AG490及5-Aza对大鼠肺动脉平滑肌细胞增殖的影响及分子机制

Effects of AG490 and 5-Aza on proliferation of rat pulmonary artery smooth muscle cells and its molecular mechanism

目的探讨JAK2/STAT3通路抑制剂AG490及DNA甲基化抑制剂5-Aza对血小板源性生长因子-BB(PDGF-BB)诱导的大鼠肺动脉平滑肌细胞(PASMCs)增殖的影响及意义。方法采用CCK8检测不同浓度AG490及5-Aza对PDGF-BB诱导PASMCs增殖的影响;将细胞分为对照组(正常的PASMCs)、0μM组(予30μg/L PDGF-BB孵育48h)和25μM/1μM组、50μM/3μM组、100μM/5μM组(25μM/1μM组、50μM/3μM组、100μM/5μM组分别给予25/1、50/3、100/5μmol/L AG490/5-Aza共同孵育细胞)。采用RT-qPCR检测IL-6、GATA6、JAK2及STAT3 mRNA表达水平;Western blot检测GATA6、p-JAK2及p-STAT3蛋白的表达。结果与对照组比较,0μM AG490组IL-6、JAK2、STAT3mRNA表达均增加,GATA6mRNA及蛋白水平降低,p-JAK2/JAK2和p-STAT3/STAT3表达升高(P<0.05)。与0μM AG490组比较,25、50、100μM AG490组细胞增殖减少,IL-6、JAK2和STAT3mRNA的表达均降低,GATA6mRNA及蛋白表达升高,p-JAK2/JAK2水平降低(均P<0.05),50和100μM组中p-STAT3/STAT3水平降低(均P<0.05);与0μM 5-Aza组相比,1μM、3μM、5μM 5-Aza组细胞增殖减少,IL-6mRNA表达降低,GATA6蛋白、p-STAT3/STAT3表达增高,p-JAK2/JAK2水平降低(均P<0.05),JAK2、STAT3mRNA水平无显著差异(P>0.05);3μM和5μM 5-Aza组中GATA6mRNA表达增高(P<0.05)。结论AG490及5-Aza对PDGF-BB诱导的PASMCs增殖具有抑制作用,该作用可能与阻断JAK2/STAT3信号通路,抑制IL-6调节GATA6启动子甲基化有关。

Objective To explore the effect and significance of JAK2/STAT3 pathway inhibitor AG490 and DNA methylation inhibitor 5-Aza on proliferation of rat pulmonary artery smooth muscle cells(PASMCs)induced by platelet-derived growth factor-BB(PDGF-BB).Methods CCK8 was used to detect the effects of different concentrations of AG490 and 5-Aza on PDGF-BB-induced proliferation of PASMCs.The cells were divided into 5 groups,control group:normal PASMCs,0μM group:30ng/mL PDGF-BB incubation for 48h,25μM(1μM),50μM(3μM),100μM(5μM)groups:25μm/L AG490(5-Aza)was co-incubated with 25(1),50(3),100(5)on the 0μM group.RT-qPCR was used to detect the expression of IL-6,GATA6,JAK2 and STAT3 mRNA.Western blot was used to detect the expression of GATA6,p-JAK2 and p-STAT3 protein.Results Compared with the control group,the proliferation of PASMCs was significantly increased after 30 ng/mL PDGF-BB induction,IL-6,JAK2,and STAT3 mRNA expressions increased,GATA6 mRNA levels decreased,p-JAK2/JAK2 and p-STAT3/STAT3 expression increased,and GATA6 protein levels decreased(P<0.05).AG490 and 5-Aza can inhibit PDGF-BB-induced proliferation of PASMCs,with IC 50 of 61.269μmol/L and 3.507μmol/L,respectively.Compared with the 0μM group,the expression of IL-6,JAK2 and STAT3mRNA in 25,50 and 100μM groups were all reduced,GATA6mRNA level was increased,p-JAK2/JAK2 level was decreased,GATA6 protein level was increased,and p-STAT3/STAT3 level was decreased in 50 and 100μM groups(P<0.05).IL-6 mRNA and p-JAK2/JAK2 levels decreased in 25μM,50μM,and 100μM groups,p-STAT3/STAT3 and GATA6 protein levels increased,and GATA6 mRNA expression increased in 50μM and 100μM groups(P<0.05).Conclusion AG490 and 5-Aza have an inhibitory effect on PDGF-BB-induced proliferation of PASMCs,which may be related to blocking the JAK2/STAT3 signaling pathway and inhibiting IL-6 from regulating GATA6 promoter methylation.