目的 探討nNOS選擇性抑制劑亞胺基烯丁基-L-鳥氨酸（L-VNIO）對心肌缺血再灌注（I/R）損傷的影響及機(jī)制。方法 構(gòu)建SD大鼠離體心臟I/R模型和H9c2細(xì)胞缺氧/復(fù)氧（H/R）模型；nNOS抑制劑L-VNIO（10 μmol·L^-1）持續(xù)給藥整個(gè)再灌注或復(fù)氧過程。TTC染色測定心肌梗死面積；流式細(xì)胞術(shù)檢測H9c2細(xì)胞凋亡率；Fluo-3/AM Ca²⁺熒光探針通過流式細(xì)胞儀檢測H9c2細(xì)胞內(nèi)Ca²⁺濃度；試劑盒法測定離體心臟灌流液乳酸脫氫酶（LDH）、丙二醛（MDA）水平以及H9c2細(xì)胞MDA水平和超氧化物歧化酶（SOD）活性；離體心臟提取肌漿網(wǎng)，試劑盒法檢測肌漿網(wǎng)Ca²⁺-ATP酶（SERCA）活性，Western blotting檢測肌漿網(wǎng)SERCA蛋白表達(dá)；Western blotting檢測離體心臟中受磷蛋白（PLB）和蘭尼堿受體2（RyR2）蛋白表達(dá)水平和磷酸化水平。結(jié)果 與I/R或H/R模型組相比，L-VNIO顯著降低細(xì)胞凋亡率，減少心肌梗死面積，降低LDH、MDA水平，提高SOD活性，差異均有統(tǒng)計(jì)學(xué)意義（P<0.05）；此外，與I/R或H/R模型組相比，L-VNIO組明顯降低細(xì)胞內(nèi)Ca²⁺超載，增高PLB磷酸化水平，降低RyR2磷酸化水平，增強(qiáng)SERCA活性（P<0.05）。結(jié)論 nNOS抑制劑L-VNIO可以減輕I/R損傷，機(jī)制與調(diào)節(jié)Ca²⁺轉(zhuǎn)運(yùn)相關(guān)蛋白而降低I/R引起的Ca²⁺超載相關(guān)。

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

南京醫(yī)科大學(xué)科技發(fā)展基金（NMUB2020039）