同型半胱氨酸在心肌细胞凋亡和增殖中的作用及其机制  

Effects and mechanism of Hcy on apoptosis and proliferation of cardiomyocytes

作　　者：刘达越 董小艳 徐灵博 张慧萍 全尚琨[1,2] 马胜超 白永生[2,4] 杨勇 李桂忠 Liu Da-Yue;Dong Xiao-Yan;Xu Ling-Bo;Zhang Hui-Ping;Quan Shang-Kun;Ma Sheng-Chao;Bai Yong-Sheng;Yang Yong;Li Gui-Zhong(School of Basic Medical Sciences,Ningxia Medical University,Yinchuan,Ningxia 750004,China;National Health Council Key Laboratory of Metabolic Cardiovascular Diseases Research/Ningxia Key Lab of Vascular Injury and Repair Research,Yinchuan,Ningxia 750004,China;Prenatal Diagnosis Center,General Hospital of Ningxia Medical University,Yinchuan,Ningxia 750004,China;Department of Neurology,People's Hospital of Ningxia Hui Autonomous Region,Yinchuan,Ningxia 750004,China;Nuclear Medicine Department,People's Hospital of Ningxia Hui Autonomous Region,Yinchuan,Ningxia 750004,China)

机构地区：[1]宁夏医科大学基础医学院,宁夏银川750004 [2]国家卫生健康委员会代谢性心血管疾病研究重点实验室/宁夏血管损伤与修复研究重点实验室,宁夏银川750004 [3]宁夏医科大学总医院产前诊断中心,宁夏银川750004 [4]宁夏回族自治区人民医院神经内科,宁夏银川750004 [5]宁夏回族自治区人民医院核医学科,宁夏银川750004

出　　处：《解放军医学杂志》2022年第10期984-991,共8页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金青年基金(81900273);国家自然科学基金地区项目(81860044,8176009);宁夏回族自治区重点研发计划重点项目(2019BFG02004)。

摘　　要：目的探讨同型半胱氨酸(Hcy)对大鼠心肌细胞凋亡、增殖的作用及其可能机制。方法将大鼠心肌细胞分为对照组与Hcy组(用终浓度为3 mmol/L的Hcy刺激24 h),采用Western blotting检测TLR4、髓样分化因子(MyD88)、NF-κB、磷酸化的NF-κB(p-NF-κB),凋亡相关蛋白Bax、cleaved-caspase-3及抗凋亡蛋白Bcl-2的表达情况,流式细胞术检测心肌细胞凋亡率,EdU染色观察心肌细胞增殖情况。将TLR4干扰片段转染心肌细胞,采用Western blotting检测TLR4表达情况,对干扰片段进行筛选和验证。将心肌细胞分为干扰片段空载体组(si-NC+Hcy组)与干扰TLR4组(si-TLR4+Hcy组),并加入3 mmol/L的Hcy干预24 h,采用Western blotting检测心肌细胞MyD88、NF-κB、p-NF-κB,凋亡相关蛋白Bax、cleavedcaspase-3及抗凋亡蛋白Bcl-2的表达情况,流式细胞术检测心肌细胞凋亡率,EdU染色观察心肌细胞增殖情况。结果Western blotting检测结果显示,与对照组比较,Hcy组TLR4、MyD88、p-NF-κB及凋亡相关蛋白Bax、cleaved-caspase-3表达水平升高(P<0.01),抗凋亡蛋白Bcl-2表达水平降低(P<0.01);与si-NC+Hcy组比较,si-TLR4+Hcy组TLR4、MyD88、p-NF-κB及凋亡相关蛋白Bax、cleaved-caspase-3的表达水平降低(P<0.01),抗凋亡蛋白Bcl-2表达水平升高(P<0.01)。流式细胞术检测结果显示,与对照组比较,Hcy组细胞凋亡率明显增高(P<0.01);与si-NC+Hcy组比较,si-TLR4+Hcy组细胞凋亡率明显降低(P<0.01)。EdU染色结果显示,与对照组比较,Hcy组EdU阳性细胞率明显降低(P<0.01);与si-NC+Hcy组比较,si-TLR4+Hcy组EdU阳性细胞率明显增高(P<0.01)。结论Hcy可诱导大鼠心肌细胞凋亡,抑制心肌细胞增殖,其机制可能与TLR4/NF-κB信号通路有关。Objective To investigate the effect and mechanism of homocysteine on apoptosis and proliferation of cardiomyocytes.Methods Cardiomyocytes were stimulated with homocysteine at the concentration of 3 mmol/L for 24 h.Cells were divided into control group and homocysteine group(Hcy group).Western blotting was detected the expression of TLR4,Myeloid differentiation factor 88(MyD88),NF-κB,p-NF-κB,the expression of apoptosis related proteins Bax,cleaved-caspase-3 and anti-apoptotic protein Bcl-2;Flow cytometry was detected the apoptosis rate of cardiomyocyte,EdU staining was observed the proliferation of cardiomyocytes.After TLR4 interference fragment was transfected into cardiomyocytes,the interference fragment was verified by Western blotting.The cells were transfected with si-NC or si-TLR4 and treated with Hcy.Western blotting was detected the expression of MyD88,NF-κB and p-NF-κB,the expression of apoptosis related protein and anti-apoptosis protein in cardiomyocytes;The apoptosis rate of cardiomyocytes was detected by flow cytometry;The apoptosis of cardiomyocytes was observed by EdU staining.Results Compare with control group,Western blotting showed that TLR4,MyD88 and p-NF-κB significantly increased in Hcy group(P<0.01);The expression of apoptosis related proteins Bax and cleaved-caspase-3 increased and the expression of anti-apoptotic protein Bcl-2 was decreased(P<0.01);Compared with si NC+Hcy group,the expression of TLR4,MyD88 and p-NF-κB decreased(P<0.01),the expression of apoptosis related protein decreased(P<0.01),and the expression of anti-apoptosis protein increased in si-TLR4+Hcy group(P<0.01);Compare with control group,the results of flow cytometry showed that the apoptosis rate of cardiomyocytes increased in Hcy group(P<0.01);Compared with si-NC+Hcy group,the apoptosis rate of si-TLR4+Hcy group decreased(P<0.01).Compared with control group,EdU staining showed that the number of positive cells decreased in Hcy group(P<0.01);Compared with si-NC+Hcy group,the number of positive cells increased in si-

关 键 词：同型半胱氨酸 TLR4/NF-κB信号通路 心肌细胞 细胞凋亡 增殖 

分 类 号：R364.5[医药卫生—病理学]