摘要
目的通过体外构建心肌细胞缺氧复氧模型(H/R)模拟体内心肌细胞缺血再灌注,验证阿利吉仑(Aliskiren)对于改善心肌细胞缺血再灌注的药物效果,同时探究细胞凋亡在其中的机制。方法将细胞实验分为四组:正常氧供应组即对照组(Control)、缺氧复氧组(H/R)、阿利吉仑+缺氧复氧组(阿利吉仑+H/R)、NF-κB P65特异抑制剂+缺氧复氧组(bay11-7082+H/R)。使用CCK-8检测不同浓度阿利吉仑处理的心肌细胞存活率,ELISA检测各实验组炎症因子肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)水平。Hoechst33258染色、Annexin V/PI双染流式细胞仪检测各组心肌细胞凋亡比例,JC-1试剂盒测量线粒体膜电位及心肌细胞ATP含量。同时采用Caspase-3试剂盒检测各组心肌细胞凋亡蛋白酶的活性。结果阿利吉仑小于20 mmol/L时,与心肌细胞活性存在正相关关系,而在20 mmol/L和80 mmol/L之间,两者之间存在负相关关系,文章中阿利吉仑的最佳处理浓度是20 mmol/L,此时的心肌细胞活性最高(76.40%±1.64%)。相比H/R组,阿利吉仑能降低TNF-α和IL-6水平[(129.33±5.86)ng/L比(319.00±4.58)ng/L,P<0.05;(29.67±1.53)ng/L比(64.67±2.08)ng/L,P<0.05],同时显著降低心肌细胞的凋亡率[(7.23%±1.14%)比(32.25%±3.15%),P<0.05],并具有降低能量代谢障碍心肌细胞所占比例[(6.9%±1.6%)比(13.5%±1.7%),P<0.05]、稳定线粒体膜电位的功能[(3.90±0.60)比(1.80±0.16),P<0.05]。另外,抑制凋亡蛋白酶Caspase-3的活性[(2.26±0.35)比(3.26±0.62),P<0.05],且阿利吉仑+H/R组与bay11-7082+H/R组的各项实验结果无统计学差异。结论阿利吉仑可以通过抑制炎症反应、调控线粒体受体介导的凋亡,改善缺血心肌细胞缺血再灌注损伤,且阿利吉仑的调控凋亡作用可能与NF-κB表达抑制有关。
Aim Through simulating the ischemia/reperfusion of cardiomyocytes in vivo by constructing a model of cardiomyocyte hypoxia and reoxygenation in vitro,to verify the effect of Aliskiren on improving the myocardial ischemia/reperfusion injury and to explore its mechanism in regulating apoptosis.Methods Cell experiments were divided into four groups:normal oxygen supply group(Control),hypoxia/reoxygenation group(H/R),Aliskiren+H/R group,NF-κB P65 specific inhibitor(bay11-7082)+H/R group.CCK-8 was used to detect the survival rate of cardiomyocytes pretreated with different concentrations of Aliskiren,and ELISA was performed to determine the levels of inflammatory factors(TNF-α,IL-6)in each experimental group.Hoechst33258 staining and Annexin V/PI dual staining flow cytometry were conducted to evaluate the myocardial cell apoptosis ratio in each group.The JC-1 kit was used to measure mitochondrial membrane potential and cardiomyocyte ATP content.Meanwhile,the Caspase-3 activity kit was performed to detect the activity of apoptotic proteases in each group.Results When the concentration of Aliskiren was less than 20 mmol/L,a positive correlation with cardiomyocyte activity was showed,and when between 40 mmol/L and 80 mmol/L,there was a negative correlation.The optimal concentration of Aliskiren was 20 mmol/L at which the myocardial cell activity is the highest.Compared with the H/R group,Aliskiren can decrease the expression of TNF-αand IL-6((129.33±5.86)ng/L vs(319.00±4.58)ng/L,P<0.05;(29.67±1.53)ng/L vs(64.67±2.08)ng/L,P<0.05),and significantly reduce the apoptosis rate of cardiomyocytes((7.23%±1.14%)vs(32.25%±3.15%),P<0.05),and reduced the proportion of cardiomyocytes with energy disorders((6.9%±1.6%)vs(13.5%±1.7%),P<0.05),what’s more,Aliskiren can gain the function of stabilizing mitochondrial membrane potential((3.90±0.60)vs(1.80±0.16),P<0.05)and inhibit the activity of apoptotic protease Caspase-3((2.26±0.35)vs(3.26±0.62),P<0.05).It is of great importance that there was no statistical difference in
作者
古钎林
吴冰璇
黄振华
陈伟栋
韦秋霞
詹红
GU Qianlin;WU Bingxuan;HUANG Zhenhua;CHEN Weidong;WEI Qiuxia;ZHAN Hong(The First Affiliated Hospital of Sun Yat-sen University,Guangzhou,Guangdong 510080,China)
出处
《中国动脉硬化杂志》
CAS
2020年第7期559-565,共7页
Chinese Journal of Arteriosclerosis
基金
国家自然科学基金青年项目(81801948)
广州市科技项目(201804010007)。
