和血柔肝方通过PI3K/Akt/NF-κB信号通路调控自噬相关蛋白改善大鼠肝纤维化机制研究

Mechanism of Hexue Rougan decoction in relieve liver fibrosis in rats by regulating autophagy-related proteins through PI3K/Akt/NF-κB signaling pathway

目的:探讨和血柔肝方通过调控磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)/核因子-κB(NF-κB)信号通路调控自噬相关蛋白改善大鼠肝纤维化的作用机制。方法:将40只SPF级SD雄性大鼠随机分为空白对照组、模型组、秋水仙碱组与和血柔肝方最佳剂量组,每组各10只。除空白对照组外,其余各组均以四氯化碳(CCl_(4))构建肝纤维化大鼠模型。造模8周后,空白对照组、模型组采用0.9%氯化钠溶液灌胃;秋水仙碱组采用秋水仙碱灌胃;和血柔肝方最佳剂量组以和血柔肝方灌胃。灌胃结束后,取各组大鼠血清及肝脏组织。生化检测各组大鼠血清天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)水平;HE及Masson染色评估大鼠肝纤维化情况。RT-qPCR法检测大鼠肝组织PI3K、Akt、NF-κB以及NF-κB下游自噬相关蛋白微管相关蛋白轻链3Ⅱ(LC3-Ⅱ)、Beclin-1的mRNA的表达水平;蛋白免疫印迹(Western blot)检测大鼠肝组织中PI3K、p-PI3K、Akt、p-Akt、NF-κB P65、p-NF-κB P65、LC3-Ⅱ、Beclin-1蛋白表达水平。结果:与模型组比较,和血柔肝方最佳剂量组血清ALT、AST水平明显降低(P<0.05)。与模型组比较,和血柔肝方最佳剂量组大鼠肝脏病理受损可见明显缓解。与模型组比较,和血柔肝方最佳剂量组大鼠肝组织PI3K、Akt、NF-κB、LC3-Ⅱ、Beclin-1基因表达水平明显降低(P<0.05)。与模型组比较,和血柔肝方最佳剂量组的PI3K、Akt、p-Akt、NF-κB P65、p-NF-κB P65、LC3-Ⅱ、Beclin-1蛋白表达Western blot结果均显著降低(P<0.05)。结论:和血柔肝方可通过抑制肝纤维化大鼠肝组织PI3K/Akt/NF-κB信号通路的表达以降低自噬相关蛋白活化,从而改善大鼠肝脏炎症,减缓肝脏病理损害,改善肝纤维化。

Objective:To investigate the mechanism of Hexue Rougan decoction in improving liver fibrosis in rats by regulating phosphatidylinositol 3 kinase/protein kinase B/nuclear factor-κB signaling pathway and regulating autophagy-related proteins.Methods:Forty SPF male SD rats were randomly divided into blank control group,model group,colchicine group and optimal dose of Hexue Rougan decoction group,with 10 rats in each group.Except for the blank control group,the rats models of liver fibrosis were established with carbon tetrachloride(CCl_(4)) in the other groups.After 8 weeks of modeling,the blank control group and the model group were given normal saline by gavage.The mice in the colchicine group were treated with colchicine by gavage.The optimal dose of Hexue Rougan decoction group was gavaged with Hexue Rougan decoction.At the end of gavage,serum and liver tissue were collected from each group.The serum levels of aspartate aminotransferase(AST) and alanine aminotransferase(ALT) were detected by biochemical methods.HE and Masson staining were used to evaluate liver fibrosis in rats.RT-qPCR was used to detect the mRNA expression levels of PI3K,Akt,NF-κB and NF-κB downstream autophagy-related proteins microtubule-associated protein light chain 3Ⅱ(LC3-Ⅱ) and Beclin-1 in rat liver tissue.Western blot was used to detect the protein expression levels of PI3K,p-PI3K,Akt,p-Akt,NF-κB P65,p-NF-κB P65,LC3-Ⅱ and Beclin-1 in rat liver tissue.Results:Compared with the model group,the levels of ALT and AST in the optimal dose of Hexue Rougan decoction group were significantly decreased(P<0.05).Compared with the blank control group,the liver pathological damage of the optimal dose of Hexue Rougan decoction group was significantly alleviated.Compared with the model group,the expression levels of PI3K,Akt,NF-κB,LC3-Ⅱ and Beclin-1 genes in liver tissue of rats in the optimal dose of Hexue Rougan decoction group were significantly decreased(P<0.05).Compared with the model group,PI3K,Akt,p-Akt,NF-κB P65,p-NF-κB P65,LC3-Ⅱ and Beclin-1 in the optimal dose of Hexue Rougan decoction group were significantly decreased(P<0.05).Conclusion:Hexue Rougan decoction can inhibit the expression of PI3K/Akt/NF-κB signaling pathway in liver tissue of liver fibrosis rats and reduce the expression of autophagy-related proteins,thereby improving liver inflammation,reducing liver pathological damage and liver fibrosis in rats.