mTOR/HIF-1α信号通路在布-加综合征肝纤维化中的作用机制研究

Study on the mechanism of mTOR/HIF-1αsignaling pathway in Budd-Chiari syndrome liver fibrosis

目的探讨mTOR/HIF-1α信号通路在布-加综合征(B-CS)肝纤维化中的作用机制。方法雄性C57小鼠20只按随机数字表法分为假手术(Sham)组、假手术+雷帕霉素(Sham+Ra)组、B-CS组、B-CS+雷帕霉素(B-CS+Ra)组,每组均5只。于肝后段下腔静脉(IVC)部分结扎构建B-CS小鼠模型;Sham组未结扎IVC。Sham+Ra和B-CS+Ra组小鼠隔天腹腔注射雷帕霉素(2 mg/kg,5%DMSO溶液配制),Sham组和B-CS组注射同等剂量5%DMSO溶液。6周后取材,部分肝脏组织用于制作石蜡切片进行苏木精-伊红、天狼星红染色观察病理变化,免疫组织化学染色检测肝组织中α-SMA、Fibrinogen的表达;新鲜肝脏组织提取蛋白和RNA,分别用Western-blot检测α-SMA、Fibrinogen、p-mTOR、mTOR、HIF-1α、CollagenⅠ、VEGF蛋白水平,实时荧光定量PCR检测mTOR、HIF-1α、VEGF mRNA水平。计量资料用均数±标准差(±s)表示,组间的比较采用单因素ANOVA检验。结果病理染色结果显示,B-CS组小鼠肝脏中央静脉周围及肝窦淤血严重,窦间隙增宽,胶原沉积增加,表明本研究成功建立小鼠B-CS肝纤维化模型。Sham组纤维化指标α-SMA、CollagenⅠ蛋白,mTOR通路相关指标p-mTOR、HIF-1α蛋白,微血栓指标Fibrinogen蛋白表达量分别为0.027±0.012、0.337±0.008、0.138±0.024、0.296±0.113、0.733±0.192;B-CS组分别为0.986±0.001、0.927±0.055、0.936±0.044、1.693±0.443、1.612±0.068,两组相比差异有统计学意义(P<0.05)。B-CS+Ra组表达量分别为0.707±0.078、0.311±0.024、0.332±0.094、0.254±0.117、0.569±0.075,与B-CS组相比,差异具有统计学意义(P<0.05)。结论 mTOR/HIF-1α信号通路在小鼠B-CS肝纤维化中被显著激活,该通路可通过调节微血栓形成,参与肝纤维化发展。

Objective To explore the mechanism of mTOR/HIF-1αsignaling pathway in Budd-Chiari syndrome(B-CS)liver fibrosis.Methods Twenty male C57 mice were randomly divided into Sham operation group(Sham),sham operation+rapamycin(Sham+Ra)group,B-CS group,B-CS+rapamycin(B-CS+Ra)Group,5 in each group.The B-CS mouse model was constructed by partial ligation of the inferior vena cava(IVC)at the posterior segment of the liver;IVC was not ligated in the Sham group.Mice in Sham+Ra and B-CS+Ra groups were intraperitoneally injected with rapamycin(2 mg/kg,5%DMSO solution preparation)every other day,Sham group and B-CS group were injected with the same dose of 5%DMSO solution.After 6 weeks,samples were taken,and part of the liver tissue was used to make paraffin sections for hematoxylin-eosin(HE)and Sirus Red staining to observe the pathological changes,and immunohistochemical staining to detect the expression ofα-SMA and Fibrinogen in liver tissues;Protein and RNA were extracted from fresh liver tissue,and Western-blot was used to detectα-SMA,Fibrinogen,p-mTOR,mTOR,HIF-1α,Collagen I,and VEGF protein levels.Real-time fluorescent quantitative PCR was used to detect mTOR,HIF-1α,CollagenⅠ,VEGF mRNA levels.Measurement data were expressed as mean±standard deviation(±s),and the comparison between groups was performed by one-way ANOVA test.Results The results of pathological staining showed that in the B-CS group,there was severe congestion around the central vein of the liver and sinusoids,widening of the sinus space,and increased collagen deposition,indicating that this study successfully established a mouse B-CS liver fibrosis model.The expression levels of fibrosis indicatorsα-SMA and Collagen I protein,mTOR pathway related indicators p-mTOR and HIF-1αprotein,and microthrombus indicator Fibrinogen protein in the Sham group were 0.027±0.012,0.337±0.008,0.138±0.024,0.296±0.113,0.733±0.192;B-CS group were 0.986±0.001,0.927±0.055,0.936±0.044,1.693±0.443,1.612±0.068,and the differences were statistically significant(P<0.05).The expression levels of B-CS+Ra group were 0.707±0.078,0.311±0.024,0.332±0.094,0.254±0.117,0.569±0.075,which were statistically significant compared with B-CS group(P<0.05).Conclusions The mTOR/HIF-1αsignaling pathway is significantly activated in mouse B-CS liver fibrosis.This pathway may participate in the development of liver fibrosis by regulating microthrombosis.