文献类型：期刊文献

中文题名：黄芪甲苷改善辐射旁效应引起的心肌成纤维细胞损伤及对miR-134-5p/BDNF/TGF-β1信号通路的影响

英文题名：AstragalosideⅣameliorates cardiac fibroblast injury induced by the radiation bystander effect and modulates the miRNA-134-5p/BDNF/TGF-β1 signaling pathway

作者：顾静[1];方丹[1];付力文[1];荆爽[1];侯敏[1];颉亚辉[1]

第一作者：顾静

机构：[1]甘肃中医药大学基础医学院,兰州730000

第一机构：甘肃中医药大学基础医学院（敦煌医学研究所）

年份：2026

卷号：54

期号：5

起止页码：570

中文期刊名：中华心血管病杂志

外文期刊名：Chinese Journal of Cardiology

收录：;北大核心:【北大核心2023】；

基金：国家自然科学基金(82360878);甘肃省自然科学基金(24JRRA560)。

语种：中文

中文关键词：黄芪;黄芪甲苷;微小RNA-134-5p;辐射旁效应;心肌成纤维细胞;脑源性神经营养因子;转化生长因子-β1

外文关键词：Astragalus membranaceus;AstragalosideⅣ;MicroRNA-134-5p;Radiation-induced bystander effect;Cardiac fibroblasts;Brain-derived neurotrophic factor;Transforming growth factor-β1

摘要：目的探讨微小RNA(miRNA)-134-5p/脑源性神经营养因子(BDNF)/转化生长因子-β1(TGF-β1)信号通路介导的辐射旁效应对心肌成纤维细胞的影响,以及黄芪甲苷对纤维化损伤的防护作用。方法选取大鼠心肌成纤维细胞,利用Transwell共培养板,将经2 Gy X射线辐照1 min的心肌成纤维细胞与正常心肌成纤维细胞共培养,构建辐射旁效应体系。实验分为9组:对照组(正常心肌成纤维细胞,无额外干预)、黄芪甲苷组(正常心肌成纤维细胞经5.1μmol/L黄芪甲苷溶液预处理2 h)、X-co组(经X射线辐照的心肌成纤维细胞与正常心肌成纤维细胞共培养)、X-IN-co组(经X射线辐照且转染miRNA-134-5p抑制剂的心肌成纤维细胞与正常心肌成纤维细胞共培养)、X-inNC-co组(经X射线辐照且转染miRNA-134-5p抑制剂阴性对照的心肌成纤维细胞与正常心肌成纤维细胞共培养)、X-AST-co组(经X射线辐照且经5.1μmol/L黄芪甲苷预处理2 h的心肌成纤维细胞与正常心肌成纤维细胞共培养)、MI-co组(转染miRNA-134-5p模拟剂的心肌成纤维细胞与正常心肌成纤维细胞共培养)、miNC-co组(转染miRNA-134-5p模拟剂阴性对照的心肌成纤维细胞与正常心肌成纤维细胞共培养)、AST-MI-co组(转染miRNA-134-5p模拟剂的心肌成纤维细胞与经5.1μmol/L黄芪甲苷预处理2 h的心肌成纤维细胞共培养)。采用流式细胞术检测各组心肌成纤维细胞中活性氧水平;采用细胞计数试剂盒-8检测不同时间点(0、6、12、24、48 h)细胞增殖能力;采用硫代巴比妥酸法测定丙二醛浓度;采用水溶性四氮唑法检测超氧化物歧化酶(SOD)活性;采用实时荧光定量逆转录聚合酶链式反应检测miRNA-134-5p表达水平;采用Western blot法检测BDNF、α-平滑肌肌动蛋白(α-SMA)、TGF-β1、Sma和Mad相关蛋白2(Smad2)及Ⅰ型胶原蛋白表达水平。结果细胞计数试剂盒-8检测结果显示,各组细胞在6 h时活性均较低,24 h时达到峰值。与对照组相比,X-co组辐射旁效应细胞中miRNA-134-5p表达水平较高,BDNF水平较低,活性氧及丙二醛含量较高,而SOD活性较低(P均<0.05);同时,X-co组辐射旁效应细胞中α-SMA、TGF-β1、Smad2及Ⅰ型胶原蛋白水平均高于对照组(P均<0.05)。与X-co组相比,X-IN-co组辐射旁效应细胞中miRNA-134-5p表达水平、活性氧水平、丙二醛含量较低,BDNF水平及SOD活性较高,α-SMA、TGF-β1、Smad2及Ⅰ型胶原蛋白等纤维化因子水平较低(P均<0.05)。X-AST-co组辐射旁效应细胞中上述氧化应激损伤及纤维化因子水平均低于X-co组(P均<0.05)。此外,与对照组相比,MI-co组心肌成纤维细胞中miRNA-134-5p、α-SMA及TGF-β1水平较高,BDNF水平较低(P均<0.05);与MI-co组相比,AST-MI-co组心肌成纤维细胞中miRNA-134-5p、TGF-β1及α-SMA水平较低,而BDNF水平较高(P均<0.05)。结论miRNA-134-5p/BDNF/TGF-β1通路可介导辐射旁效应,促进心肌成纤维细胞氧化应激、转分化激活及促纤维化损伤;黄芪甲苷可通过调控该通路,防护辐射旁效应引起的心脏纤维化损伤。
Objective To investigate the effects of radiation bystander effect mediated by microRNA(miRNA)-134-5p/brain-derived neurotrophic factor(BDNF)/transforming growth factor-β1(TGF-β1)pathway on cardiac fibroblasts,as well as the protective effect of astragalosideⅣ(AST)against fibrotic injury.Methods Rat cardiac fibroblasts were used,and a radiation bystander effect model was established via co-culturing cardiac fibroblasts irradiated with 2 Gy X-ray for 1 min with normal cardiac fibroblasts in a Transwell co-culture plate.The experiment was divided into 9 groups:control group(normal cardiac fibroblasts without additional intervention),AST group(normal cardiac fibroblasts pretreated with 5.1μmol/L AST solution for 2 h),X-co group(irradiated cardiac fibroblasts co-cultured with normal cardiac fibroblasts),X-IN-co group(irradiated cardiac fibroblasts transfected with miRNA-134-5p inhibitor co-cultured with normal cardiac fibroblasts),X-inNC-co group(irradiated cardiac fibroblasts transfected with miRNA-134-5p inhibitor negative control co-cultured with normal cardiac fibroblasts),X-AST-co group(irradiated cardiac fibroblasts pretreated with 5.1μmol/L AST for 2 h co-cultured with normal cardiac fibroblasts),MI-co group(cardiac fibroblasts transfected with miRNA-134-5p mimic co-cultured with normal cardiac fibroblasts),miNC-co group(cardiac fibroblasts transfected with miRNA-134-5p mimic negative control co-cultured with normal cardiac fibroblasts),and AST-MI-co group(cardiac fibroblasts transfected with miRNA-134-5p mimic co-cultured with cardiac fibroblasts pretreated with 5.1μmol/L AST for 2 h).Flow cytometry was used to detect the reactive oxygen species(ROS)level in cardiac fibroblasts of each group;Cell Counting Kit-8 assay was used to measure cell proliferation capacity at different time points(0,6,12,24,and 48 h);the thiobarbituric acid method was used to determine malondialdehyde(MDA)concentration;the water-soluble tetrazolium salt method was applied to detect superoxide dismutase(SOD)activity;quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR)was used to detect the expression level of miRNA-134-5p;and Western blot was performed to measure the protein expression levels of BDNF,α-smooth muscle actin(α-SMA),TGF-β1,mothers against decapentaplegic homolog 2(Smad2),and typeⅠcollagen.Results Cell Counting Kit-8 assay showed that the cell viability in all groups was low at 6 h and peaked at 24 h.Compared with the control group,the bystander cells in the X-co group had higher expression levels of miRNA-134-5p,higher levels of ROS and MDA,lower levels of BDNF,and lower SOD activity(all P<0.05);meanwhile,the protein levels ofα-SMA,TGF-β1,Smad2 and typeⅠcollagen in the bystander cells of the X-co group were higher than those in the control group(all P<0.05).In comparison with the X-co group,the bystander cells in the X-IN-co group exhibited lower expression of miRNA-134-5p,reduced ROS level and MDA content,elevated BDNF level and SOD activity,as well as decreased protein levels of fibrotic factors includingα-SMA,TGF-β1,Smad2 and typeⅠcollagen(all P<0.05).The levels of the aforementioned oxidative stress injury indicators and fibrotic factors in the bystander cells of the X-AST-co group were all lower than those in the X-co group(all P<0.05).In addition,compared with the control group,the cardiac fibroblasts in the MI-co group had higher levels of miRNA-134-5p,α-SMA and TGF-β1,and lower BDNF level(all P<0.05);while in comparison with the MI-co group,the cardiac fibroblasts in the AST-MI-co group presented with lower levels of miRNA-134-5p,TGF-β1 andα-SMA,and higher BDNF level(all P<0.05).Conclusion The miRNA-134-5p/BDNF/TGF-β1 pathway can mediate the radiation bystander effect and promote oxidative stress,transdifferentiation and profibrotic injury in cardiac fibroblasts.AST exerts a protective effect against radiation bystander effect-induced cardiac fibrotic injury by regulating this signaling pathway.