瓜蒌皮水提物通过PI3K/Akt/eNOS信号通路抑制缺血缺氧大鼠原代心肌细胞的凋亡

Water extract of Trichosanthis pericarpium inhibits the apoptosis of ischemic and hypoxic primary cardiomyocytes in rat by regulating PI3K/Akt/eNOS signaling pathway

以激活磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/内皮型一氧化氮合酶(eNOS)信号通路、抑制心肌细胞凋亡为切入点,探究瓜蒌皮水提物(TP-W)保护心肌缺血的机制.分离大鼠心肌细胞,通过缺氧气体+无血清培养液培养制造缺血缺氧损伤模型,以模拟急性心肌缺血时的体内心肌细胞环境;同时添加PI3K特异性抑制剂(LY294002)和TP-W处理细胞,利用倒置相差显微镜观察细胞形态,噻唑兰法、末端标记法分别检测细胞存活率及凋亡率,免疫荧光联合免疫印迹方法定位、定量地检测细胞内PI3K、Akt及eNOS蛋白表达及磷酸化水平.结果显示:对上述缺血缺氧损伤的心肌细胞,TP-W可显著改善其生长状态、提高存活率、降低凋亡率;同时显著上调上述3种蛋白表达水平及磷酸化水平,但磷酸化水平均可被LY294002处理显著削弱.可见,激活PI3K/Akt/eNOS信号通路、抑制心肌细胞凋亡可能是TP-W抗心肌缺血的重要机制,也是中药“开胸除痹”的关键生物学内涵.

This study focused on the activation of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/endothelial nitric oxide synthase(eNOS)signaling pathway and the inhibition of myocardial apoptosis to explore the mechanism of water extract of Trichosanthis pericarpium(TP-W)protecting myocardial ischemia.In order to simulate the living environment of cardiomyocytes during acute myocardial ischemia(AMI)in vivo,rat cardiomyocytes were isolated and cultured in anoxic gas in serum-free medium to create the model of ischemia-hypoxia injury.The cells were treated with PI3K specific inhibitor(LY294002)and TP-W at the same time.The morphological changes of the cells were observed by inverted phase contrast microscopy.The survival rate of cells was measured by Thiazolyl Blue(MTT)method,and the apoptosis rate of cells was detected by terminal uridine nucleotide end labeling(TUNEL)staining.Immunofluorescence and Western blotting were used to detect the expression of PI3K,Akt and eNOS proteins and their phosphorylation levels locally and quantitatively.The results showed that TP-W could significantly improve the growth state,increase survival rate and reduce apoptosis rate of cardiomyocytes injured by ischemia and hypoxia.Meanwhile,the expression and phosphorylation levels of the above three proteins were significantly up-regulated.However,these effects can be significantly reduced by LY294002 treatment.In conclusion,activation of PI3K/Akt/eNOS signaling pathway and inhibition of cardiomyocyte apoptosis may be an important mechanism of TP-W against myocardial ischemia,as well as the key biological connotation of traditional Chinese medicine"opening the chest and removing arthralgia".