TRPC6通过AKT/GSK-3β和ERK信号通路抑制心肌纤维化的形成

TRPC6 Inhibits Formation of Myocardial Fibrosis through AKT/GSK-3βand ERK Signaling Pathways

目的探讨瞬时受体电位通道6(transient receptor potential canonical 6,TRPC6)介导的蛋白激酶B(protein kinase B,AKT)/糖原合酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)和胞外信号调节激酶(extracellular signal-regulated kinase,ERK)蛋白信号通路在异丙肾上腺素(isoprenaline,ISO)诱导心肌纤维化中的作用。方法取野生型(wild type,WT)和TRPC6基因敲除型(TRPC6 knockout,TRPC6-/-)小鼠各40只,分别使用生理盐水(Saline)和ISO进行注射,根据基因型和注射药物的不同将实验小鼠随机分为以下4组:WT Saline组;WT ISO组;TRPC6-/-Saline组;TRPC6-/-ISO组;用组织免疫荧光技术检测TRPC6通道蛋白在心肌组织中表达水平的差异;用苏木精-伊红(hematoxylin-eosin,HE)、Masson、天狼猩红等组织化学染色法检测心肌组织的纤维化表现;用Western blot(WB)检测小鼠心肌组织蛋白中纤维化及相关通路指标。结果WT ISO组小鼠心肌细胞内出现明显空泡坏死,细胞间炎性细胞浸润,心肌细胞排列紊乱、胶原蛋白纤维沉积明显增加,与之相比,TRPC6-/-ISO组小鼠心肌细胞坏死及炎性细胞浸润较少,胶原纤维沉积显著低于WT ISO组;WB结果显示,WT ISO组小鼠心肌组织内TRPC6表达水平升高;与相应的Saline组相比,WT ISO和TRPC6-/-ISO组小鼠心肌组织中α-平滑肌动蛋白(α-smooth muscle actin,α-SMA)、Ⅰ型胶原蛋白(collagenⅠ,COL-1)、磷酸化蛋白激酶B(phosphorylated-protein kinase B,p-AKT)、磷酸化糖原合酶激酶-3β(phosphorylated-glycogen synthase kinase-3β,p-GSK-3β)及磷酸化胞外信号调节激酶(phosphorylated-extracellular signal-regulated kinase,p-ERK)蛋白表达量明显增加,但TRPC6-/-ISO组表达水平较WT ISO组明显降低。结论TRPC6基因敲除可通过下调AKT/GSK-3β及ERK信号通路的活性,减轻ISO诱导的心肌纤维化。

Objective To study the functions of protein kinase B(AKT)/glycogen synthase kinase-3β(GSK-3β)and extracellular signal-regulated kinases(ERK)signaling pathways mediated by the transient receptor potential canonical 6(TRPC6)in myocardial fibrosis induced by isoprenaline(ISO).Methods Forty wild type(WT)and forty TRPC6 knockout(TRPC6-/-)mice were selected and injected with saline and ISO,respectively.The mice were randomly divided into the following four groups according to the genotypes and drugs of injection:WT Saline group;WT ISO group;TRPC6-/-Saline group;TRPC6-/-ISO group.Immunofluorescence technique was used to observe the expression level of TRPC6 channel protein in myocardial tissue.Hematoxylin-eosin(HE)staining,Masson trichrome staining,and Sirius red staining were conducted to assess the fibrotic lesions of heart tissue.Western blot(WB)was utilized to evaluate fibrotic lesions and indicators of related signaling molecules in myocardial tissue of experimental mice.Results In the WT ISO group,obvious vacuolar necrosis,intercellular inflammatory cell infiltration,disordered arrangement of myocardial cells,and significantly increased deposition of collagen fiber were detected in myocardial tissue.In comparison,TRPC6-/-group exhibited less myocardial cell necrosis and inflammatory cell infiltration than that in WT ISO group.In addition,the deposition of collagen fiber in TRPC6-/-ISO group was significantly lower than that in the WT ISO group.WB results showed that the expression level of TRPC6 protein was increased in WT ISO group.The protein expression levels ofα-smooth muscle actin(α-SMA),collagen I(COL-1),phosphorylated-protein kinase B(p-AKT),phosphorylated-glycogen synthase kinase-3β(p-GSK-3β)and phosphorylated-extracellular signal-regulated kinase(p-ERK)were significantly increase in both WT ISO group and TRPC6-/-ISO group as compared with saline group,but such enhancement of these signaling molecules expression in TRPC6-/-ISO group were marginal,compared to that in WT ISO group.Conclusion TRPC6 gene knockout mitigate ISO-induced myocardial fibrosis through down-regulating the activity of AKT/GSK-3βand ERK signaling pathway.