虫草菌丝逆转二甲基亚硝胺诱导大鼠肝纤维化的有效组分及其作用机制

Actions of Cordyceps sinensis Berk. Sacc and its Effective Components on Reversing Liver Fibrosis Induced by Dimethylnitrosamine in Rats

目的:研究虫草菌丝治疗二甲基亚硝胺(DMN)模型大鼠肝纤维化的有效组分及其作用机制。方法:大鼠ipDMN 5 mg.kg-1,每周连续3d,共4周。造模结束后,模型大鼠随机分组,治疗组分别以虫草菌丝(CsB)及其组分(C12)以生药800,5 mg.kg-1 ig,每日1次,共2周;正常组及模型组以等量生理盐水ig。分别检测血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)活性,检测肝组织羟脯氨酸(Hyp)、丙二醛(MDA)含量,超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)和谷胱甘肽-S转移酶(GST)活性,观察肝组织胶原沉积变化,免疫组织化学和蛋白印迹法(Western)检测α-平滑肌肌动蛋白(α-SMA),转化生长因子-β1(TGF-β1)和图像分析肿瘤坏死因子-α(TNF-α)阳性面积百分比,观察肝组织病理及胶原沉积变化。结果:与模型组比较,CsB及C12治疗组大鼠血清ALT,AST活性显著降低(P<0.05)。与6周模型组比较,CsB及C12治疗组大鼠肝组织Hyp含量显著降低(P<0.05)。模型组大鼠肝组织可见炎性细胞浸润,胶原增生明显,大多数形成较厚的完全间隔,假小叶形成。CsB及C12治疗组的坏死出血、炎性细胞浸润、胶原增生、纤维间隔形成有所改善。与6周模型组相比,CsB及C12治疗组的SOD,GSH-Px活性显著升高,MDA含量,GST活性显著降低(P<0.01)。与6周模型组比较,CsB及C12治疗组大鼠肝组织α-SMA,TGF-β1及TNF-α的蛋白表达显著减少(P<0.01)。结论:CsB及C12能够显著改善肝功能,有显著抗肝纤维化作用。CsB及C12能够显著减轻过氧化损伤,保护肝细胞,减少胶原生成,阻断和逆转二甲基亚硝胺大鼠肝纤维化。CsB和C12抑制库普弗细胞(KCs)活化和细胞因子TNF-α,TGF-β1的释放,抑制肝星状细胞(HSCs)活化,合成和分泌细胞外基质。

Objective：To study the effect and mechanism of Cordyceps sinensis Berk Sacc（CsB） and its effective components on liver fibrosis induced by dimethylnitrosamine（DMN） in rats.Method： From the 1st to 4th week,rats were treated with intraperitoneal injection of DMN（5 mg.kg-1） for three continuous days each week.After modeling,normal group and model group were given saline（10 mL.kg-1） intragastrically,and rats in drug treatment group were treated separately with CsB and its componentsC12（800 mg.kg-1.d-1and 5 mg.kg-1.d-1） for 2 weeks.All the rats were sacrificed to harvest blood and liver tissue sample.Serum alanine aminotransferase（ALT） and aspartate transferase（AST） were detected,maleic dialdehyde（MDA）,superoxide dismutase（SOD）,glutathione peroxidase（GSH-Px） and glutathiones s transferase（GST） in liver tissue were assayed biochemically.Content of hydroxyproline（Hyp） in liver tissue was assayed biochemically;Histological changes with collagen deposition with sirus red staining in rat liver tissue were observed.Expression of α-smooth muscle action（α-SMA）,transforming growth factor-β1（TGF-β1）,hepatocyte growth factor（HGF-α） and tumor necrosis factor-α（TNF-α） were detected immunohistologically.Result： Compared to 6 wk model group,serum ALT and AST in CsB and C12 group were lowered（P 0.05）.Compared to 6 wk model group,Hyp content in liver tissue in CsB and C12 group was decreased significantly（P 0.05）.Large area of hemorrhage and necrosis,swelling of large amount of hepatocytes,widened portal area,inflammatory cells infiltration,increased collagen deposition,and pseudolobule were seen in liver tissue in the model group.Some histological improvement was seen in CsB group and C12 group.Compared to 6 wk model group,CsB and C12 could increase SOD and GSH-Px activity,meanwhile decrease MDA and GST activity（P 0.01）.Compared to 6w model group,expression of α-SMA,TGF-β1 and TNF-α in CsB and C12 was decreased obviously（P 0.01）.Conclusion：CsB and C12 blocks development and formation of liver fibrosis induced by dimethylnitrosamine,through protecting hepatocytes to resist liver injury,decreasing synthesis of collagen,inhibiting oxidative stress.CsB and C12 inhibit KCs activation and release of cytokines,which further inhibits HSCs’activation,synthesis and secretion of ECM.