基于miRNA-134-5p/BDNF/Akt信号通路探讨X线辐射致大鼠心肌细胞凋亡的机制  

Mechanism of X-ray radiation-induced rat myocardial cell apoptosis based on miRNA-134-5p/BDNF/Akt signaling pathway

作　　者：顾静 付力文 韩晓斐 方丹 金戈 董晓丽 颉亚辉 侯敏 GU Jing;FU Liwen;HAN Xiaofei;FANG Dan;JIN Ge;DONG Xiaoli;XIE Yahui;HOU Min(Department of Physiology,Basic Medical College,Gansu University of Chinese Medicine,Lanzhou 730000,Gansu,China;Department of Mathematics and Health Statistics,School of Public Health,Gansu University of Chinese Medicine,Lanzhou 730000,Gansu,China;Department of Anatomy and Histoembryology,Basic Medical College,Gansu University of Traditional Chinese Medicine,Lanzhou 730000,Gansu,China)

机构地区：[1]甘肃中医药大学基础医学院生理学教研室,兰州730000 [2]甘肃中医药大学公共卫生学院数学与卫生统计学教研室,兰州730000 [3]甘肃中医药大学基础医学院解剖学与组织胚胎学教研室,兰州730000

出　　处：《海军军医大学学报》2024年第11期1352-1361,共10页Academic Journal of Naval Medical University

基　　金：国家自然科学基金(82360878);甘肃省中医药管理局重点项目(GZKZ-2020-10)。

摘　　要：目的探讨X线辐射对大鼠心肌细胞凋亡的影响及相关机制。方法将大鼠H9C2心肌细胞分为空白对照组、X线照射组(X-ray组)、X线照射miRNA-134-5p抑制剂组(X-inhibitor组)、X线照射miRNA-134-5p抑制剂阴性对照组(X-NC组)。最终选择以6 Gy X线辐照大鼠H9C2心肌细胞,48 h后检测各项指标变化。采用CCK-8法检测细胞存活率,用流式细胞术和Hoechst 33342染色检测细胞凋亡率,用DCFH-DA荧光探针法检测细胞内活性氧(ROS)水平,用JC-1法检测细胞线粒体膜电位变化,用超氧化物歧化酶(SOD)和丙二醛(MDA)检测试剂盒分别测定细胞中SOD活性、MDA水平,用qPCR法检测细胞中miRNA-134-5p表达,用蛋白质印迹法检测细胞中脑源性神经营养因子(BDNF)、Akt、磷酸化Akt(p-Akt)、Bcl2、Bax蛋白的表达。结果与空白对照组相比,X-ray组大鼠心肌细胞中ROS和MDA水平上升、SOD活性下降、线粒体膜电位下降百分比增加、DNA损伤微核形成数增加、细胞凋亡率升高(均P<0.01);与X-ray组相比,X-inhibitor组上述各项指标均有所逆转(P<0.05或P<0.01),而X-NC组的以上各指标差异均无统计学意义(均P>0.05)。与空白对照组相比,X-ray组大鼠心肌细胞中miRNA-134-5p水平上调,BDNF表达及Bcl2/Bax、p-Akt/Akt比值下降(均P<0.01);与X-ray组相比,X-inhibitor组大鼠心肌细胞中miRNA-134-5p水平下降,BDNF表达及Bcl2/Bax、p-Akt/Akt比值升高(均P<0.01),X-NC组各项指标差异均无统计学意义(均P>0.05)。结论X线照射诱导了大鼠心肌细胞发生氧化应激、线粒体损伤及DNA损伤,最终导致细胞凋亡,其发生机制可能涉及miRNA-134-5p/BDNF/Akt信号通路。Objective To investigate the effect of radiation on cardiomyocyte apoptosis and its related mechanism.Methods Rat H9C2 cardiomyocytes were divided into blank control group,X-ray irradiation group(X-ray group),X-ray irradiation+microRNA(miRNA)-134-5p inhibitor group(X-inhibitor group)and X-ray irradiation+miRNA-134-5p inhibitor negative control group(X-NC group).H9C2 cardiomyocytes were irradiated with 6 Gy X-ray,and the changes of various indexes were detected 48 h after irradiation.Cell viability was detected by cell counting kit 8 assay.The apoptosis rate was detected by flow cytometry and Hoechst 33342 staining.The level of reactive oxygen species(ROS)in cells was detected by DCFH-DA fluorescence probe.The mitochondrial membrane potential was detected by JC-1 method.The activity of superoxide dismutase(SOD)and the level of malondialdehyde(MDA)in cells were measured by kits.The expression of miRNA-134-5p was detected by quantitative polymerase chain reaction.The protein expression of brain-derived neurotrophic factor(BDNF),protein kinase B(Akt),phosphorylated Akt(p-Akt),Bcl2 and Bax was detected by Western blotting.Results Compared with the blank control group,in the X-ray group the levels of ROS and MDA were significantly increased,the activity of SOD was significantly decreased,the decreased percentage in mitochondrial membrane potential was significantly increased,the number of micronuclei of DNA damage was significantly increased,and the apoptosis rate was significantly increased(all P<0.01).Compared with the X-ray group,all the indexes of the X-inhibitor group were reversed(P<0.05 or P<0.01),while there was no significant difference in the above parameters in the X-NC group(all P>0.05).Compared with the blank control group,the X-ray group had a significant increase in the miRNA-134-5p level and significant reductions in the protein level of BDNF,Bcl2/Bax ratio,and p-Akt/Akt ratio(all P<0.01).Compared with the X-ray group,the X-inhibitor group had a significant reduction in the level of miRNA-134-5p and sig

关 键 词：放射性心脏损伤 辐射 心肌细胞 微RNA-134-5p 细胞凋亡 

分 类 号：R818[医药卫生—放射医学]