nNOS抑制剂亚胺基烯丁基-L-鸟氨酸对心肌缺血再灌注损伤的影响及机制  

Effect of nNOS inhibitor N-5-(1-imino-3-butenyl)-L-ornithine on myocardial ischemia-reperfusion injury and its mechanism

作　　者：周露[1] 李冰艳[1] 顾霞飞 唐广胜 ZHOU Lu;LI Bingyan;GU Xiafei;TANG Guangsheng(Kangda College of Nanjing Medical University,Lianyungang 222000,China)

机构地区：[1]南京医科大学康达学院,江苏连云港222000

出　　处：《药物评价研究》2023年第10期2159-2164,共6页Drug Evaluation Research

基　　金：南京医科大学科技发展基金(NMUB2020039)。

摘　　要：目的探讨nNOS选择性抑制剂亚胺基烯丁基-L-鸟氨酸(L-VNIO)对心肌缺血再灌注(I/R)损伤的影响及机制。方法构建SD大鼠离体心脏I/R模型和H9c2细胞缺氧/复氧(H/R)模型;nNOS抑制剂L-VNIO(10μmol·L^(−1))持续给药整个再灌注或复氧过程。TTC染色测定心肌梗死面积;流式细胞术检测H9c2细胞凋亡率;Fluo-3/AM Ca^(2+)荧光探针通过流式细胞仪检测H9c2细胞内Ca^(2+)浓度;试剂盒法测定离体心脏灌流液乳酸脱氢酶(LDH)、丙二醛(MDA)水平以及H9c2细胞MDA水平和超氧化物歧化酶(SOD)活性;离体心脏提取肌浆网,试剂盒法检测肌浆网Ca^(2+)-ATP酶(SERCA)活性,Western blotting检测肌浆网SERCA蛋白表达;Western blotting检测离体心脏中受磷蛋白(PLB)和兰尼碱受体2(RyR2)蛋白表达水平和磷酸化水平。结果与I/R或H/R模型组相比,L-VNIO显著降低细胞凋亡率,减少心肌梗死面积,降低LDH、MDA水平,提高SOD活性,差异均有统计学意义(P<0.05);此外,与I/R或H/R模型组相比,L-VNIO组明显降低细胞内Ca^(2+)超载,增高PLB磷酸化水平,降低RyR2磷酸化水平,增强SERCA活性(P<0.05)。结论nNOS抑制剂L-VNIO可以减轻I/R损伤,机制与调节Ca^(2+)转运相关蛋白而降低I/R引起的Ca^(2+)超载相关。Objective To investigate the effect of N-5-(1-imino-3-butenyl)-L-ornithine(L-VNIO),a selective inhibitor of nNOS,on myocardial I/R injury and its mechanism.Methods The ischemia/reperfusion(I/R)model of isolated hearts and the hypoxia/reoxygenation(H/R)model of H9C2 cells were established.The nNOS inhibitor L-VNIO was added throughout the reperfusion or reoxygenation process.Infarction size was measured by TTC staining.Cell apoptosis was measured by Annexin V⁃FITC staining with a flow cytometer.Intracellular concentration of Ca^(2+)was determined by Fluo-3/AM as a fluorescent signals using a flow cytometer.The levels of LDH,malondialdehyde(MDA)and the activities of superoxide dismutase(SOD)were detected by the kit.Myocardial sarcoplasmic reticulum was isolated from refused rat hearts and sarcoplasmic reticulum Ca^(2+)-ATPase(SERCA)activity was detected with a kit.The expression of SERCA,phospholamban(PLB),p-PLB,ryanodine receptor 2(RyR2)and p-RyR2 were detected by Western blotting.Results Compared with I/R or H/R model group,L-VNIO significantly decreased the rate of apoptosis,the size of myocardial infarction and the levels of LDH and MDA,while enhanced the activity of SOD(P<0.05).In addition,L-VNIO significantly reduced intracellular Ca^(2+)overload,increased PLB phosphorylation,decreased RyR2 phosphorylation and enhanced SERCA activity compared with I/R or H/R group(P<0.05).Conclusion The nNOS inhibitor L-VNIO can reduce I/R injury and reduce I/R-induced Ca^(2+)overload by regulating Ca^(2+)transport-related proteins.

关 键 词：心肌缺血再灌注损伤 nNOS抑制剂 亚胺基烯丁基-L-鸟氨酸 Ca^(2+)超载 肌浆网Ca^(2+)-ATP酶(SERCA) 受磷蛋白(PLB) 兰尼碱受体 

分 类 号：R965[医药卫生—药理学]