miR-34c介导的辐射旁效应对心肌成纤维细胞的影响及黄芪甲苷的防护

Influence of MiR-34c-Mediated Radiation-Induced Bystander Effect on Cardiac Fibroblasts and Intervention of AstragalosideⅣ

目的:探讨miR-34c/Notch1介导的辐射旁效应对心肌成纤维细胞(CFs)的影响和黄芪甲苷的干预效应。方法:将miR-34c inhibitor和miR-34c inhibitor NC转染至CFs中分别构建(miR-34c-inhibitor)-CFs和(miR-34c-inhibitor NC)-CFs,用黄芪甲苷5.1μmol/L预先2 h处理CFs构建黄芪甲苷-CFs,以2 Gy X线照射,并与正常CFs共培养48 h建立辐射旁效应体系。试验分为:正常对照组、模型对照组、miR-34c-inhibitor NC组、miR-34c-inhibitor组、黄芪甲苷5.1μmol/L组;以qRT-PCR、Western blot、流式细胞术、CCK-8法、划痕实验法等检测分析旁效应CFs中miR-34c和Notch1分子的表达、CFs转分化和致纤维化能力的变化。结果:与照射前相比,照射后CFs中miR-34c表达逐渐上调,24 h时最高(P<0.01),而旁效应CFs中miR-34c的表达也随之上调,48 h达高峰(P<0.01)。与正常对照组相比,模型对照组旁效应CFs中miR-34c表达显著上调,Notch1蛋白表达显著下调,α-SMA表达和α-SMA阳性细胞比例显著增加,CFs增殖活性和迁移率显著升高、TGF-β1和ColⅠ蛋白及mRNA表达上调(P<0.01);与模型对照组相比,miR-34c-inhibitor组和黄芪甲苷5.1μmol/L组中旁效应细胞miR-34c和Notch1的差异表达被逆转,旁效应CFs的α-SMA表达、α-SMA阳性细胞比例和细胞增殖、迁移率均显著降低,TGF-β1和ColⅠ蛋白及mRNA表达显著下调(P<0.01)。结论:X线诱导CFs中miR-34c表达上调,并上调旁效应CFs的miR-34c,进而通过负性调控Notch1信号通路,促进旁效应CFs转分化,增强其致纤维化能力;而黄芪甲苷通过调控miR-34c/Notch1介导的辐射旁效应对CFs的转分化和促纤维化具有抑制效应,从而发挥防护放射性心脏纤维化损伤的作用。

Objective:To explore the influence of miR-34c/Notch1-mediated radiation-induced bystander effect on cardiac fibroblasts(CFs)and the intervention of AstragalosideⅣ(AST).Methods:CFs were transfected with miR-34c inhibitor and miR-34c inhibitor NC to establish(miR-34c-inhibitor)-CFs and(miR-34c-inhibitor NC)-CFs,respectively.CFs pretreated with 5.1μmol/L AST for 2 hours were used to create AST-CFs.These CFs were then irradiated with 2 Gy X-rays and co-cultured with normal CFs for 48 hours to establish a model of radiation-induced bystander effect.The experiment included five groups:normal control,model control,miR-34c-inhibitor NC,miR-34c-inhibitor,and AST 5.1μmol/L.Techniques such as qRT-PCR,Western Blot,flow cytometry,CCK-8 assay,and scratch assay were used to analyze the expressions of miR-34c and Notch1,the transdifferentiation of CFs,and changes in fibrogenic capability.Results:After irradiation,miR-34c in CFs increased,peaking at 24 hours(P<0.01),and it was also elevated in CFs with bystander effect,reaching a maximum at 48 hours(P<0.01).Compared with the normal control group,the model control group had higher miR-34c(P<0.01)and lower Notch1(P<0.01),and there was a significant rise inα-SMA andα-SMA-positive cells(P<0.01).In addition,the proliferation activity and migration rate were elevated(P<0.01),and fibrosis markers TGF-β1 and ColⅠwere up-regulated(P<0.01).No significant differences were observed in the miR-34c-inhibitor NC group compared with the model control group.In contrast,the miR-34c-inhibitor and AST 5.1μmol/L groups showed reversed expression of miR-34c and Notch1 in CFs with bystander effect,and there were remarkable reductions inα-SMA,α-SMA-positive cells,as well as proliferation and migration(P<0.01).TGF-β1 and ColⅠwere also down-regulated(P<0.01).Conclusion:X-ray exposure increased miR-34c in CFs and subsequently up-regulated miR-34c in CFs with bystander effect.This upregulation negatively regulated the Notch1 signaling pathway,promoting the transdifferentiation of CFs and enhancing their fibrogenic ability.AST exerted a protective role against radiation-induced cardiac fibrosis by inhibiting the miR-34c/Notch1-mediated radiation-induced bystander effects and thus inhibiting the transdifferentiation and fibrogenic processes of CFs.