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（10 μmol·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 Ca²⁺荧光探针通过流式细胞仪检测H9c2细胞内Ca²⁺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Ca²⁺-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Ca²⁺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Ca²⁺转运相关蛋白而降低I/R引起的Ca²⁺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 Ca²⁺ 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 Ca²⁺-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 Ca²⁺ 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 Ca²⁺ overload by regulating Ca²⁺ transport-related proteins.

R965

南京医科大学科技发展基金（NMUB2020039）