Hepatoprotective effect of ginsenoside Rg1 from Panax ginseng on carbon tetrachloride‐induced acute liver injury by activating Nrf2 signaling pathway in mice

Oxidative stress and inflammatory response are well known to be involved in the pathogenesis of acute liver injury. This study was performed to examine the hepatoprotective effect of ginsenoside Rg1 (Rg1) against CCl₄‐induced acute liver injury, and further to elucidate the involvement of Nrf2 signaling pathway in vivo and in vitro. Mice were orally administered Rg1 (15, 30, and 60 mg/kg) or sulforaphane (SFN) once daily for 1 week prior to 750 μL/kg CCl₄ injection. The results showed that Rg1 markedly altered relative liver weights, promoted liver repair, increased the serum level of TP and decreased the serum levels of ALT, AST and ALP. Hepatic oxidative stress was inhibited by Rg1, as evidenced by the decrease in MDA, and increases in GSH, SOD, and CAT in the liver. Further research demonstrated that Rg1 suppressed liver inflammation response through repressing the expression levels of inflammation‐related genes including TNF‐α, IL‐1β, IL‐6, COX‐2, and iNOS. In addition, Rg1 enhanced antioxidative stress and liver detoxification abilities by up‐regulating Nrf2 and its target‐genes such as GCLC, GCLM, HO‐1, NQO1, Besp, Mrp2, Mrp3, Mrp4, and down‐regulating Cyp2e1. However, the changes in Nrf2 target‐genes, as well as ameliorative liver histology induced by Rg1 were abrogated by Nrf2 antagonist all‐transretinoic acid in vivo and Nrf2 siRNA in vitro. Overall, the findings indicated that Rg1 might be an effective approach for the prevention against acute liver injury by activating Nrf2 signaling pathway.     

Ginsenoside Rg3 enhances the chemosensitivity of tumors to cisplatin by reducing the basal level of nuclear factor erythroid 2-related factor 2-mediated heme oxygenase-1/NAD(P)H quinone oxidoreductase-1 and prevents normal tissue damage by scavenging cisplatin-induced intracellular reactive oxygen species

The clinical use of cisplatin (cis-diamminedichloroplatinum II) has been limited by the frequent emergence of cisplatin-resistant cell populations and numerous other adverse effects. Therefore, new agents are required to improve the therapy and health of cancer patients. Oral administration of ginsenoside Rg3 significantly inhibited tumor growth and promoted the anti-neoplastic efficacy of cisplatin in mice inoculated with CT-26 colon cancer cells. In addition, Rg3 administration remarkably inhibited cisplatin-induced nephrotoxicity, hepatotoxicity and oxidative stress. In cell-based experiments, Rg3 inhibited cisplatin-induced cytotoxicity in LLC-RK1 kidney and NCTC1469 liver cells but not in CT-26 cancer cells and significantly decreased cisplatin-induced intracellular ROS levels in these cells. In normal cells with cytoplasmically localized Nrf2 and negligible levels of HO-1 and NQO-1, Rg3 substantially decreased cisplatin-induced elevation in HO-1/NQO-1 levels and inhibited cisplatin-induced translocation of Nrf2 into the nucleus. In chemoresistant cancer cells with high levels of HO-1/NQO-1 and nuclear Nrf2, both basal and cisplatin-induced levels of HO-1/NQO-1 and nuclear Nrf2 were decreased by Rg3 treatment, thereby enhancing the susceptibility of cancer cells to cisplatin. Collectively, Rg3 promotes the efficacy of cisplatin by inhibiting HO-1 and NQO-1 expression in cancer cells and protects the kidney and liver against tissue damage by preventing cisplatin-induced intracellular ROS generation.     

Enhanced expression of Nrf2 in mice attenuates the fatty liver produced by a methionine- and choline-deficient diet

Oxidative stress has been proposed as an important promoter of the progression of fatty liver diseases. The current study investigates the potential functions of the Nrf2-Keap1 signaling pathway, an important hepatic oxidative stress sensor, in a rodent fatty liver model. Mice with no (Nrf2-null), normal (wild type, WT), and enhanced (Keap1 knockdown, K1-kd) expression of Nrf2 were fed a methionine- and choline-deficient (MCD) diet or a control diet for 5 days. Compared to WT mice, the MCD diet-caused hepatosteatosis was more severe in the Nrf2-null mice and less in the K1-kd mice. The Nrf2-null mice had lower hepatic glutathione and exhibited more lipid peroxidation, whereas the K1-kd mice had the highest amount of glutathione in the liver and developed the least lipid peroxidation among the three genotypes fed the MCD diet. The Nrf2 signaling pathway was activated by the MCD diet, and the Nrf2-targeted cytoprotective genes Nqo1 and Gstα1/2 were induced in WT and even more in K1-kd mice. In addition, Nrf2-null mice on both control and MCD diets exhibited altered expression profiles of fatty acid metabolism genes, indicating Nrf2 may influence lipid metabolism in liver. For example, mRNA levels of long chain fatty acid translocase CD36 and the endocrine hormone Fgf21 were higher in livers of Nrf2-null mice and lower in the K1-kd mice than WT mice fed the MCD diet. Taken together, these observations indicate that Nrf2 could decelerate the onset of fatty livers caused by the MCD diet by increasing hepatic antioxidant and detoxification capabilities.     

Participation of nuclear factor (erythroid 2-related), factor 2 in ameliorating lithocholic acid-induced cholestatic liver injury in mice

BACKGROUND AND PURPOSE

Lithocholic acid (LCA), the most toxic bile acid, induces cholestatic liver injury in rodents. We previously showed that LCA activates the oxidative stress-responsive nuclear factor (erythroid-2 like), factor 2 (Nrf2) in cultured liver cells, triggering adaptive responses that reduce cell injury. In this study, we determined whether Nrf2 protects the liver against LCA-induced toxicity in vivo.

EXPERIMENTAL APPROACH

Nrf2 disrupted (Nrf2^−/−) and wild-type mice were treated with LCA (125 mg·kg⁻¹ body weight) to induce liver injury. Levels of mRNA, protein and function of important Nrf2 target genes coupled with liver histology and injury biomarkers of mice were examined.

KEY RESULTS

In 4 day LCA treatments, we observed a significantly higher hepatic induction of Nrf2 target, cytoprotective genes including thioredoxin reductase 1, glutamate cysteine ligase subunits, glutathione S-transferases, haeme oxygenase-1 and multidrug resistance-associated proteins 3 and 4 in the wild type as compared with the Nrf2^−/− mice. Moreover, basal and LCA-induced hepatic glutathione and activities of glutathione S-transferases and thioredoxin reductases were higher in wild-type than in Nrf2^−/− mice. This reduced production of cytoprotective genes against LCA toxicity rendered Nrf2^−/− mice more susceptible to severe liver damage with the presence of multifocal liver necrosis, inflamed bile ducts and elevation of lipid peroxidation and liver injury biomarkers, such as alanine aminotransferase and alkaline phosphatase.

CONCLUSIONS AND IMPLICATIONS

Nrf2 plays a crucial cytoprotective role against LCA-induced liver injury by orchestrating adaptive responses. The pharmacological potential of targeting liver Nrf2 in the management of cholestatic liver diseases is proposed.     

Protective effect of isochlorogenic acid B on liver fibrosis in non‐alcoholic steatohepatitis of mice

Liver fibrosis is a common symptom of non‐alcoholic steatohepatitis (NASH) and a worldwide clinical issue. The miR‐122/HIF‐1α signalling pathway is believed to play an important role in the genesis of progressive fibrosis. Isochlorogenic acid B (ICAB), naturally isolated from Laggera alata, is verified to have antioxidative and hepatoprotective properties. The aim of this study was to investigate the effect of ICAB on liver fibrosis in NASH and its potential protective mechanisms. NASH was induced in a mouse model with a methionine‐ and choline‐deficient (MCD) diet for 4 weeks, and ICAB was orally administered every day at three doses (5, 10 and 20 mg/kg). Pathological results indicated that ICAB significantly improved the pathological lesions of liver fibrosis. The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and hepatic hydroxyproline (Hyp), cholesterol (CHO) and triglyceride (TG) were also significantly decreased by ICAB. In addition, ICAB inhibited hepatic stellate cells (HSCs) activation and the expressions of hepatic genes involved in liver fibrosis including LOX, TGF‐β1, MCP‐1, COL1α1 and TIMP‐1. ICAB also attenuated liver oxidative stress through Nrf2 signalling pathway. What is more, the decreased levels of miR‐122 and over‐expression of hepatic HIF‐1α could be reversed by ICAB treatment. These results simultaneously confirmed that ICAB had a significant protective effect on fibrosis in NASH by inhibiting oxidative stress via Nrf2 and suppressing multiple profibrogenic factors through miR‐122/HIF‐1α signalling pathway.     

Nrf2 pathway activation contributes to anti-fibrosis effects of ginsenoside Rg1 in a rat model of alcohol- and CCl4-induced hepatic fibrosis

Aim:

To investigate the anti-fibrosis effects of ginsenoside Rg1 on alcohol- and CCl₄-induced hepatic fibrosis in rats and to explore the mechanisms of the effects.

Methods:

Rats were given 6% alcohol in water and injected with CCl₄ (2 mL/kg, sc) twice a week for 8 weeks. Rg1 (10, 20 and 40 mg/kg per day, po) was administered in the last 2 weeks. Hepatic fibrosis was determined by measuring serum biochemical parameters, HE staining, Masson''s trichromic staining, and hydroxyproline and α-SMA immunohistochemical staining of liver tissues. The activities of antioxidant enzymes, lipid peroxidation, and Nrf2 signaling pathway-related proteins (Nrf2, Ho-1 and Nqo1) in liver tissues were analyzed. Cultured hepatic stellate cells (HSCs) of rats were prepared for in vitro studies.

Results:

In the alcohol- and CCl₄-treated rats, Rg1 administration dose-dependently suppressed the marked increases of serum ALT, AST, LDH and ALP levels, inhibited liver inflammation and HSC activation and reduced liver fibrosis scores. Rg1 significantly increased the activities of antioxidant enzymes (SOD, GSH-Px and CAT) and reduced MDA levels in liver tissues. Furthermore, Rg1 significantly increased the expression and nuclear translocation of Nrf2 that regulated the expression of many antioxidant enzymes. Treatment of the cultured HSCs with Rg1 (1 μmol/L) induced Nrf2 translocation, and suppressed CCl₄-induced cell proliferation, reversed CCl_4- induced changes in MDA, GPX, PCIII and HA contents in the supernatant fluid and α-SMA expression in the cells. Knockdown of Nrf2 gene diminished these actions of Rg1 in CCl₄-treated HSCs in vitro.

Conclusion:

Rg1 exerts protective effects in a rat model of alcohol- and CCl₄-induced hepatic fibrosis via promoting the nuclear translocation of Nrf2 and expression of antioxidant enzymes.     

Ginsenoside Rg1 prevents acetaminophen-induced oxidative stress and apoptosis via Nrf2/ARE signaling pathway

Inappropriate use of acetaminophen (APAP) can lead to morbidity and mortality secondary to hepatic necrosis. Ginsenoside Rg1 is a major active ingredient in processed Panax ginseng, which is proved to elicit biological effects. We hypothesized the beneficial effect of Rg1 on APAP-mediated hepatotoxicity was through Nrf2/ARE pathway. The study was conducted in cells and mice, comparing the actions of Rg1. Rg1 significantly improved cell survival rates and promoted the expression of antioxidant proteins. Meanwhile, Rg1 reduced the excessive ROS and the occurrence of cell apoptosis, which were related to Nrf2/ARE pathway. Expression of Nrf2 has a certain cell specificity.

     

Role of Nrf2 in preventing ethanol-induced oxidative stress and lipid accumulation

Oxidative stress and lipid accumulation play important roles in alcohol-induced liver injury. Previous reports showed that, in livers of nuclear factor erythroid 2-related factor 2 (Nrf2)-activated mice, genes involved in antioxidant defense are induced, whereas genes involved in lipid biosynthesis are suppressed. To investigate the role of Nrf2 in ethanol-induced hepatic alterations, Nrf2-null mice, wild-type mice, kelch-like ECH-associated protein 1-knockdown (Keap1-KD) mice with enhanced Nrf2, and Keap1-hepatocyte knockout (Keap1-HKO) mice with maximum Nrf2 activation, were treated with ethanol (5 g/kg, po). Blood and liver samples were collected 6 h thereafter. Ethanol increased alanine aminotransferase and lactate dehydrogenase activities as well as thiobarbituric acid reactive substances in serum of Nrf2-null and wild-type mice, but not in Nrf2-enhanced mice. After ethanol administration, mitochondrial glutathione concentrations decreased markedly in Nrf2-null mice but not in Nrf2-enhanced mice. H₂DCFDA staining of primary hepatocytes isolated from the four genotypes of mice indicates that oxidative stress was higher in Nrf2-null cells, and lower in Nrf2-enhanced cells than in wild-type cells. Ethanol increased serum triglycerides and hepatic free fatty acids in Nrf2-null mice, and these increases were blunted in Nrf2-enhanced mice. In addition, the basal mRNA and nuclear protein levels of sterol regulatory element-binding protein 1(Srebp-1) were decreased with graded Nrf2 activation. Ethanol further induced Srebp-1 mRNA in Nrf2-null mice but not in Nrf2-enhanced mice. In conclusion, Nrf2 activation prevented alcohol-induced oxidative stress and accumulation of free fatty acids in liver by increasing genes involved in antioxidant defense and decreasing genes involved in lipogenesis.     

The pathogenesis of ethanol versus methionine and choline deficient diet-induced liver injury

The differences and similarities of the pathogenesis of alcoholic (ASH) and non-alcoholic steatohepatitis (NASH) were examined. Mice (six/group) received one of four Lieber-Decarli liquid diets for 6 weeks: (1) paired-fed control diet; (2) control diet with ethanol (ethanol); (3) paired-fed methionine/choline deficient (MCD) diet; and (4) MCD plus ethanol (combination). Hepatotoxicity, histology, and gene expression changes were examined. Both MCD and ethanol induced macrovesicular steatosis. However, the combination diet produced massive steatosis with minor necrosis and inflammation. MCD and combination diets, but not ethanol, induced serum ALT levels by 1.6- and 10-fold, respectively. MCD diet, but not ethanol, also induced serum alkaline phosphatase levels suggesting bile duct injury. Ethanol increased liver fatty acid binding protein (L-FABP) mRNA and protein levels. In contrast, the combination diet decreased L-FABP mRNA and protein levels and increased hepatic free fatty acid and lipid peroxide levels. Ethanol, but not MCD, reduced hepatic S-adenosylmethionine (SAM) and GSH levels. Hepatic TNFalpha protein levels were increased in all treatment groups, however, IL-6, a hepatoprotective cytokine which promotes liver regeneration was increased in ethanol-fed mice (2-fold), but decreased in the combination diet-treated mice. In addition, the combination diet reduced phosphorylated STAT3 and Bcl-2 levels. While MCD diet might cause bile duct injury and cholestasis, ethanol preferentially interferes with the SAM-GSH oxidative stress pathway. The exacerbated liver injury induced by the combination diet might be explained by reduced L-FABP, increased free fatty acids, oxidative stress, and decreased IL-6 protein levels. The combination diet is an efficient model of steatohepatitis.     

Protective mechanisms of Aralia continentalis extract against tert-butyl hydroperoxide-induced hepatotoxicity: in vivo and in vitro studies

In the present work, we investigated the protective effects of the ethanol extract of Aralia continentalis roots (AC) on tert-butyl hydroperoxide (t-BHP)-induced hepatotoxicity in a cultured Hepa1c1c7 cell line and in mouse liver. Pretreatment with AC prior to the administration of t-BHP significantly prevented the increase in serum levels of hepatic enzyme markers (ALT, AST) and lipid peroxidation and reduced oxidative stress, as measured by glutathione content, in the liver. Histopathological evaluation of the livers also revealed that AC reduced the incidence of liver lesions. The in vitro study showed that AC significantly reduced t-BHP-induced oxidative injury in Hepa1c1c7 cells, as determined by cell cytotoxicity, intracellular glutathione content, lipid peroxidation, reactive oxygen species (ROS) levels, and caspase-3 activation. Also, AC up-regulated phase II genes including heme oxygenase-1 (HO-1), NAD(P)H:quinone reductase, and glutathione S-transferase. Moreover, AC induced Nrf2 nuclear translocation and ERK1/2 and p38 activation, pathways that are involved in inducing Nrf2 nuclear translocation. Taken together, these results suggest that the protective effects of AC against t-BHP-induced hepatotoxicity may, at least in part, be due to its ability to scavenge ROS and to regulate the antioxidant enzyme HO-1 via the ERK1/2 and p38/Nrf2 signaling pathways.     

Hepatic ischemia-reperfusion induces renal heme oxygenase-1 via NF-E2-related factor 2 in rats and mice

Hepatic ischemia-reperfusion (IR) results in Kupffer cell activation and subsequent tumor necrosis factor (TNF) alpha release, leading to localized hepatic injury and remote organ dysfunction. Heme oxygenase (HO)-1 is an enzyme that is induced by various stimuli, including proinflammatory cytokines, and exerts antioxidative and anti-inflammatory functions. Up-regulation of HO-1 is known to protect against hepatic IR injury, but the effects of hepatic IR on the kidney are poorly understood. Thus, the purpose of this study was to determine whether hepatic IR and resultant Kupffer cell activation alters renal HO-1 expression. Male Sprague-Dawley rats and wild-type and NF-E2-related factor 2 (Nrf2)-null mice were subjected to 60 min of partial hepatic ischemia, and at various times thereafter, blood, liver, and kidneys were collected. After reperfusion, 1) creatinine clearance decreased; 2) HO-1 mRNA and protein expression in liver and kidney markedly increased; 3) renal NAD(P)H: quinone oxidoreductase 1 mRNA expression was induced; 4) serum TNFalpha levels increased; 5) Nrf2 translocation into the nucleus of renal tissue increased; and 6) renal and urinary 15-deoxy-Delta(12,14)-prostaglandin J2 (15-d-PGJ2) levels increased. Kupffer cell depletion by pretreating with gadolinium chloride 1) attenuated increased mRNA expression of HO-1 in kidney; 2) attenuated the increase in TNFalpha; 3) inhibited the increase in Nrf2 nuclear translocation; and 4) tended to attenuate renal 15-d-PGJ2 levels. Whereas renal HO-1 mRNA expression increased in wild-type mice, it was attenuated in Nrf2-null mice. These results suggest that renal HO-1 is induced via Nrf2 to protect the kidney from remote organ injury after hepatic IR.     

Metadoxine prevents damage produced by ethanol and acetaldehyde in hepatocyte and hepatic stellate cells in culture.

Metadoxine (pyridoxine-pyrrolidone carboxylate) has been reported to improve liver function tests in alcoholic patients. In the present work we have investigated the effect of metadoxine on some parameters of cellular damage in hepatocytes and hepatic stellate cells in culture treated with ethanol and acetaldehyde. HepG2 and CFSC-2G cells were treated with 50 mM ethanol or 175 microM acetaldehyde as initial concentration in the presence or absence of 10 microg ml(-1) of metadoxine. Twenty-four hours later reduced and oxidized glutathione content, lipid peroxidation damage, collagen secretion and IL-6, IL-8 and TNF- alpha secretion were determined. Our results suggest that metadoxine prevents glutathione depletion and the increase in lipid peroxidation damage caused by ethanol and acetaldehyde in HepG2 cells. In hepatic stellate cells, metadoxine prevents the increase in collagen and attenuated TNF- alpha secretion caused by acetaldehyde. Thus, metadoxine could be useful in preventing the damage produced in early stages of alcoholic liver disease as it prevents the redox imbalance of the hepatocytes and prevents TNF- alpha induction, one of the earliest events in hepatic damage.     

Free radicals,antioxidants, nuclear factor-E2-related factor-2 and liver damage

Oxidative/nitrosative stress is proposed to be a critical factor in various diseases, including liver pathologies. Antioxidants derived from medicinal plants have been studied extensively and are relevant to many illnesses, including liver diseases. Several hepatic disorders, such as viral hepatitis and alcoholic or nonalcoholic steatohepatitis, involve free radicals/oxidative stress as agents that cause or at least exacerbate liver injury, which can result in chronic liver diseases, such as liver fibrosis, cirrhosis and end-stage hepatocellular carcinoma. In this scenario, nuclear factor-E2-related factor-2 (Nrf2) appears to be an essential factor to counteract or attenuate oxidative or nitrosative stress in hepatic cells. In fact, a growing body of evidence indicates that Nrf2 plays complex and multicellular roles in hepatic inflammation, fibrosis, hepatocarcinogenesis and regeneration via the induction of its target genes. Inflammation is the most common feature of chronic liver diseases, triggering fibrosis, cirrhosis and hepatocellular carcinoma. Increasing evidence indicates that Nrf2 counteracts the proinflammatory process by modulating the recruitment of inflammatory cells and inducing the endogenous antioxidant response of the cell. In this review, the interactions between antioxidant and inflammatory molecular pathways are analyzed.     

Magnolia officinalis Reverses Alcoholic Fatty Liver by Inhibiting the Maturation of Sterol Regulatory Element–Binding Protein-1c

The generally accepted hypothesis for the pathogenesis of alcoholic liver disease (ALD) is the two-hit model, which proposes that fat accumulation in the liver increases the sensitivity of the liver to a second hit that leads to inflammatory liver cell damage. In this study we evaluated the effects of Magnolia officinalis (MO), which contains honokiol and magnolol as the primary pharmacological components, to eradicate fatty liver in rats fed an ethanol diet. In vitro studies showed that MO was able to protect RAW 264.7 cells from ethanol-induced production of tumor necrosis factor-α, reactive oxygen species, and superoxide anion radicals; the activation of NADPH oxidase; and subsequent cell death. We also investigated the therapeutic effects of MO on alcoholic fatty liver in Lieber-DeCarli ethanol diet–fed rats. MO treatment of the rats for the last 2 weeks of ethanol feeding completely reversed all the serum, hepatic parameters, and fatty liver changes. The increased maturation of sterol regulatory element– binding protein-1c in the liver by ethanol treatment was completely inhibited by treatment with MO. Therefore, MO may be a promising candidate for development as a therapeutic agent for ALD.[Supplementary Figures: available only at http://dx.doi.org/10.1254/jphs.08182FP]     

Panax notoginseng saponins protect against chronic ethanol-induced hepatic steatosis

Chronic alcohol consumption induces hepatic steatosis, the early stage of alcoholic liver disease (ALD). The aim of present study is to investigate the protective effect of Panax notoginseng saponins (PNS) against chronic ethanol-induced hepatic steatosis in vivo. Mice were pair-fed a modified Lieber-DeCarli liquid diet containing alcohol or isocaloric maltose dextrin as control diet with or without PNS (200 mg/kg, BW) for 8 weeks. Animals supplemented with PNS were protected against hepatic lipid accumulation induced by chronic ethanol exposure. Accordingly, PNS could significantly decrease the elevation of plasma triglyceride, plasma enzyme activities, i.e. alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and hepatic TNF-α and IL-6 levels which were induced by chronic alcohol exposure. In addition, PNS markedly reduced the lipolysis of white adipose tissue (WAT) that stimulated by alcohol feeding through the inhibiting protein expression of phosphorylation of hormone-sensitive lipase (p-HSL), rather than total HSL. Furthermore, alcohol exposure also enhanced fatty acid uptake capacity in liver by elevated hepatic CD36 expression, which could attenuated by PNS treatment. These results demonstrate that PNS supplementation protects against chronic ethanol-induced hepatic steatosis, which is associated with ameliorating dysfunctional lipid metabolism of WAT and the reduced inflammatory cytokines. Our findings suggested that PNS might be potential to be developed as an effective agent for the treatment of chronic alcoholic steatosis.     

Activation of Nrf2 pathway and inhibition of NLRP3 inflammasome activation contribute to the protective effect of chlorogenic acid on acute liver injury

Chlorogenic acid (CGA), a kind of polyphenol found in coffee, fruits and vegetables, has potent anti-oxidant and anti-inflammatory properties. Our previous studies showed CGA could efficiently alleviate liver fibrosis in rats. However, whether CGA regulates nuclear factor erythroid-2-related factor 2 (Nrf2) anti-oxidant pathway and NLRP3 inflammasome activation and protects against carbon tetrachloride (CCl₄)-induced acute liver injury are unknown. We found that CGA could increase Nrf2 activation and expression of Nrf2-related anti-oxidant genes, including HO-1, NQO1 and GCLC. Pretreatment with CGA could reduce CCl₄-induced elevation of serum transaminases and alleviate liver pathological abnormalities. CGA also reversed CCl₄-induced increase in MDA level and decrease in the levels of GSH, SOD and CAT in liver tissues. Meanwhile, CGA inhibited NLRP3 inflammasome activation, as indicated by the reduced protein expression of NLRP3, Pro-Caspase-1, Caspase-1, Pro-IL-1β and IL-1β. Moreover, CGA reduced serum levels and liver mRNA expression of TNF-α, IL-6 and IL-1β. These results demonstrate that CGA protects against CCl₄-induced acute liver injury probably through enhancing Nrf2-mediated anti-oxidant pathway and inhibiting NLRP3 inflammasome activation.