The Protective Effect Of Schaftoside On Liver Injury Induced By APAP Or High Fat Diet Through Farnesoid X Receptor

Objecti veAs a widely used antipyretic analgesic,acetaminophen(APAP)is the main cause of acute liver failure due to its hepatotoxicity.The high-calorie and high-fat diet(HFD)has caused the prevalence of non-alcoholic fatty liver(NAFL)damage in worldwide.However,oxidative stress,metabolic disorder,inflammation and other reactions occur in liver injury induced by APAP or HFD to vary-ing degrees.In the exploration of the pathological process of APAP and NAFL injury,it is particularly important to clarify its mechanism for the determination of therapeutic strategies.Rcecent studies have shown that farnesoid X receptor(FXR)is a key target of drug-induced liver injury,liver and metabolic diseases,and its agonist is expected to be a potential therapeutic agent for a variety of liver diseases.Schiavoside(SS)is the main flavonoid active component of Desmodium styracifolium(Osb.)merr.,with anti-inflammatory,antioxidant,liver-protecting and liver-protecting pharmacological effects.In the pharmacological study of SS,this study found that SS can activate FXR and its target genes,but it is not clear whether FXR,as a hotspot gene in liver disease research,is significant in the efficacy of SS.In this study,the protective effect of SS on APAP-induced toxic liver injury and HFD-induced NAFL injury was studied,and the relevant molecular mechanism was elaborated.Methods1.SS as the ligand of FXR activates FXR and its target genesThe combination of SS and FXR-LBD was preliminarily evaluated by molecular docking.Then FXR(Nr1h4)expression plasmid and BSEP promoter plasmid were constructed and transfected into HEK293T cells.After SS administration,promoter activity detected by using dual luciferase reporter gene.Whether SS could excite FXR and its target genes was confirmed by qRT-PCR,western blot and cellular immunofluorescence.Finally,FXR was silenced by hFXR siRNA,mRNA and protein expression levels detected by qRT-PCR,western blot and immunofluorescence to verified again the significance of FXR in SS activation of downstream target genes.2.The protective effect and molecular mechanism of SS on APAP-induced inflammation and liver injuryMale C57BL/6 mice were randomly divided into 6 groups(n=6),including the normal group,the model group,the obeticholic acid group(OCA,20mg/kg),the low-dose medium-dose and the high-dose of SS group(40mg/kg,80mg/kg,160mg/kg).In addition to the normal group and the model group,each group received oral administration once a day for 7 days.APAP(300mg/kg)was administered to all groups except the normal group for 12h after fasting for 12h on 7th day.The liver damage index alanine aminotransferase(ALT),aspertate aminotransferase(AST),lactate dehydrogenase(LDH)were detected,H&E histopathological staining,TUNEL assay were used for pathologyical analysis,then the related to oxidative stress,inflammation,metabolism of APAP indices,gene mRNA and protein expression were analysed by qRT-PCR,western blot,immunofluorescence,immunohistochemical,LC-MS/MS detection experiment et al.Primary mouse hepatocytes were isolated and cultured.APAP-induced cell damage model was established,the cell injury indices(ALT,AST)were detected,and the protective effect of SS on APAP-induced cell damage and death was evaluated by CCK-8 cell proliferation toxicity test,TUNEL assay and mitochondrial membrane potential(JC-1)assay.The concentration of glutathione(GSH)was detected;the expression of P38,JNK and their phosphorylated protein were detected,and ROS levels were detected by immunofluorescence to evaluate the protective effect of SS on APAP-induced oxidative stress.LPS-induced cellular inflammation model was established.And the inhibitory effect of SS on inflammatory cytokines was evaluated by detecting the content of cytokine by ELISA and the expression of its mRNA by qRT-PCR.Plasmids loaded with NF-?B or-(and)FXR+NF-?B were transfected into HEK293T cells,induced by LPS,the activity of NF-?B was detected by double luciferase reporter genes,to evaluate the anti-inflammatory effect of SS through FXR and NF-?B,and western blot and immunofluorescence were used for further support this.3.To prove the significance of FXR in SS ameliorates APAP-induced inf lammation and liver injurySix C57BL/6 mice(WT mice)and six Nr1h4 knockout mice(FXR-/-mice)were administered with APAP(300mg/kg)for 24h to observe the survival rate.Twenty-four SPF male C57BL/6 mice(WT mice)were randomly divided into 4 groups(n=6),including the normal group,the model group,the OCA group(20mg/kg),and the SS group(160mg/kg).Twenty-four Nrlh4 knockout mice(FXR-/-mice)were randomly divided into 4 groups(n=6),including the normal group,the model group,the OCA group(20mg/kg),and the SS group(160mg/kg).In addition to the normal group and the model group,each group received oral administration once a day for 7 days.APAP(300mg/kg)was administered to all groups except the normal group for 6h after fasting for 12h on 7th day.ALT,AST,LDH,H&E histopathological staining and TUNEL assay were used for pathological analysis,and then the related to oxidative stress,inflammation,metabolism of APAP indices,gene mRNA and protein expression were analysed by qRT-PCR,western blot,immunofluorescence,immunohistochemical,LC-MS/MS detection expreriment et al.4.The protective effect and molecular mechanism of SS on HFD-induced NAFL injury.Seventy-two male C57BL/6 mice were randomly divided into 6 groups,including the normal group,the model group,ursodeoxycholic acid(UDCA)group(60 mg/kg),OCA group(20mg/kg),the low-dose of SS group(80 mg/kg),and the high-dose of SS group(160 mg/kg).The normal group was fed normal diet every day,and the rest group were given HFD or(and)the simultaneous oral administration of SS or UDCA or OCA for 4 weeks.The mice were weighed every 7 days.Samples were taken after ? 12h fast on day 28.Serum triglycerides(TG),cholesterol(Ch),high-density lipoprotein cholesterol(HFD-C),and low-density lipoprotein cholesterol(LDL-C)were detected.ALT and AST were detected,and H&E histopathological staining was used for liver histopathological analysis.The mRNA and proteins of related pathway genes were detected by qRT-PCR,western blot and immunodouble fluorescence.Oleic acid(OA)was used to establish a cell lipid accumulation model,after SS administration,intracellular TG content were deteced,oil red and hematoxylin double staining was used to evaluate the lipid clearance,and the expression of mRNA and protein in lipid-related pathways were analysed by RT-PCR and wstern blot.Results1.SS can interact with FXR-LBD,activate FXR to up-regulate the mRNA and protein expression of BSEP and down-regulate the mRNA and protein expression of CYP7A1.After silencing FXR,SS could not effectively regulate the mRNA and protein expression of BSEP and CYP7A1,indicating the importance of FXR in regulating the downstream target genes of SS.2.APAP-induced hepatic injury model was established in mice.SS can reduce the APAP-induced elevation of AST and LDH content(p<0.01 or 0.05),reduce the degree of liver tissue inflammatory infiltration and necrosis,and have a great protective effect on liver function recovery and liver injury.The depletion of GSH is the initial start of liver injury caused by excessive NAPQI generated by APAP metabolism.SS increase the content of GSH and SOD and reduce the content of MDA and 8-OHdG(p<0.01 or 0.05),enhance the expression of Gclc and Gclm,inhibit the phosphorylation of JNK and P38,and protect APAP-induced oxidative stress liver injury through antioxidant capacity.Arachidonic acid metabolism inflammation mediator,which is the important mechanism of the inflammatory response,SS regulating arachidonic acid metabolism of APAP mediated inflammatory mediators,inhibits the production and mRNA expression of inflammatory factors such as IL-1?,TNF-?,and IL-6(p<0.01 or 0.05),inhibits the expression of IL-6,CRP,F4/80+/CD11b+/CD45+in liver tissues,inhibits APAP-induced nuclear transfection of NF-?B,and protects APAP-induced inflammatory liver injury by mediating the inflammatory response in liver tissues.Furthermore,SS can activation of FXR,enhance or increase the expression of ? phase metabolic enzymes and transporters,such as BSEP,UGT1A1,SLC10a1,SLCO1a1,GSTA1 and OST?,rather than I phase CYP3A11 and CYP2E1 enzyme metabolism,improve the toxic liver damage induced by APAP.In vitro experiments,APAP-induced mouse primary hepatocyte injury model,APAP-induced cell death rate was 70%for 24h,and SS intervention improved cell survival rate.APAP-induced primary hepatocytes resulted in significantly higher ALT and AST levels(p<0.01),but SS effectively reduced AST levels(p<0.01 or p<0.05).SS also reduce DNA fragments,increase APAP-induced mitochondrial membrane potential decline,and protect APAP-induced primary hepatocytes injury and death in mice.APAP-induced mouse primary hepatocytes significantly consumed GSH and increased ROS production,while SS significantly increased the content of GSH and decreased ROS production(p<0.01 or p<0.05).Next,we investigated the protective mechanism of SS against hepatocyte injury.The phosphorylation of JNK and P38 protein was increased and the MAPK pathway was activated,which was induced by APAP for 24h,while SS intervention significantly inhibited the phosphorylation of JNK and P38.SS alleviates APAP-induced cytotoxicity by reducing cell death and oxidative stress.In addition,LPS-induced cellular inflammatory model was established.The increase of IL-1?,TNF-?,IL-6 and IL-10 content in mouse primary hepatocytes was induced by LPS(p<0.01),while SS intervention reduced the production of IL-1 ?,TNF-?,IL-6(p<0.01 or 0.05),with no significant effect on IL-10.Next,the effect of SS on the expression of NF-?B indicated that SS could inhibit the expression of NF-?B induced by LPS and inhibit NF-?B nuclear translocation induced by LPS.SS inhibit LPS-induced NF-?B activation and nuclear translocation,reduce the production of inflammatory cytokines and protect cells from inflammatory injury.3.APAP-induced liver injury model was established in FXR-/-mice to investigate the protective role of FXR.In the observation of the survival rate of mice for 24h,given 300mg/kg APAP,the survival rate of WT mice at 24h was 83.33%,while the survival rate of FXR-/-mice at 12h was only 16.67%,and all died after 18h,indicating that FXR is very important in the protective effect of APAP-induced death of mice.After 6h of APAP administration in mice,APAP-induced the content of AST and LDH increased(p<0.01 or 0.05),leading to severe liver tissue inflammatory infiltration and necrosis.In FXR-/-mice,SS could not alleviate this phenomenon,losing the role of liver function recovery and liver injury protection.The depletion of GSH is the initial start of liver injury caused by excessive NAPQI generated by APAP metabolism.The decrease of GSH and SOD content and the increase of MDA and 8-0HdG content induced by APAP(p<0.01 or 0.05).In FXR-/-mice,SS cannot reverse the decrease of GSH and SOD and the increase of MDA and 8-0HdG content,nor can it inhibit the phosphorylation of JNK and P38.In FXR-/-mice,SS loses its role in protecting APAP-induced oxidative stress liver injury through antioxidant capacity.Arachidonic acid metabolism inflammation medium,which is the important mechanism of the inflammatory response,APAP mediated inflammatory mediators arachidonic acid metabolism,meanwhile increase the production and mRNA expression of inflammation factors such as IL-l?,TNF a,IL-6(p<0.01 or 0.05),increase the expression of IL-6,CRP,F4/80+CDllb+/CD45+in liver tissue,however,in FXR-/-mice,SS did not increase the inflammation factors increase.In addition,APAP-induced nuclear transposition of NF-?B in FXR-/-mice,SS fails to inhibit nuclear transposition of NF-?B and loses its role in protecting APAP-induced inflammatory liver injury by mediating the inflammatory response in liver tissue.In addition,in FXR-/-mice,SS can't activate the expression of FXR pathway,losing some regulation of ? phase metabolic enzymes and transporters such as BSEP,LGT1A1,SLC10al,SLC0lal,GSTA1 and OST?,not by I phase metabolic enzymes,couldn't improve APAP toxicity induced by liver damage.4.HFD-induced mouse NAFL model in mice was established,that induce the increase of TG and Ch in serum,while the intervention of SS decreased the increase of TG and Ch(p<0.01 or 0.05).Therefore,SS reduced the increase of lipid content in mice induced by HFD.HFD significantly increased the content of ALT and AST in serum(p<0.01 or 0.05),and increased hepatic steatosis and inflammatory cell infiltration.After SS intervention,the content of AST in serum was significantly reduced(p<0.01 or 0.05),which alleviated liver steatosis and inflammatory cell infiltration,and protected liver tissue injury induced by HFD.Next,the protective mechanism of SS was evaluated.The expression of SREBP1 mRNA and protein in liver tissues of mice was reduced by HFD.However,after SS intervention,FXR was activated in liver tissues to inhibit the expression of SREPB1 and play a role in improving HFD-induced NAFL injury.In addition,OA-induced lipid accumulation model in Huh-7 cells was established,in which the intracellular TG content increased and the lipid droplets significantly aggregated.After the intervention of SS,the increased intracellular TG content induced by OA was reduced(p<0.01 or 0.05),and the Huh-7 cell lipid droplet accumulation induced by OA was reduced.In addition,SS activates FXR to inhibit the expression of SREPB1,which plays a role in reducing intracellular lipid accumulation.ConclusionSS can inhibit the activity of JNK and ROS release by reducing APAP-induced oxidative stress.Activation of FXR inhibited the activity of NF-?B,reduced the production of inflammatory factors and inhibited excessive inflammatory response,providing a protective effect for APAP-induced hepatotoxic therapy.In addition,as an FXR agonist,SS can reduce the expression of SREBP1 by activating the FXR signaling pathway,reduce the HFD-induced TG and Ch content,reduce metabolic stress,and reduce liver damage.