| ObjectiveAcetaminophen（APAP）is one of the most commonly used clinical antipyretic and analgesic drugs for its analgesic and antipyretic properties,which are safe and effective at recommended doses,while overdose may lead to hepatotoxicity and acute liver failure（ALF）.n-Acetyl-L-cysteine（NAC）,a known scavenger of reactive oxygen species（ROS）,is recommended by the FDA as the only clinically effective treatment for APAP-induced acute liver failure.N-Acetyl-L-cysteine（NAC）,a known scavenger of reactive oxygen species（ROS）,is recommended by the FDA as the only clinically effective treatment for APAP-induced acute liver injury;however,this drug has significant limitations with significant side effects and a narrow therapeutic window.Therefore,there is a need to develop new drugs that outperform NAC in terms of efficacy and treatment timeframe.Numerous studies have shown that APAP-induced hepatocyte necrosis is often accompanied by inflammation,and ginsenoside Rc（Ginsenoside Rc）,as one of the most representative and specific biomarkers in ginseng,has good antioxidant stress,anti-inflammatory,neuroprotective,and bioactive effects such as regulating glycolipid metabolism and hepatoprotection,but its therapeutic effects on acute liver injury caused by acetaminophen overdose are not yet However,the therapeutic effects on acute liver injury caused by acetaminophen overdose are not clear.Therefore,the present study was conducted to further investigate the protective effects of FXR（Farnesoid X Receptor）,a key target of hepatic metabolic regulation,on acetaminophen-induced acute hepatocyte injury,oxidative stress and inflammatory response,as well as on APAP-induced acute liver injury.Methods1.Validation of the molecular mechanism of action of ginsenoside Rc on APAP-induced liver injury in WT（Wild Type）mice and primary hepatocytesAPAP acute liver injury model was established and administered in vitro in primary hepatocytes of C57BL/6 mice and mice.The primary mouse liver hepatocytes were divided into 5 groups（n=4）:Control group,model group（APAP）,ginsenoside Rc+APAP group（Rc 50μM）,ginsenoside Rc+APAP group（Rc 25μM）,ginsenoside Rc+APAP group（Rc12.5μM）group,and 10m M APAP was added to each dosing group and injury model group,respectively.An equal volume of DMSO lysate was added to the blank control group and induced for 24h.The mouse models were randomly divided into 4 groups（n=8）by selecting SPF grade male C57BL/6 mice,including normal control group,APAP（300 mg/kg）liver injury model group,APAP（300 mg/kg）+Rc low dose group（5 mg/kg/d）,and APAP（300 mg/kg）+Rc high dose group（10 mg/kg/d）.Each group was given ginsenoside Rc by intraperitoneal injection according to the dose,and the normal control group was given an equal volume of saline.The drug was administered continuously for 10 d,once a day.The standard injection solution of APAP at a concentration of 30 mg/ml was prepared,and the standard solution was rapidly aspirated into the peritoneal cavity of mice with a l ml syringe.Except for the normal control group,acute liver injury was induced by intraperitoneal injection of APAP（300 mg/kg）in all groups.The control group was given equal volume of saline according to body weight.The expression changes of genes related to APAP metabolism,apoptosis,oxidation and inflammation in hepatocytes were detected by q PCR,Western Blotting,ELISA and immunofluorescence,the liver damage indexes such as ALT and AST in hepatocytes/liver were detected by kits,the changes in liver tissue morphology were detected by HE staining,the apoptotic status of hepatocytes was detected by TUNEL method,and the apoptotic status of hepatocytes was detected by GSH and SOD kits.GSH and SOD kits were used to detect the expression of antioxidant in mouse liver.2.To investigate the mechanism of Rc on APAP-induced liver injury in FXR（Nrlh4）knockout mice and primary hepatocytesWe used molecular docking to verify whether ginsenoside Rc could dock with FXR,and established an acute liver injury model of APAP on FXR-/-mouse primary hepatocytes and FXR-/-mice and administered the drug.FXR-/-mouse liver primary hepatocytes were divided into 3 groups（n=4）:Control group,model group（APAP）,and ginsenoside Rc+APAP group（Rc 50μM）.10m M APAP was added to each administration group and injury model group,and an equal volume of DMSO lysate was added to the blank control group,and after 24h induction,the next experiment was performed.FXR knockout mice were randomly divided into 3 groups（n=8）,including normal control group,APAP（300mg/kg）liver injury model group,and APAP（300mg/kg）+Rc group（10mg/kg/d）.Each group was given ginsenoside Rc by intraperitoneal injection according to the dose,and the normal control group was given an equal volume of saline.The drug was administered continuously for 10 d,once a day.The standard injection solution of APAP at a concentration of 30 mg/ml was prepared,and the standard solution was rapidly aspirated into the peritoneal cavity of mice with a l ml syringe.Except for the normal control group,acute liver injury was induced by intraperitoneal injection of APAP（300 mg/kg）in all groups.The control group was given equal volume of saline according to body weight.The expression changes of genes related to APAP metabolism,apoptosis,oxidation and inflammation in hepatocytes/liver were detected by q PCR,Western Blotting,ELISA and immunofluorescence,and liver damage indicators such as ALT and AST in hepatocytes were detected by kits.GSH and SOD kits were used to detect the expression of antioxidant in mouse liver.The differences between the groups were compared and the hepatoprotective pathway of Rc was further verified.Results1.In primary mouse liver cells,treatment with ginsenoside Rc effectively mitigated APAP-induced inflammation and ROS production,and alleviated APAP-induced apoptosis and NAPQI accumulation in MPHs.Western blotting results demonstrated that the relative expression levels of the apoptotic gene Bcl2/Bax m RNA were upregulated in the APAP model group,and ginsenoside Rc treatment could successfully reverse the down-regulation of Bcl2/Bax m RNA relative expression level.The number of red fluorescence-labeled positive apoptotic cells was significantly reduced in TUNEL staining following ginsenoside Rc treatment in comparison to the injury model group.q PCR results showed that ginsenoside Rc administration had a significant inhibitory effect on TNF-α,IL-β,and IL-6.The NAPQI kit demonstrated that ginsenoside Rc treatment was effective in reducing NAPQI levels.2.In mice with an acute liver injury model of APAP,administration of ginsenoside Rc significantly attenuated hepatotoxicity,as evidenced by increased survival rates and repair of liver dysfunction.（1）The results of ALT and AST kits showed that the administration of ginsenoside Rc significantly reduced serum ALT and AST levels,and the lethality rate was significantly reduced.（2）HE staining revealed that the necrotic area of the liver was significantly reduced after ginsenoside Rc administration,and apoptosis of hepatocytes was significantly improved.（3）GSH kit showed that Rc treatment enhanced the expression of GSH serum levels,and m RNA demonstrated enhanced expression of antioxidant genes.3.Ginsenoside Rc was found to upregulate m RNA expression of FXR in primary mouse liver cells as well as in FXR knockout mice.Combined with molecular docking and KEGG results,these findings suggest that ginsenoside Rc may exert relevant effects by mediating FXR proteins.After FXR knockout,ginsenoside Rc lost its protective effects against APAP-induced inflammatory response,apoptosis,and oxidative stress.Ginsenoside also lost its anti-inflammatory and antioxidant regulatory effects on FXR-/-MPHs cells.ConclusionGinsenoside Rc,a compound isolated from ginseng,has been found to ameliorate apoptosis induced by APAP in mouse liver primary cells.Furthermore,it improves their oxidative stress status and attenuates inflammation.Notably,the protective effects of ginsenoside Rc against APAP-induced liver injury are dependent on the activation of FXR nuclear receptors.Knockdown of FXR led to the loss of protective effects of ginsenoside Rc on the liver.These findings suggest that ginsenoside Rc may mediate its effects by modulating FXR. |
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