三七总皂苷抑制心肌细胞凋亡的作用机制研究

目的探讨三七总皂苷（Ponax notoginsengsaponin，PNS）对无糖无血清／缺氧诱导的心肌细胞凋亡的抑制作用。方法将心肌H9c2细胞株分为正常对照组（control）、模型组（model）和PNS不同浓度（0．05、0．25、2．25g／L）组。对照组采用含10％胎牛血清（FBS）的DMEM／F12培养基于普通二氧化碳培养箱培养，模型组采用无糖无血清培养基培养，同时进行缺氧处理诱导细胞凋亡，给药组按照模型组方法处理的同时给予不同浓度的PNS。细胞处理结束后收集细胞．Annexin V／PI染色后流式细胞仪检测不同浓度PNS干预后各组细胞的凋亡率。DCFH-DA荧光探针检测细胞内活性氧的水平。Western blot检测Akt和磷酸化Akt（P—Akt）的表达水平。结果PNS能够抑制缺血环境诱导的心肌细胞凋亡．且呈剂量依赖性。对照组、模型组和PNS处理组细胞凋亡率分别为（8．01±1．44）％、（65．71±3．36）％（vs control P〈0．001）、（65．00±1．24）％、（52．51±1．76）％（vs model P〈0．01）、（23．99±0．76）％（vs model P〈0．001）。DCFH—DA染色结果显示，模型组细胞能够观察到明显的绿色荧光信号，而PNS组荧光信号与模型组比较明显减弱．说明PNS具有清除ROS的作用。Western blot结果显示与对照组比较，模型组p-Akt蛋白表达水平明显降低；与模型组比较，PNS组p-Akt蛋白表达水平显著升高，而P13K特异性抑制剂LY294002明显抑制PNS促进p-Akt表达的作用。结论PNS通过促进p-Akt的活性从而抑制缺血诱导的心肌细胞凋亡。

The Anti-Apoptotic Effect of Panax notoginseng saponin on Rat Cardiomyocytes

Objective To investigate the protective effect of Panax notoginseng saponin （PNS） on apoptosis of rat cardiomyocytes induced by serum, glucose and oxygen deprivation （SGOD）. Method Rat cardiomyocyte H9c2 were divided into normal control group, model group, and PNS （0.05 g/L, 0.25 g/L, 2.25 g/L） groups. The cells in control group were cultured in DMEM/F12 supplemented with 10%FBS in a humidified atmosphere of 5% CO2. Model group cells were cultured in SGOD DMEM. PNS group cells were cultured as model group and treated with PNS at the same time. After treatments, cells were collected and used for apoptotic assay using flow cytometer （FCM） after stained by Annexin V/PI. DCFH-DA was used to detect reactive oxygen species （ROS） in cytoplasm. Akt and Phosphorylated Akt （p-Akt） levels were detected by Western blot assay. Results The FCM analysis showed that PNS could protect H9c2 cell apoptosis induced by ischemia and hypoxia. The apoptotic ratios of normal control group, model group and PNS groups were （8.01±1.44）%,（65.71±3.36）%（vs control P〈0.001）,（65.00±1.24）%,（52.51± 1.76）%（vs model P〈0.01） and （23.99±0.76）%（vs model P〈0.001）, respectively. DCFH-DA staining indicated that SGOD significantly increased cytoplastic ROS. After treatment with PNS cytoplastic ROS significantly decreased. Western blot assay showed that SGOD could significantly decrease the expression of p-Akt compared with normal control group. After treatment with PNS, p-Akt level was significantly increased. This anti-apoptosis effect of PNS was inhibited by LY294002, a PI3K specific inhibitor. Conclusion PNS can inhibit apoptosis of H9c2 induced by ischemia and hypoxia by promoting p-Akt activity.