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目的:建立体外缺氧/复氧(hypoxia/reoxygenation,H/R)模型,以模拟在体心肌细胞缺血/再灌注损伤,观察附子多糖(FPS)对H/R后心肌细胞凋亡的影响,并以Smac/Diablo为切入点探讨其作用机制。方法:采用原代培养的乳鼠心肌细胞建立缺氧/复氧模型,随机分为:正常对照组(Control)、缺氧/复氧组(H/R)、附子多糖(0.1、1、10mg/mL)组。MTT法测定细胞的存活率,流式细胞仪测定心肌细胞凋亡率,荧光定量PCR检测BCL-2 mRNA的表达量,Western blot检测胞浆Smac/Diablo蛋白的表达。结果:与对照组比较,H/R组细胞存活率降低,细胞凋亡率显著增加,BCL-2 mRNA的表达量减少,胞浆中Smac/Diablo的表达增加。与H/R组比较,经附子多糖处理24 h后,附子多糖可呈剂量依赖性增加心肌细胞的存活率,抑制细胞凋亡率,促进BCL-2 mRNA的表达,降低胞浆中Smac/Diablo的表达,在10 mg/mL浓度时保护效应达到峰值。结论:FPS可抑制缺氧复氧后心肌细胞凋亡的发生,作用机制可能与其促进BCL-2 mRNA的表达,保护线粒体,抑制Smac/Diablo的释放,从而阻碍细胞凋亡的线粒体信号转导有关。
OBJECTIVE: To establish a hypoxia / reoxygenation (H / R) model in vitro to simulate cardiomyocyte ischemia / reperfusion injury and to observe the effect of monkshood polysaccharide (FPS) on cardiomyocyte apoptosis after H / R , And Smac / Diablo as a breakthrough point to explore its mechanism of action. Methods: Hypoxia / reoxygenation model was established by primary cultured neonatal rat cardiomyocytes and randomly divided into control group, H / R group, monkshood polysaccharide (0.1, 1, 10 mg / mL) group. Cell viability was measured by MTT assay. Cardiomyocyte apoptosis rate was determined by flow cytometry. The expression of BCL-2 mRNA was detected by real-time quantitative PCR. The expression of Smac / Diablo protein was detected by Western blot. Results: Compared with the control group, the survival rate of H / R group was decreased, the apoptosis rate was significantly increased, the expression of BCL-2 mRNA was decreased and the expression of Smac / Diablo in cytoplasm was increased. Compared with the H / R group, the aconite polysaccharide could increase the survival rate of cardiomyocytes, inhibit the apoptosis rate, promote the expression of BCL-2 mRNA and decrease the expression of Smac / Diablo in the cytoplasm The protective effect peaked at a concentration of 10 mg / mL. Conclusion: FPS can inhibit cardiomyocyte apoptosis after hypoxia-reoxygenation and its mechanism may be related to its role in promoting the expression of BCL-2 mRNA, protecting mitochondria, inhibiting the release of Smac / Diablo, and thus impeding the apoptosis of mitochondria. .