清心饮抑制CVB3感染心脏内皮细胞发生EndMT的作用机制研究

Mechanism of Qingxinyin ininhibiting endothelial-to-mesenchymal transition of cardiac microvascular endothelial cells following CVB3 infection

目的探讨清心饮含药血清通过调控Pyk2-PI3K-AKT信号通路抑制柯萨奇B3病毒(CVB_3)感染心脏微血管内皮细胞(CMVECs)向间充质细胞转分化(EndMT)的作用。方法选取稳定培养的心脏CMVECs,以100TCID_(50)CVB_3进行感染,采用20%清心饮含药血清进行干预(期间设立Pyk2抑制剂-TAE226和PI3K抑制剂-LY294002进行干预)。通过免疫荧光(IF)、蛋白质印迹(Western blot)、聚合酶链式反应(PCR)等实验方法检测内皮细胞标志物血小板/内皮细胞粘附分子-1(CD31)和间充质细胞标志物α-平滑肌肌动蛋白(α-SMA),富含脯氨酸的酪氨酸激酶2(Pyk2)、磷脂酰肌醇3激酶(PI3K)、蛋白激酶B(AKT)蛋白及基因的表达。结果与空白对照组比较,模型组CD31蛋白表达下调,α-SMA蛋白表达上调(P<0.05),Pyk2,PI3K,AKT蛋白均表达下调(P<0.05)。与空白血清组比较,20%清心饮含药血清组CD31蛋白表达上调,α-SMA蛋白表达下调(P<0.05);Pyk2,PI3K,AKT表达上调(P<0.05)。与20%清心饮含药血清组比较,Pyk2抑制剂组PI3K,AKT表达下调(P<0.05),CD31蛋白表达下调,α-SMA蛋白表达上调(P<0.05);PI3K抑制剂组AKT表达下调(P<0.05),CD31蛋白表达下调,α-SMA蛋白表达上调(P<0.05),而Pyk2表达无明显变化(P>0.05)。通过PCR亦发现,Pyk2,PI3K,AKT mRNA与Western blot法蛋白表达趋势一致。结论20%清心饮含药血清可通过调控Pyk2-PI3K-AKT信号通路抑制CVB_3感染CMVECs发生EndMT。

Objective To explore the effectivenss and mechanism of Qingxinyin(Heart-clearing Formula)-containing serum(QXY serum)in inhibiting the endothelial-to-mesenchymal transition(EndMT)of cardiac microvascular endothelial cells(CMVECs)following CVB3 infection by regulating the Pyk2-PI3 K-AKT signal pathway.Methods Stably cultured CMVECs were infected with 100 TCID50 CVB3,and intervened by 20%QXY serum(intervened by Pyk2 inhibitor-TAE226 and PI3 K inhibitor-LY294002 in other groups).Edothelial cells marker-CD31,mesenchymal cells marker-α-SMA,and protein and gene expression of Pyk2,PI3 K,AKT were tested by using immunofluorescence(IF),Western blot(WB)and Polymerase Chain Reaction(PCR).Results Compared with the blank control group,the expressions of Pyk2,PI3 K,AKT(P<0.05)and CD 31 were decreased(P<0.05),but that ofα-SMA was increased(P<0.05)in the model group.Compared with the blank serum group,the expressions of Pyk2,PI3 K,AKT(P<0.05)and CD 31 were increased(P<0.05),but that ofα-SMA was decreased(P<0.05)in the QXY group.Moreover,compared with the QXY group,in the Pyk2 inhibited group,PI3 K,AKT(P<0.05)and CD31 showed lower expression,andα-SMA showed higher expression(P<0.05);while in the PI3 K inhibited group,AKT(P<0.05)and CD31 showed lower expression,α-SMA showed higher expression(P<0.05),while Pyk2 showed no significant change(P>0.05).Besides,the expression trends of Pyk2,PI3 K,AKT and mRNA according to PCR were consistent with western blot.Conclusion 20%QXY-containing serum seems to inhibit the EndMT of CMVECs following CVB3 infection,and one of the possible mechanisms could be through the Pyk2-PI3 K-AKT signaling pathway during that process.