Notch1减轻心肌缺血再灌注损伤作用的研究

The effect of Notchl on myocardial ischemia reperfusion injury

目的探讨Notch1减轻心肌缺血再灌注损伤作用的分子机制。方法采用Noteh1胞内结构域（NIICD）慢病毒表达载体（Lv—N1ICD）及N1ICD慢病毒干扰载体（Lv—N1ICD—shRNA）分别感染H9e2心肌细胞，建立体外心肌缺血再灌注（H／R）、缺血预适应（IPC）及缺血后适应（IPost）模型，从而分为对照组、缺氧／复氧（H／R）组、缺氧／复氧＋N1ICD感染（H／R＋N1ICD）组、缺血预适应（IPC）组、缺血预适应＋NIICD干扰（IPC＋N1ICD—shltNA）组、缺血后适应（IPost）组、缺血后适应＋N1ICD干扰（IPost＋N1ICD-shRNA）组。采用凋亡检测试剂盒、活性氧（ROS）检测试剂盒分别检测细胞凋亡、细胞内ROS；采用免疫印迹法（Western blot）检测磷酸化糖原合成酶激酶-3β／糖原合成酶激酶-3β（p-GSK-3β／GSK-3B）的表达。结果心肌细胞凋亡率在对照组、H／R组、H／R＋N1ICD组、IPC组、IPC＋N1ICD—shRNA组、IPost组、IPost＋N1ICD—shRNA组分别为（5．34±1．70）％、（47．03±4．10）％、（33．89±12．25）％、（35．85±3．17）％、（51．12±8．22）％、（37．35±3．82）％、（47．90±3．08）％，差异有统计学意义（F=18．47，P〈0．05）。心肌细胞ROS在对照组、H／R组、H／R＋N1ICD组、IPC组、IPC＋N1ICD—shRNA组、IPost组、IPost＋N1ICD—shRNA组分别为624．66±79．52、1221．87±63．66、913．12±115．82、935．85±201．62、1204．03±113．82、967．15±106．11、1296．59±222．38，差异有统计学意义（F：8．77，P〈0．05）；心肌细胞p-GSK-3β／GSK-3β比值在对照组、H／R组、H／R＋N1ICD组、IPC组、IPC＋N1ICD—shRNA组、IPost组、IPost＋N1ICD-shRNA组分别为0．58±0．12、0．62±0．20、1．24±0．09、1．16±0．12、0．72±0．15、1．16±0．12、0．75±0．12，差异有统计学意义（F=11．21，P〈0．05）。结论Notchl作为内源性心肌保护因子，通过促进GSK-3磷酸化，抑制心肌细胞凋亡，减轻心肌细胞ROS形成，从而减轻心肌缺血再灌注损伤。

Objective To investigate the protective effects of Notchl on myocardial ischemia reperfusion injury and explore underlying molecular mechanism. Methods H9c2 cells were exposed to hypoxia/reoxygenation （H/R）, isehemic preconditioning （IPC） and ischemic posteonditioning （IPost） treatment following infection with lentiviral vector-Notchl intracellular domain （Lv-N11CD ） or Lv-N11CD- shRNA, and divided into control group, H/R group, H/R ＋ NIICD group, IPC group, IPC ＋ NIICD- shRNA group, IPost group, IPost ＋ NlICD-shRNA group, respectively. Apoptosis was detected by Annexin V/propidium iodide （PI）, and reactive oxygen species （ROS） was detected by 2＇, 7＇ dichlorofluorescin- diacetate （DCFH-DA） . The expression of p-glycogen synthase kinase-3β （p-GSK-3β）/GSK-3β were detected by Western blot analysis. Results The apoptosis rate of cardiomyocytes in control group, H/R group, H/R ＋NIICD group, IPC group, IPC ＋ NIlCD-shRNA group, IPost group, IPost ＋ NIICD-shRNA group was （5.34 ±1.70）%, （47.03 ±4. 10）%, （33.89±12.25）%, （35.85 ±3. 17）%, （51.12±8.22 ）% , （37.35 ±3.82 ）% , （47.90 ± 3.08 ）% , respectively, showing significant differences in all group （F= 18.47, P 〈0.05）. ROS in control group, H/R group, H/R ＋ NIICD group, IPC group, IPC ＋N11CD-shRNA group, IPost group, IPost ＋ N IlCD-shRNA group was 624. 66 ± 79. 52, 1 221.87 ± 63.66, 913.12±115.82, 935.85 ± 201.62, 1 204. 03 ± 113.82, 967. 15 ± 106.11, 1 296. 59 ± 222. 38, respectively, showing significant differences in all group （ F = 8.77, P 〈 0. 05 ）. The ratio of p-GSK-3β to GSK-3β in control group, H/R group, I-I/R ＋ N1lCD group, IPC group, IPC ＋ NIlCD-shRNA group, IPost group, IPost ＋ NIlCD-shRNA group was 0. 58 ±0. 12, 0.62 ± 0.20, 1.24 ± 0.09, 1.16 ± 0. 12, 0.72±0. 15, 1.16 ±0. 12, 0. 75±0. 12, respectively, showing significant differences in all group （F = 11.21, P 〈 0.05 ）. Conclusion As an endogenous cardioprotective factor, Notchl reduces myocardial ischemia reperfusion injury by promoting GSK-3[5 phosphorylation, inhibiting cardiomyocyte apoptosis and reducing ROS formation.