Wang B Y, Zhao W, Niu X M, Du J H, Fu N, Zhao S X, Wang Y, Wang R Q, Zhang Y G, Nan Y M
Department of Traditional and Western Medical Hepatology, The Third Hospital of Hebei Medical University, Shijiazhuang 050051, China.
Zhonghua Gan Zang Bing Za Zhi. 2017 May 20;25(5):365-370. doi: 10.3760/cma.j.issn.1007-3418.2017.05.011.
To investigate the role and mechanism of action of Yiqi Huoxue Recipe (YQHXR) in regulating autophagy and reversing liver fibrosis in rats with carbon tetrachloride (CCl4)-induced liver fibrosis. Healthy male Wistar rats were intraperitoneally injected with a mixture of CCl4 (30%) and olive oil (70%) twice a week for 8 weeks to establish a rat model of liver fibrosis. The rats administered normal diet were used as control group. Furthermore, YQHXR or Fuzheng Huayu Recipe (FZHYR) was intragastrically administered to the rats. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using an automatic biochemical analyzer. Hematoxylin-eosin (HE) staining and Masson staining were performed to observe the degree of fibrosis in rat liver. The protein expression of α-smooth muscle actin (α-SMA) and type I collagen α1 chain (Col1A1) in liver tissue was measured by immunohistochemistry. Furthermore, the mRNA and protein expression of α-SMA, Col1A1, autophagy-related protein 7 (Atg7), microtubule-associated protein 1 light chain 3 (LC3), and ubiquitin-binding protein (SQSTM1/p62) were determined using qRT-PCR and Western blotting, respectively. Comparison between multiple groups was made by one-way analysis of variance, and comparison between any two groups was made using the LSD test. < 0.05 was considered as statistically significant. The YQHXR group and FZHYR group had significantly lower serum levels of ALT and AST than the model group (ALT: 66.8±10.42 U/L and 73.2±10.33 U/L vs 106.80±18.24 U/L, = 31.672, < 0.001; AST: 122.6±16.65 U/L and 125.4±16.92 U/L vs 278.4±66.14 U/L, = 25.539, < 0.001). The pathological grades of hepatic fibrosis were S5.64±0.22, S3.70±0.35, and S3.90±0.34 in the model group, YQHXR group, and FZHYR group, respectively ( = 362.188, < 0.001). Compared with the control group, the YQHXR group and FZHYR group had significantly reduced mRNA and protein expression of α-SMA, Col1A1, Atg7, and LC3B and significantly increased expression of p62 (all < 0.05), and the differences were greatest in the YQHXR group. YQHXR and FZHYR can prevent or reverse liver fibrosis by regulating hepatocyte autophagy and inhibiting hepatic stellate cell activation and collagen deposition.
探讨益气活血方(YQHXR)在调节四氯化碳(CCl4)诱导的肝纤维化大鼠自噬及逆转肝纤维化中的作用及作用机制。将健康雄性Wistar大鼠每周2次腹腔注射CCl4(30%)与橄榄油(70%)的混合物,连续8周,建立大鼠肝纤维化模型。给予正常饮食的大鼠作为对照组。此外,对大鼠进行YQHXR或扶正化瘀方(FZHYR)灌胃。采用自动生化分析仪测定血清丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)水平。进行苏木精-伊红(HE)染色和Masson染色以观察大鼠肝脏纤维化程度。通过免疫组织化学法检测肝组织中α-平滑肌肌动蛋白(α-SMA)和I型胶原α1链(Col1A1)的蛋白表达。此外,分别采用qRT-PCR和Western印迹法测定α-SMA、Col1A1、自噬相关蛋白7(Atg7)、微管相关蛋白1轻链3(LC3)和泛素结合蛋白(SQSTM1/p62)的mRNA和蛋白表达。多组间比较采用单因素方差分析,任意两组间比较采用LSD检验。P<0.05认为差异有统计学意义。YQHXR组和FZHYR组血清ALT和AST水平均显著低于模型组(ALT:66.8±10.42 U/L和73.2±10.33 U/L vs 106.80±18.24 U/L,F = 31.672,P<0.001;AST:122.6±16.65 U/L和125.4±16.92 U/L vs 278.4±66.14 U/L,F = 25.539,P<0.001)。模型组、YQHXR组和FZHYR组肝纤维化病理分级分别为S5.64±0.22、S3.70±0.35和S3.90±0.34(F = 362.188,P<0.001)。与对照组比较,YQHXR组和FZHYR组α-SMA、Col1A1、Atg7和LC3B的mRNA和蛋白表达均显著降低,p62表达显著升高(均P<0.05),且YQHXR组差异最明显。YQHXR和FZHYR可通过调节肝细胞自噬、抑制肝星状细胞活化和胶原沉积来预防或逆转肝纤维化。