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Title: [Mechanism of action of Yiqi Huoxue Recipe in regulating autophagy and reversing liver fibrosis].
Author: Wang BY, Zhao W, Niu XM, Du JH, Fu N, Zhao SX, Wang Y, Wang RQ, Zhang YG, Nan YM.
Journal: Zhonghua Gan Zang Bing Za Zhi; 2017 May 20; 25(5):365-370. PubMed ID: 28763844.
Abstract:
Objective: 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. Methods: 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. P < 0.05 was considered as statistically significant. Results: 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, F = 31.672, P < 0.001; AST: 122.6±16.65 U/L and 125.4±16.92 U/L vs 278.4±66.14 U/L, F = 25.539, P < 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 (F = 362.188, P < 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 P < 0.05), and the differences were greatest in the YQHXR group. Conclusion: YQHXR and FZHYR can prevent or reverse liver fibrosis by regulating hepatocyte autophagy and inhibiting hepatic stellate cell activation and collagen deposition. 目的： 探明自拟益气活血方在四氯化碳诱导的肝纤维化动物模型中调节肝脏自噬及阻止肝纤维化的作用及其机制。 方法： 选用健康雄性Wistar大鼠，以30%四氯化碳橄榄油溶液腹腔注射建立肝纤维化模型，分别以益气活血方和扶正化瘀复方冲剂灌胃进行干预实验，以正常饮食设立对照组。检测血清丙氨酸氨基转移酶、天冬氨酸氨基转移酶水平；HE、Masson染色观察大鼠肝组织纤维化程度；免疫组织化学检测肝组织α-平滑肌肌动蛋白（α-SMA）和I型胶原α1链（Col1A1）蛋白的表达；qRT-PCR和Western blot检测肝组织α-SMA、Col1A1、自噬蛋白7（Atg7）、微管相关蛋白1轻链3（LC3）及泛素结合蛋白（p62）mRNA及蛋白的表达。多组间比较采用单因素方差分析，组间两两比较采用LSD检验，P < 0.05为差异有统计学意义。 结果： 益气活血方组大鼠血清丙氨酸氨基转移酶及天冬氨酸氨基转移酶水平分别为（66.8±10.42）U/L、（122.6±16.65）U/L，扶正化瘀复方组大鼠分别为（73.2±10.33）U/L，（125.4±16.92）U/L，均明显低于模型组大鼠的（106.80±18.24）U/L、（278.4±66.14）U/L，F值分别为31.672和25.539，P值均< 0.001，差异均有统计学意义。模型组大鼠肝纤维组织增生病理分级为S5.64±0.22，与益气活血方组和扶正化瘀复方组的S3.70±0.35，S3.90±0.34比较，F = 362.188，P < 0.01，差异有统计学意义。与模型组比较，益气活血方组和扶正化瘀复方组肝纤维化相关基因α-SMA、Col1A1及肝脏自噬相关基因Atg7、LC3B的mRNA及蛋白相对表达量均显著减低，而泛素结合蛋白表达明显增高，差异均有统计学意义（P值均< 0.05），尤以益气活血方组效果明显。 结论： 益气活血方和扶正化瘀复方均可通过调节肝脏细胞自噬，抑制肝星状细胞活化及胶原沉积，阻止或逆转肝纤维化。.

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