益气活血颗粒抗肝纤维化的分子机制研究

Study on the Molecular Mechanism of Yiqi Huoxue Granules Against Hepatic Fibrosis

【目的】探讨益气活血颗粒对大鼠肝纤维化的治疗作用及机制。【方法】将60只雄性SD大鼠随机分为正常组、模型组、异甘草酸镁组、益气活血颗粒组及罗格列酮组、益气活血颗粒+罗格列酮组[后2组仅进行过氧化物酶体增殖物激活受体γ(PPARγ)蛋白检测],每组10只。除正常组,其他各组大鼠采用四氯化碳(CCl_(4))法诱导构建肝纤维化模型。对应治疗10周后,苏木素-伊红(HE)染色法观察肝组织病理变化,采用放射免疫分析法检测血清肝纤维化指标透明质酸(HA)、Ⅲ型前胶原(PCⅢ)和Ⅳ型胶原(CⅣ)水平,采用末端脱氧核苷酸标记(TUNEL)法检测肝细胞凋亡,应用试剂盒检测肝组织中氧化应激相关指标超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽(GSH)水平,Western Blot法检测肝组织内质网应激相关指标葡萄糖调节蛋白78(GRP78)、蛋白激酶R样内质网激酶(PERK)、肌醇需求酶1α(IRE1α)、活化转录因子6(ATF6)蛋白表达,免疫组织化学法检测肝组织PPARγ蛋白阳性表达。【结果】与正常组比较,模型组可见明显肝纤维化病理改变,HA、PCⅢ、CⅣ水平显著升高(P<0.05),SOD、GSH活性显著降低(P<0.05),MDA含量显著增加(P<0.05),肝细胞凋亡率升高(P<0.05),GRP78、PERK、IRE1α、ATF6蛋白表达水平升高(P<0.05),PPARγ蛋白阳性表达水平降低(P<0.05);与模型组比较,益气活血颗粒组和异甘草酸镁组大鼠肝纤维化病理得到明显改善,HA、PCⅢ、CⅣ水平显著降低(P<0.05),SOD、GSH活性显著升高(P<0.05),MDA含量显著减少(P<0.05),肝细胞凋亡率降低(P<0.05),GRP78、PERK、IRE1α、ATF6蛋白表达水平降低(P<0.05),PPARγ蛋白阳性表达水平升高(P<0.05);益气活血颗粒组上述各指标的作用效果均优于异甘草酸镁组(均P<0.05)。其中在PPARγ指标方面,益气活血颗粒+罗格列酮组其蛋白阳性表达水平高于罗格列酮组(P<0.05)。【结论】益气活血颗粒可显著改善CCl_(4)诱导的大鼠肝纤维化,其机制与抑制氧化应激、内质网应激反应及促进PPARγ活化,进而减轻肝细胞凋亡有关,体现了“扶正祛瘀”治则。

Objective To investigate the therapeutic effect and mechanism of Yiqi Huoxue Granules on rats with hepatic fibrosis.Methods Sixty male SD rats were randomly divided into normal group,model group,magnesium isoglycyrrhizinate group,Yiqi Huoxue Granules group and Rosiglitazone group,and Yiqi Huoxue Granules group+Rosiglitazone group[only peroxisome proliferator-activated receptorγ(PPARγ)protein assay was carried out for the latter two groups],with 10 rats in each group.Except for the normal group,rats in all other groups were induced by carbon tetrachloride(CCl_(4))method to construct a liver fibrosis model.After 10 weeks of corresponding treatment,pathological changes of liver tissue were observed by hematoxylin-eosin(HE)staining method,serum hepatic fibrosis indexes hyaluronic acid(HA),typeⅢpre-collagen(PCⅢ)and typeⅣcollagen(CⅣ)levels were detected by radioimmunoassay,apoptosis of hepatocytes was detected by terminal deoxyribonucleotide labelling(TUNEL)method,and oxidative stress-related indexes of superoxide dismutase(SOD),malondialdehyde(MDA),glutathione(GSH)levels in liver tissue were detected by the kit,Western Blot was used to detect endoplasmic reticulum stress-related indicators of glucose-regulated protein 78(GRP78),protein kinase R-like endoplasmic reticulum kinase(PERK),inositol-requiring enzyme 1α(IRE1α),andactivatedtranscriptionfactor 6(ATF6)protein expression in liver tissue,and immunohistochemistry was applied for the detection of positive expression of PPARγprotein in liver tissue.Results Compared with the normal group,obvious pathological changes of liver fibrosis could be seen in the model group,with significantly higher levels of HA,PCⅢand CⅣ(P<0.05),significantly lower activities of SOD and GSH(P<0.05),significantly higher MDA content(P<0.05),higher rate of apoptosis of hepatocytes(P<0.05),and elevated levels of protein expression of GRP78,PERK,IRE1αand ATF6(P<0.05),and PPARγprotein positive expression level was reduced(P<0.05);compared with the model group,the pathology of hepatic fibrosis in rats in the Yiqi Huoxue Granules group and magnesium isoglycyrrhizate group was significantly improved,with significantly reduced levels of HA,PCⅢand CⅣ(P<0.05),significantly elevated activities of SOD and GSH(P<0.05),significantly reduced MDA content(P<0.05),hepatocyte apoptosis rate was reduced(P<0.05),protein expression levels of GRP78,PERK,IRE1αand ATF6 were reduced(P<0.05),and positive expression level of PPARγprotein was increased(P<0.05);the effect of the above indicators in the group of Yiqi Huoxue Granules was superior to that of the magnesium isoglycyrrhizinate group(all P<0.05).In addition,the positive expression of PPARγprotein in the group of Yiqi Huoxue Granules+Rosiglitazone was higher than that in the Rosiglitazone group(P<0.05).Conclusion Yiqi Huoxue Granules significantly improved CCl_(4)-induced hepatic fibrosis in rats,the mechanism of which was related to the inhibition of oxidative stress,the endoplasmic reticulum stress response and the promotion of PPARγactivation,which in turn reduced the apoptosis of hepatocytes,reflecting the therapeutic principle of“supporting the healthy qi and eliminating blood stasis”.