丁苯酞激活Hedgehog信号通路抑制缺氧复氧心肌细胞氧化应激、炎症、凋亡的机制研究

DL-3-n-butylphthalide-activated Hedgehog signaling pathway in inhibiting oxidative stress,inflammation and apoptosis in hypoxic-reoxygenated cardiomyocytes:a study on mechanism

目的 探讨丁苯酞(NBP)对缺氧复氧(H/R)心肌细胞氧化应激、炎症、细胞凋亡的影响及其分子机制.方法 将心肌细胞分为空白组、H/R组、NBP-L组(NBP浓度为2μmol/L)、NBP-M组(NBP浓度为10μmol/L)、NBP-H组(NBP浓度为50μmol/L).噻唑蓝(MTT)法检测细胞增殖,流式细胞术检测细胞凋亡,酶联免疫(ELISA)法检测肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)、白介素-1β(IL-1β)水平,试剂盒检测活性氧(ROS)、超氧化物歧化酶(SOD)、丙二醛(MDA)水平,蛋白质印迹法(Western blot)检测细胞周期蛋白D1(cyclin D1)、细胞周期蛋白依赖性激酶2(CDK2)、活化的多聚ADP-核糖聚合酶(cleaved PARP)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(cleaved caspase-3)、PTCH、Smoothened(SMO)和Gli-1蛋白表达.使用Hedgehog信号通路抑制剂HPI-4处理心肌细胞,观察其对丁苯酞诱导的缺氧复氧心肌细胞氧化应激、炎症、凋亡的影响.结果 与空白组比较,H/R组心肌细胞存活率、cyclinD1、CDK2蛋白表达量、SOD活性、PTCH、SMO和Gli-1蛋白表达量明显减少(P<0.05),H/R组心肌细胞凋亡率、cleaved PARP、cleaved caspase-3蛋白表达量、TNF-α、IL-6、IL-1β、ROS、MDA水平显著增加(P<0.05).与H/R组比较,NBP-L、NBP-M、NBP-H组显著提高H/R心肌细胞存活率、cyclin D1、CDK2蛋白表达量、SOD活性(P<0.05),显著降低心肌细胞凋亡率、cleaved PARP、cleaved caspase-3蛋白、TNF-α、IL-6、IL-1β、ROS、MDA水平、PTCH、SMO和Gli-1蛋白水平(P<0.05).HPI-4部分逆转丁苯酞对缺氧复氧心肌细胞的保护作用.结论 丁苯酞通过激活Hedgehog信号通路,促进缺氧复氧心肌细胞增殖,并抑制缺氧复氧心肌细胞氧化应激、炎症、凋亡.

Objective To investigate the influence and molecular mechanism of DL-3-n-butylphthalide(NBP)in oxidative stress,inflammation and apoptosis in hypoxic-reoxygenated(H/R)cardiomyocytes.Methods Cardiomyocytes were divided into blank group,H/R group,NBP-L group(NBP dose=2μmol/L),NBP-M group(NBP dose=10μmol/L),NBP-H group(NBP dose=50μmol/L).The proliferation of cardiomyocytes was detected by using MTT,apoptosis was detected by using flow cytometry,and levels of TNF-α,IL-6 and IL-1βwere detected by using ELISA.The levels of ROS,SOD and MDA were detected by using kit.The expressions of cyclin D1,CDK2,cleaved PARP,cleaved caspase-3,PTCH,Smoothened(SMO)and Gli-1 were detected by using Western blotting assay.The cardiomyocytes were treated with HPI-4,an inhibitor of Hedgehog signaling pathway,for observing the influence of NBP on oxidative stress,inflammation and apoptosis in H/R cardiomyocytes.Results Compared with blank group,cardiomyocyte survival rate,expressions of cyclinD1 and CDK2,SOD activity,and expressions of PTCH,SMO and Gli-1 decreased significantly in H/R group(P<0.05).The apoptosis rate of cardiomyocytes,expressions of cleaved PARP and cleaved caspase-3,and levels of TNF-α,IL-6,IL-1β,ROS and MDA increased significantly in H/R group(P<0.05).Compared with H/R group,cardiomyocyte survival rate,expressions of cyclin D1 and CDK2 and SOD activity increased significantly in NBP-L group,NBP-M group and NBP-H group(P<0.05).The apoptosis rate of cardiomyocytes,and levels of cleaved PARP,cleaved caspase-3,TNF-α,IL-6,IL-1β,ROS,MDA,PTCH,SMO and Gli-1 decreased significantly in NBP-L group,NBP-M group and NBP-H group(P<0.05).HPI-4 reversed partially the protective effect of NBP on H/R cardiomyocytes.Conclusion NBP can improve the proliferation and inhibit oxidative stress,inflammation and apoptosis in H/R cardiomyocytes through activating Hedgehog signaling pathway.