清肝九味散对肝纤维化大鼠Raf/MEK/ERK信号通路的影响

Effect of Qinggan Jiuwei Powder on MMPs/TIMPs and Raf/MEK/ERK Signal Pathway in Liver Cirrhosis Rats

目的观察清肝九味散对肝纤维化大鼠的干预作用及其潜机制。方法采用随机数字表法将50只SD大鼠分为正常组、模型组、阳性对照组、蒙药低剂量组及蒙药高剂量组,每组10只。除正常组外,其余各组大鼠均通过腹腔注射四氯化碳(CCl_(4))建立肝纤维化模型。造模成功后,正常组大鼠给予蒸馏水灌胃,阳性对照组灌服水飞蓟素(SIL)10 mg/kg;蒙药低、高剂量组每日分别灌服清肝九味散水煎液0.525、4.725 g/kg。各组均每天灌胃1次,连续8周。采用全自动生化仪检测血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)活性水平;HE染色法观察肝组织病理变化;免疫组化法检测肝组织平滑肌肌动蛋白(α-SMA)表达;实时荧光定量聚合酶链反应(RT-PCR)检测基质金属蛋白酶2(MMP2)、Ⅰ型胶原α(COL1α)、基质金属蛋白酶抑制剂1(TIMP1)mRNA水平;蛋白免疫印迹(Western Blot)检测肝组织MMP2、TIMP1蛋白表达及Raf蛋白激酶1(Raf-1)、丝裂原活化细胞外信号调节激酶(MEK)、细胞外调节蛋白激酶(ERK)磷酸化水平的变化。结果与正常组比较,模型组大鼠明显消瘦、暴躁,肝脏经HE染色后可见典型纤维化病理表现;阳性对照组和蒙药低、高剂量组大鼠一般状态有明显改善,肝脏病理损伤减轻,血清ALT、AST水平较模型组显著下降(P<0.01);RT-PCR结果显示:与正常组比较,模型组MMP2、COL1α、TIMP1 mRNA表达显著升高(P<0.01);与模型组比较,阳性对照组、蒙药低、高剂量组MMP2 mRNA表达明显升高,TIMP1、COL1αmRNA表达明显降低(P<0.01)。免疫组化结果显示:与正常组比较,模型组α-SMA蛋白阳性面积显著升高(P<0.01);与模型组比较,蒙药低、高剂量组α-SMA蛋白表达均显著降低(P<0.01)。Western Blot结果显示:与正常组比较,模型组MMP2、TIMP1、p-Raf、p-MEK、p-ERK蛋白表达均显著升高(P<0.05):与模型组比较,阳性对照组及蒙药高剂量组MMP2蛋白表达明显升高,阳性对照组及蒙药各剂量组TIMP1、p-Raf、p-MEK、p-ERK蛋白表达明显下降(P<0.05,P<0.01),且蒙药高剂量组较蒙药低剂量组下降更明显(P<0.01)。结论蒙药清肝九味散对CCl_(4)诱导的肝纤维化大鼠一般状态有改善作用,能减轻大鼠肝脏的纤维增生程度,并通过调节MMPs/TIMPs体系促进细胞外基质(ECM)降解,其抗肝纤维化机制可能与抑制Raf/MEK/ERK信号通路有关。

Objective To observe the therapeutic actions and possible mechanisms of Qinggan Jiuwei Powder(QGJWP)against liver fibrosis rats.Methods Fifty SD rats were divided to normal group,model group,positive control group,and high and low dose QGJWP treatment groups,10 in each group.Except the normal group,rats in the rest 4 groups were injected with 40%carbon tetrachloride olive oil solution intraperitoneally to induce liver fibrosis model.Rats in normal group were administered with distilled water by gastrogavage.After successful modeling rats in the positive control group were administered with Silymarin(SIL)10 mg/kg by gastrogavage.Rats in high and low dose QGJWP treatment groups were administered with QGJWP 0.525 and 4.725 g/kg by gastrogavage.All administrations were performed once per day for 8 successive weeks.The activity levels of serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were detected by automatic biochemical analyzer.Pathological changes of liver tissue were observed by HE staining.The expression ofα-smooth muscle actin(α-SMA)in liver tissue was detected by immunohistochemistry.mRNA levels of matrix metalloproteinase 2(MMP2),collagen typeⅠα(COL1α),tissue inhibitor of metalloproteinase 1(TIMP1)were detected by Real-time PCR.Protein expressions of MMP2 and TIMP1,phosphorylation levels of Raf-1,mitogen-activated extracellular signal-regulated kinase(MEK),extracellular regulated protein kinases(ERK)in liver tissue were detected by Western Blot.Results Compared with the normal group,rats in the model group were obviously emaciated and irritable.Typical pathological manifestations of fibrosis could be seen after HE staining.The general state was obviously alleviated,liver pathological injury was attenuated,serum levels of ALT and AST decreased significantly in the positive control group and high and low dose QGJWP treatment groups,as compared with the model group(P<0.01).Results of Real-Time PCR showed:Compared with the normal group,mRNA expressions of MMP2,COL1α,and TIMP1 significantly increased in the model group(P<0.01).Compared with the model group,mRNA expressions of MMP2 significantly increased,mRNA expressions of COL1αand TIMP1 significantly decreased in the positive control group and high and low dose QGJWP treatment groups(P<0.01).Results of immunohistochemistry showed:Compared with the normal group,α-SMA positive area significantly increased in the model group(P<0.01).Compared with the model group,α-SMA positive area significantly decreased in high and low dose QGJWP treatment groups(P<0.01).Results of Western Blot showed:Compared with the normal group,protein expressions of MMP2,TIMP1,p-Raf,p-MEK,and p-ERK significantly increased in the model group(P<0.05).Compared with the model group,MMP2 protein expression significantly increased,protein expressions of TIMP1,p-Raf,p-ERK,and p-MEK significantly decreased in positive control group and each dose QGJWP treatment group(P<0.05,P<0.01),They desreased more obviously in high dose QGJWP treatmeat group than in low dose QGJWP treatment group(P<0.01).Conclusions QGJWP improved the general state of rats with liver fibrosis induced by carbon tetrachloride.It also attenuated the degree of hepatic fibrosis in rats.It promoted degradation of extracelllar matrix(ECM)by regulating MMPs/TIMPs systems.Its anti-hepatic fibrosis mechanism might be related to inhibiting Raf/MEK/ERK signal pathways.