The Effect And Mechanism Of Babao Dan On Chronic Liver Injury In Rats

BackgroundChronic liver disease prevalence is increasing globally.Many reason could cause chronic liver diseases,such as chronic viral hepatitis infection(HBV and HCV),alcoholic liver disease(ALD)and nonalcoholic fatty liver disease(NAFLD),and autoimmune liver diseases and drug-induced liver injury(DILI),and et al.Chronic liver disease often results in progressive fibrosis.Hepatic fibrosis,characterized by excessive accumulation of extracellular matrix(ECM)proteins.Advanced liver fibrosis results in irreversible cirrhosis,often companying with portal hypertension,liver function failure,high susceptibility to infection or to developed hepatocellular carcinoma(HCC).Thus,focusing on events that lead to the primarily accumulation of ECM help to develop new therapeutic targets to attenuate hepatic fibrosis.Quiescent hepatic stellate cells(HSCs)is located in Disse space and characterized by storage of retinoid.Activated HSCs are demonstrated as predominant cell produced ECM to participate in hepatic fibrosis.The ECM was consist of collagen?,?,?,laminin,and fibronectin.In addition,activated HSCs regulate the recruitment of inflammatory cells via secretion of chemotactic factors,such as interleukin 6(IL-6),transforming growth factor(TGF)-?1,tumor necrosis factor(TNF)-? and monocyte chemotactic protein(MCP)-1.Therefore,inhibition of the HSCs activation and proliferation may be an attractive method to anti-fibrotic therapy.Toll-like receptors(TLRs)were originally identified as pathogen-associated molecular pattern recognition receptors(PRR)and recognized exogenous ligands in response to infection.TLR4,in particular,conferred by the lymphocyte antigen 96 also known as MD-2,can specially response to bacterial lipopolysaccharide(LPS).The process of presentation LPS to MD-2 can also be facilitated by CD14 or the LPS-binding protein(LPB).LPS,located in the cytoplasm of Gram-negative bacteria,can be considerable absorbed in liver with increased permeability of the intestinal mucosal barrier in cirrhosis rats or patients[1].Importantly,LPS canenormously induce HSCs activation and aggravate liver fibrosis through TLR4 pathway,which has been proved to be an important mechanism in liver injury.During HSCs activation induced by LPS,TLR4 signal was activated via an adaptor molecule MyD88,leading to translocation of nuclear factor kappa B(NF-?B)with consequent of transition HSCs to myofibroblasts,and up-regulation of pro-fibrogenic and pro-inflammatory cytokines.Meanwhile,LPS can increase the expression of extracellular-related kinase(ERK)phosphorylation,which can regulate cell proliferation by Cyclin D1 or c-myc.It has been demonstrated that TLR4-mutation mice displayed a profound reduction in hepatic fibrogenesis.Thus,TLR4 inhibition can obviously down-regulate inflammation and fibrosis.These results confirm the critical role of TLR4 signaling in regulating HSCs activation and proliferation,which affect the progression of hepatic fibrosis.Babao Dan(BBD),a mixed powder of traditional Chinese medicine containing eight constituents,including natural calculus bovis,snake gall,antelope horn,pearl,musk,radix notoginseng and so on.The formula of BBD was protected by Chinese Food and Drug Administration(CFDA).It has been widely used as a complementary and alternative medicine to treat chronic liver diseases.However,the function and mechanism of BBD in treating hepatic fibrosis are still unclear.ObjectiveWe aim to detect the protective function of BBD to hepatic fibrosis induced by DEN.Meanwhile,we will observe the influence of BBD on live fibrosis.Subsequently,we will explore the potential molecular mechanism.MethodFirstly,we established a diethylnitrosamine(DEN)-induced hepatic fibrosis model in rats.BBD was gavaged at a dose of 0.5mg/kg bw,once every two days at the same time of DEN administration.Serum ALT and AST were detected as an index to evaluate liver injury.We next examined inflammatory cytokines such as IL-6,TGF-?1,TNF-? and MCP-1 in fresh liver tissues were detected by RT-PCR and in peripheral vein serum was also detected by ELISA to reflect the potential effect of BBD on hepatic inflammatory infiltration.We then explore the influence of BBD on hepatic fibrosis.Masson's trichrome stain and Sirius-red stain were performed to assess deposition of collagen.To explore the possible mechanism,primary HSCs were isolated and cultured in plastic cell culture dishes.In addition,we collected portal vein serum from normal rats(normal-serum)and BBD treated rats(BBD-serum).In the LPS-induced HSCs activation system in vitro,BBD-serum was added at different concentration.Cellular immunoflurescence(IF)staining was performed for HSCs activation by staining ?-SMA.In addition,the mRNA expression of ?-SMA,collagen I and inflammatory cytokines such as IL-6,TGF-?1,TNF-? and MCP-1 were detected by RT-PCR.At the same time,to determine the effects of BBD-serum on inhibition of HSCs proliferation,CCK-8 assay was performed.ResultsWe demonstrated that BBD could ameliorate liver injury and fibrosis in rat hepatic fibrosis model induced by diethylnitrosamine(DEN),and have no obvious side effect in normal rat livers.We also found that BBD did not influence the absorption of LPS in liver by analysing serum from portal vein.Meanwhile,we firstly illustrated BBD could inhibit LPS-induced HSCs activation and proliferation in vitro through TLR4 signaling pathway.ConclusionBBD could protect liver from DEN-insult,and could obviously decrease hepatic fibrosis by TLR4 pathway.Collectively,BBD may be a novel therapeutic choice for hepatic fibrosis.