下瘀血汤抑制胶质细胞源性神经营养因子抗肝纤维化的作用机制

目的探讨下瘀血汤是否通过抑制胶质细胞源性神经营养因子(GDNF)发挥抗肝纤维化的作用。方法24只C57BL/6小鼠随机分为对照组、模型组、下瘀血汤组,每组各8只。模型组和下瘀血汤小鼠腹腔注射10%CCl4,第4周开始下瘀血汤组小鼠给予0.4678 g/kg下瘀血汤灌胃。检测肝功能指标ALT、AST水平,观测肝脏组织病理形态学。免疫组化检测平滑肌肌动蛋白(α-SMA)及GDNF蛋白表达。GDNF(10 ng/ml)处理GFP-Col-HSC和人原代肝星状细胞(HSC),检测HSC活化。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果模型组ALT和AST水平较对照组显著升高,下瘀血汤组ALT和AST水平较模型组显著降低(P值均<0.01)。肝组织病理学显示,模型组炎症细胞浸润明显,增生的胶原纤维形成纤维间隔,下瘀血汤组胶原纤维间隔较疏松及炎症细胞浸润减轻。免疫组化显示,与对照组相比,模型组α-SMA及GDNF阳性表达显著升高(P值均<0.01),均分布在纤维间隔,下瘀血汤组α-SMA与GDNF表达较模型组均显著降低(P值均<0.05)。免疫印迹结果显示,对照小鼠肝组织GDNF表达比较低,CCl4造模6周肝纤维化形成,GDNF表达上调10倍左右,下瘀血汤显著抑制模型小鼠GDNF蛋白表达(P值均<0.01);α-SMA和Ⅰ型胶原α1(Col1)表达在肝纤维化模型小鼠显著上调,下瘀血汤处理后α-SMA与Col1显著下降(P值均<0.01)。体外结果显示,GDNF可诱导HSC细胞α-SMA及Ⅰ型胶原α1蛋白表达显著上调,而下瘀血汤对此有显著抑制作用(P值均<0.01)。结论肝纤维化形成中GDNF表达显著上调,GDNF可诱导HSC活化,下瘀血汤可抑制GDNF从而抗肝纤维化。

Mechanism of action of Xiayuxue decoction in inhibiting liver fibrosis by regulating glial cell line-derived neurotrophic factor

Objective To investigate whether Xiayuxue decoction exerts an anti-liver fibrosis effect by inhibiting glial cell line-derived neurotrophic factor(GDNF).Methods A total of 24 C57BL/6 mice were randomly divided into control group,model group,and Xiayuxue decoction group.The mice in the model group and the Xiayuxue decoction group were given intraperitoneal injection of 10%CCl4,and those in the Xiayuxue decoction group were given 0.4678 g/kg Xiayuxue decoction by gavage since week 4.The liver function parameters alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were measured,and liver histopathology was observed.Immunohistochemistry was used to measure the protein expression of alpha-smooth muscle actin(α-SMA)and GDNF.GFP-Col-HSC and human primary hepatic stellate cells(HSCs)were treated with GDNF(10 ng/ml),and HSC activation was measured.A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the least significant difference t-test was used for further comparison between two groups.Results Compared with the control group,the model group had significant increases in the levels of ALT and AST,and compared with the model group,the Xiayuxue decoction group had significant reductions in the levels of ALT and AST(all P<0.01).Liver histopathology showed that the model group had marked inflammatory cell infiltration and formation of fibrous septa by proliferated collagen fibers,and the Xiayuxue decoction group had loose fibrous septa and alleviated inflammatory cell infiltration.Immunohistochemistry showed that compared with the control group,the model group had significant increases in the expression ofα-SMA and GDNF(both P<0.01),which were observed in fibrous septa,and compared with the model group,the Xiayuxue decoction group had significant reductions in the expression ofα-SMA and GDNF(both P<0.05).Western blotting showed that the control group had relatively low expression of GDNF in liver tissue,the formation of liver fibrosis at week 6 of CCl4 modeling,and an around 10-fold increase in the expression of GDNF,and the Xiayuxue decoction group had significantly inhibited protein expression of GDNF(P<0.01);there were significant increases in the expression ofα-SMA and collagen type Iα1(Col1)in mice with liver fibrosis,with significant reductions inα-SMA and Col1 after treatment with Xiayuxue decoction(all P<0.01).The in vitro experiment showed that GDNF induced the significant increases in the protein expression ofα-SMA and Col1 in HSCs,which was significantly inhibited by Xiayuxue decoction(all P<0.01).Conclusion The expression of GDNF is significantly upregulated in the formation of liver fibrosis.GDNF can induce HSC activation,and Xiayuxue decoction can exert an anti-liver fibrosis effect by inhibiting GDNF.