Department of Neurosurgery, Zhejiang Provincial Hospital of Traditional Chinese Medicine, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, China.
Eur Rev Med Pharmacol Sci. 2019 Feb;23(4):1789-1796. doi: 10.26355/eurrev_201902_17142.
To elucidate the protective role of resveratrol (RSV) in myocardial apoptosis induced by ischemia-reperfusion injury in rats with acute myocardial infarction (AMI), and to explore its underlying mechanism.
The AMI rat model was successfully established by ligation of the left anterior descending coronary artery. Rat cardiomyocytes were isolated and cultured. Cells were divided into four groups, including: control group (no specific treatment), AMI group (acute ischemia-reperfusion treatment), AMI+RSV group (RSV pretreatment for 24 h before acute ischemia-reperfusion) and AMI+ RSV+LY group (RSV pretreatment combined with 40 μmol/L phosphatidylinositide 3-kinases (PI3K) pathway inhibitor LY294002 for 24 h before acute ischemia-reperfusion). Morphology of apoptotic cardiomyocytes in each group was observed by Hoechst staining. The proliferation, apoptosis and cell cycle progression of cardiomyocytes were determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling (TUNEL) assay and flow cytometry, respectively. Finally, the protein levels of genes relative to PI3K/Akt/eNOS pathway were detected by Western blot.
Hoechst staining showed a large number of necrotic cells, cell retraction, enhanced refractive index and enlarged cell gap in AMI group. A small number of necrotic cells were found in AMI+RSV group, which was significantly fewer than that of AMI group. Meanwhile, remaining cells presented normal morphology. However, a great number of necrotic cells were observed in AMI+RSV+LY group, which was obviously more than that of AMI+RSV group. Compared with control group, cells in AMI group showed significantly decreased proliferative rate, increased early phase, late phase and total one of apoptosis. In AMI group, the ratio of G0/G1 phase was remarkably increased, whereas those of S and G2/M phases were decreased. Moreover, the expression levels of phosphorylated Akt (p-Akt) and phosphorylated e-NOS (p-eNOS) were significantly downregulated in AMI group. In AMI+RSV group, cell apoptosis, cell cycle progression and levels of p-Akt and p-eNOS showed the opposite trends as those of AMI group. However, LY294002 pretreatment reversed the protective role of RSV in cellular behaviors of cardiomyocytes.
RSV protects cardiomyocyte apoptosis from ischemia-reperfusion injury through regulating phosphorylation levels of proteins relative to PI3K/Akt/e-NOS pathway.
阐明白藜芦醇（RSV）在大鼠急性心肌梗死（AMI）缺血再灌注损伤诱导的心肌细胞凋亡中的保护作用，并探讨其作用机制。
结扎大鼠左前降支成功建立 AMI 大鼠模型，分离培养大鼠心肌细胞。将细胞分为四组：对照组（无特殊处理）、AMI 组（急性缺血再灌注处理）、AMI+RSV 组（RSV 预处理 24 h 后再进行急性缺血再灌注）和 AMI+RSV+LY 组（RSV 预处理 24 h 后再用 40 μmol/L 磷脂酰肌醇 3-激酶（PI3K）通路抑制剂 LY294002 处理）。Hoechst 染色观察各组凋亡心肌细胞形态。MTT（3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴盐）法检测细胞增殖，末端脱氧核苷酸转移酶（TdT）介导的 dUTP 缺口末端标记（TUNEL）法和流式细胞术检测细胞凋亡和细胞周期进程，Western blot 检测与 PI3K/Akt/eNOS 通路相关的基因蛋白水平。
Hoechst 染色显示，AMI 组可见大量坏死细胞，细胞收缩，折光性增强，细胞间隙增大。AMI+RSV 组可见少量坏死细胞，明显少于 AMI 组，而存活细胞形态正常。但 AMI+RSV+LY 组可见大量坏死细胞，明显多于 AMI+RSV 组。与对照组相比，AMI 组细胞增殖率明显降低，早期、晚期和总凋亡期增加。AMI 组 G0/G1 期比例显著升高，而 S 期和 G2/M 期比例降低。此外，AMI 组磷酸化 Akt（p-Akt）和磷酸化内皮型一氧化氮合酶（p-eNOS）表达水平明显下调。在 AMI+RSV 组，细胞凋亡、细胞周期进程以及 p-Akt 和 p-eNOS 的水平均呈现与 AMI 组相反的趋势。然而，LY294002 预处理逆转了 RSV 对心肌细胞行为的保护作用。
RSV 通过调节与 PI3K/Akt/eNOS 通路相关蛋白的磷酸化水平来保护心肌细胞免受缺血再灌注损伤诱导的凋亡。
