敲除TRPC1基因抑制心肌纤维化发生的机制研究

Study on the mechanism of TRPC1 gene knockout inhibiting myocardial fibrosis

目的研究瞬时受体电位通道-1(TRPC1)对心肌梗死小鼠心肌纤维化的影响及其可能机制。方法野生型小鼠与TRPC1^(-/-)小鼠各20只,分为4组,野生对照组(WT对照组):只穿线,不结扎;野生实验组(WT实验组),结扎冠状动脉左前降支并缝合;TRPC1^(-/-)对照组,只穿线,不结扎;TRPC1^(-/-)实验组,结扎冠状动脉左前降支并缝合。每组各10只。处死后分离得到左心室组织。使用反转录-聚合酶链式反应(RT-PCR)、免疫蛋白印记(Westernblot)方法检测并比较四组小鼠心肌组织中TRPC1 mRNA和蛋白表达,以及蛋白激酶B(AKT)、磷酸化蛋白激酶B(p-AKT)、糖原合成酶激酶3β(GSK-3β)、磷酸化糖原合成酶激酶3β(p-GSK3β)、细胞外调节蛋白激酶(ERK)包括ERK1和ERK2、Ⅰ型胶原蛋白(Collagen-1)、α-平滑肌肌动蛋白(α-SMA)蛋白表达的差异。结果RT-PCR检测和Westernblot检测表明:WT实验组TRPC1 mRNA和蛋白均较WT对照组表达量增多(P<0.05),p-AKT、p-GSK-3β、p-ERK蛋白表达量明显减低(P<0.05);TRPC1^(-/-)实验组TRPC1 mRNA表达量和蛋白均较WT实验组降低(P<0.05),p-AKT、p-GSK-3β、p-ERK蛋白表达量明显增多(P<0.01)。WT实验组与WT对照组相比,WT实验组的Collagen-1、α-SMA表达明显增加(P<0.01);WT对照组与TRPC1^(-/-)对照组相比,WT对照组的Collagen-1、α-SMA表达明显增加(P<0.01);TRPC1^(-/-)对照组与TRPC1^(-/-)实验组相比,TRPC1^(-/-)实验组Collagen-1表达量增加(P<0.01);WT实验组与TRPC1^(-/-)实验组相比,WT实验组的Collagen-1、α-SMA表达明显增加(P<0.01)。结论心肌梗死后加重心肌纤维化(MF),敲除TRPC1基因后显著抑制了MF的发生,其可能是通过Akt/GSK3β及ERK信号通路起到抑制MF的作用。

Objective This research aims to study the effect of transient receptor potential channel-1(TRPC1)on myocardial fibrosis in mice with myocardial infarction and its possible mecha‐nism.Methods Twenty wild-type mice and Twenty TRPC1^(-/-)mice were divided into four groups:Wild-type control group(WT Control):only threading,no ligation;Wild-type experimental group(WT Exper‐iment),ligation of the left anterior descending coronary artery and su‐ture;TRPC1^(-/-)control group,only threading,no ligation;only thread‐ing,no ligation;TRPC1^(-/-)experimental group,ligation of the left ante‐rior descending coronary artery and suture.There were 10 mice in each group.The left ventricular tissues were isolated after the mice werekilled.Reverse transcription-polymerase chain reaction(RT-PCR)and Western blot analysis were used to detect and compare the expression of TRPC1 mRNA and protein in the myocardial tissue of the four groups of mice,as well as the protein expression of AKT,p-AKT,GSK3β,p-GSK3β,ERK1/2,p-ERK1/2,Collagen1,andα-SMA.Results RT-PCR and Western blot tests showed that the expression of TRPC1 mRNA and protein in the WT experimental group was higher than that in the WT control group(P<0.05),and the expression of p-AKT,p-GSK-3β,and p-ERK proteins was signif‐icantly reduced(P<0.05).The expression of TRPC1 mRNA and protein in the TRPC1^(-/-)experimental group was lower than that in the WT experimental group(P<0.05),and the expression of p-AKT,p-GSK-3β,and p-ERK proteins was significantly increased(P<0.01).Compared with the WT control group,the expression of Collagen-1 andα-SMA in the WT experimental group was significantly increased(P<0.01);compared with the TRPC1^(-/-)control group,the expression of Collagen-1 andα-SMA in the WT control group was significantly increased(P<0.01).Compared with the TRPC1^(-/-)experimental group,the expression of Collagen-1 in the TRPC1^(-/-)exper‐imental group was increased in the TRPC1^(-/-)experimental group(P<0.01);compared with the TRPC1^(-/-)experimental group,the ex‐pression of Collagen-1 andα-SMA in the WT experimental group was significantly increased(P<0.01).Conclusion Myocardial fi‐brosis(MF)is aggravated after myocardial infarction.Knocking out the TRPC1 gene significantly inhibits the occurrence of MF,which may inhibit MF through the Akt/GSK3βand ERK signaling pathways.