. Rutin, a bioactive flavonoid with known antioxidant and anti-inflammatory properties, has not been thoroughly investigated for its potential anti-fibrotic mechanisms. This study aimed to elucidate the role of Rutin in liver fibrosis and its underlying molecular pathways. A carbon tetrachloride (CCl₄)-induced murine liver fibrosis model was employed. Liver injury, fibrotic deposition, inflammatory response, and oxidative stress were evaluated through histopathological examination, Western blotting, quantitative real-time PCR, and RNA sequence. The involvement of immune-responsive gene 1 (IRG1) was investigated using IRG1-knockout mice, while molecular docking and cellular thermal shift assay (CETSA) were performed to assess Rutin-IRG1 binding. The results showed that Rutin treatment significantly attenuated CCl₄-induced hepatic injury and collagen accumulation, accompanied by reduced markers of fibrosis. Mechanistically, Rutin activated the IRG1-itaconate axis, leading to a notable decrease in reactive oxygen species and pro-inflammatory cytokine release by Nrf2 activation and NLRP3 inflammasome containment. Molecular analyses confirmed direct binding of Rutin to IRG1, stabilizing its structure and enhancing its functional activity. The protective effects of Rutin were abolished in IRG1-deficient mice, underscoring the essential role of IRG1 in its anti-fibrotic action. In conclusion, Rutin ameliorates liver fibrosis by mitigating oxidative stress and suppressing NLRP3 inflammasome activation through targeting the IRG1/itaconate pathway, revealing a novel immunometabolic mechanism for its hepatoprotective effect.\u003c/p\u003e","manuscriptTitle":"Rutin Attenuates Liver Fibrosis by the IRG1-Itaconate-Nrf2 Axis: Modulation of Oxidative Stress and NLRP3 Inflammasome Activation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-24 17:55:18","doi":"10.21203/rs.3.rs-8939517/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-12T17:27:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-28T23:35:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-28T23:34:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Inflammation Research","date":"2026-02-22T13:50:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"inflammation-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inre","sideBox":"Learn more about [Inflammation Research](http://link.springer.com/journal/11)","snPcode":"11","submissionUrl":"https://submission.nature.com/new-submission/11/3","title":"Inflammation Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"782c7f1c-a2b0-4842-964d-7aa65f2408af","owner":[],"postedDate":"March 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T18:24:06+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-24 17:55:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8939517","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8939517","identity":"rs-8939517","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}