Inhibitive Effect Of Sophocarpine On Hepatic Fibrosis In Vivo And In Vitro

Background and objectivesHepatic fibrosis is the critical stage of chronic liver diseases.Factors including viruses, alcohol,parasites,copper and iron deposition,toxicants and cholestatis chronically affect liver,consequently cause chronic hepatic injury.The persistent injuries induce activation of hepatic stellate cells(HSCs),which transform into myofibroblast(MFB) and cause an increasing synthesis of extracellular matrix(ECM) mainly including collagenâ… ,â…¢. Excess deposition of ECM causes hepatic fibrosis.Transforming growth factor-β₁(TGF-β₁) play an important role in the activation of HSC and deposition of ECM.Recent studies on reversion of hepatic fibrosis developed dramatically.Professor Roking pointed out that human typical hepatic fibrosis could be reversed.Basing on this viewpoint,studies have been focused on the mechanism of hepatic fibrosis and treatment of hepatic fibrosis in this decade.There are few effective drugs to attenuate hepatic fibrosis.We still could not make sure of the therapeutic effect of colchicin,glucocorticoid, penicillamine on hepatic fibrosis with severe adverse reactions.Some studies on Chinese traditional herbs and their monomers to treat hepatic fibrosis have been highly evaluated.It is found that some herbs could inhibit hepatic fibrosis.Sophocarpine(SC) is a kind of sophorae alkaloids derived from foxtail-like sophora herb and seed.Sophorae alkaloids are widely clinically used and have a significantly inhibitive effect on cirrhotic pathogenesis,virus replication and liver inflammation. Recently,Studies have proved that SC can inhitive Coxsackie Virus B replication and reinforce immune function.In vitro SC of above 100ug/ml have a notable effect against SARS virus.In addition,an intensively inhibitive effect of SC has been observed on the secretion of both HBsAg and HBeAg in HepG2.2,15 in vitro,which is better in inhibition ratio compared with Lamivudine.Whereas,there are few reports about anti-fibrosis of SC in liver.This study was to investigate the conceivable function and latent mechanism of SC in preventing both DMN- and BDL-induced hepatic fibrosis in rats.Methods1.experiments in vivoHepatic fibrosis models in rats were induced by Dimethyl nitrosamine(DMN) and bile duct ligation(BDL).Both model rats were treated with SC,and then sacrificed at three weeks after the treatment.The value of alanine aminotransferase(ALT) and total bilirubin (TB) were detected in the serums of each group.The contents of Hydroxyproline(HP) in hepatic tissue were detected by alkaline hydrolysis.Liver tissue samples were used for histopathological examinations by staining of HE,for determining the content of collagen fiber by staining of Masson.Protein expression of a-smooth muscle actin(a-SMA) and collagen I was examinated by immunohistochemistry.The mRNA expression of alpha-SMA,TGF-β₁and alph 1 typeâ… procollagen were messured by reverse transcriptase PCR(RT-PCR).2.experiments in vitroHepatic stellate cells(HSCs) in rats were isolated with collagenase in situ liver recirculating perfusion established by Pro Fredmen with some modifications. Immunocytochemistry of Desmin staining was used to detect the purity of primarily isolated HSCs.The mRNA expression of a-SMA,TGF-β₁and alph 1 typeâ… procollagen were messured by reverse transcriptase PCR(RT-PCR).The proliferation of passaged HSCs was detected by Cell Counting Kit-8(CCK-8) and 5-bromo 2-deoxyuridine(BrdU).Results1.The serum levels of ALT and AST in the SC-treated rats were lower than those in model rats(P<0.05).2.The grade of hepatic fibrosis was indicated by HE staining.The grade of SC-treated rats was lower than that of model rats(P<0.05).The contents of collagen fiber determined by staining of Masson in SC-treated rats were lower than those of model rats(area ratio:19.73%±1.39%in DMN model group v.s 8.76%±0.73%in SC-treated DMN group, P<0.05;16.45%±1.57%in BDL model group v.s 9.98%±0.71%in SP-treated BDL group, P<0.05).The contents of Hydroxyproline(HP) in SC-treated rats were lower than those in model rats(361.17±20.55ug/g in DMN model group v.s 261.17±20.45 ug/g in SC-treated DMN group,P<0.05;259.33±23.18 ug/g in BDL model group v.s 163±17.03ug/g in SC-treated BDL group,P<0.05).3.The mRNA expression of alpha -SMA,TGF-β₁ and alpha 1 typeâ… procollagen messured by RT-PCR were reduced in SC-treated groups(P<0.05).Protein expression of alpha -SMA examinated by immunohistochemistry was declined in SC-treated groups.4.Compared with HSCs stimulated by TGF-β₁,SC at both 200mg/L and 400mg/L inhibited the gene expressions of alpha-SMA,TGF-β₁ and alpha 1 typeâ… procollagen (P<0.05).Protein expression of alpha-SMA in SC-treated group was lower than control group.5.SC at 400～1000mg/L inhibited the proliferation of passaged HSCs messured by CCK-8 and BrdU,compared with control HSCs.Conclusions1.Sophocarpinc could allievate liver injuries of hepatic cirrhotic rat and attenuated the pathogenesis of hepatic fibrosis.2.Sophocarpine could inhibit the expression of alpha-SMA,TGF-β₁ and alpha 1 typeâ… procollagen in cirrhotic liver and HSCs.Sophocarpine could inhibite the proliferation of activated HSCs.3.The inhibitive effects on activation and proliferation of HSCs perhaps were the cirrhotic-attenuated mechanism of Sophocarpine.