Dangfei Liganning Capsules Attenuate The Susceptibility Of Liver Injury In The Rat With Nonalcoholilc Fatty Liver Disease To Carbon Tetrachloride Toxicity Through Regulation Of SIRT1 Signaling Pathway

Objective:Based on the SIRT1 signaling pathway,this study is to investigate the underlying mechanism of the improving effect of Dangfei Liganning Capsules on the susceptibility of liver injury in the rat with nonalcoholilc fatty liver disease?NAFLD?to carbon tetrachloride?CCl₄?toxicity.Methods:Male Wistar rats were randomly divided into normal control?N?group,normal+CCl₄?N+CCl₄?group,NAFLD model?HF?group,NAFLD+CCl₄ model?HF+CCl₄?group and Dangfei Liganning Capsules treatment?DF?group.Rats of C group and N+CCl₄group were fed with normal diet,while rats of other group were fed with high fat diet?88% nomal chow+10% lard+2% cholesterol?.At the same time,Dangfei Liganning Capsules were administered through gavage to the rats of DF group everyday with equivalent dose conversion for adult.After 8 weeks,rats of N+CCl₄ and NF+CCl₄group were intraperitoneally injected with low dose of CCl₄.All rats were sacrificed after 48 hours.Serum and liver tissues were collected.The changes of liver histopathology,as well as serum alamine-aminotrasferas?ALT?,aspartateaminotransferase?AST?,total cholesterol?TC?,triglyceride?TG?,high density lipid cholesterol?HDL-c?and low density lipid cholesterol?LDL-c?were detected.Hepatic mRNA expression of IL-1?,MCP-1,SUV39H2 was detected by q RT-PCR assay.Protein expression of SUV39H2,SIRT1,p65,acetylated p65,tumor suppressor factor p53,acetylated P53,cleaved PARP protein,pro-apoptotic protein PUMA and antiapoptotic protein BCL-x L were detected by Western blot.Results:HE staining of liver tissue showed that small dose CCl₄ did not cause pathological changes in normal rat liver,large area fatty degeneration of hepatic lobules appeared in NAFLD model group,and steatosis was more serious in HF+CCl₄ group,with hepatocyte balooning and inflammatory cell infiltration.The steatosis,ballooning degeneration and inflammatory infiltration in DF group were obviously alleviated.Oil red staining of liver tissue also showed that the accumulation of lipid in HF and HF+CCl₄ group.The lipid accumulation was improved in DF group.The level of serum ALT and AST showed no obvious difference in rats of N and N+CCl₄ group,but higher in rats of HF+CCl₄ than HF group?P<0.05?.With treatment of DF,rats showed decreased serum ALT and AST than rats from HF+CCl₄ group?P<0.01?.The results suggested that the sensitivity of NAFLD rats to toxic substances was increased and Dangfei Liganning Capsules could improve the decrease of liver function induced by CCl₄ induced by NAFLD.There was no significant difference in serum HDL-c level between normal group and normal CCl₄ group.The level of HDL-c in HF group,HFCCl₄ group and DF group was lower than that in N group?P<0.05?,but there was no significant difference between DF group and HF-CCl₄ group.The serum levels of TG,LDL-c in HF group and HF-CCl₄ group were higher than those in N group and N-CCl₄ group,and the serum TG,LDL-c level in DF group was lower than that in HF-CCl₄ group compared with HF-CCl₄ group?P<0.05?.The levels of serum TC in HF group and HF-CCl₄ group were higher than those in N group and N-CCl₄ group?P<0.05?.The pro-inflammatory factors IL-1? and MCP-1 mRNA levels in liver tissues of HF and HF+CCl₄ group was significantly higher than N and N+CCl₄ group?P<0.05?,while they were down-regulated in DF group.The hepatic expression of SUV39H2 mRNA showed no obvious difference in the rats of versus N group,but higher in the rats of HF and HF+CCl₄ group compared with N group.And it was significantly reduced in DF group.Western blot results displayed the protein level of SIRT1 in liver tissues of HF and HF+CCl₄ group was decreased compared with N and N+CCl₄ group,while up-regulated significantly by DF treatment.In addition,the liver expression of p65,p53 and acetylated p65,acetylated p53 protein in the rats of NF and NF+CCl₄ group was obviously higher than N and N+CCl₄ group,and acetylated p65,acetylated p53 was even higher in HF+CCl₄ group?P<0.05?.Both p65,p53 and acetylated p65,acetylated p53 were decreased significantly in DF group?P<0.05?.At the same time,the expression of PUMA,apoptosis marker CleavedPARP and anti-apoptosis factor BCL-x L protein downstream of SIRT1/p53 signaling pathway was further investigated.It was found that PUMA protein and Cleaved-PARP were highly expressed in HF group and HF+CCl₄ group.The expression of PUMA and Cleaved PARP protein in DF group was lower than that in HF+CCl₄ group.The expression level of BCL-x L protein in HF group and HF+CCl₄ group was lower than that in N group and N+CCl₄ group?P<0.05?,and DF group could up-regulate Expression level of BCL-x L protein in HF+CCl₄ groups?P<0.05?.Conclusions:Dangfei Liganning Capsules could improve the susceptibility of rat with NAFLD to toxicants.Down-regulating SUV39H2,up-regulating SIRT1 and thereby inhibiting activation of NF-?b signaling pathway and p53 signaling pathway may partly contribute to the underlying mechanism.