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目的:观察附子多糖后处理对缺氧复氧心肌细胞的保护,并以锰超氧化物歧化酶(MnSOD)表达为着眼点,探讨其作用机制。方法:建立乳鼠心肌细胞缺氧/复氧模型,将乳鼠心肌细胞分为正常对照组、缺氧/复氧组、缺氧后适应组、附子多糖(0.1mg/ml、1mg/ml、10mg/ml)后处理组。缺氧/复氧组给予心肌细胞缺氧3h后复氧6h;缺氧后适应组在细胞缺氧3h后,复氧前即给予3个循环的5min,复氧/5min缺氧,随后复氧6h;附子多糖后处理组在缺氧3h后,将心肌细胞换入含附子多糖浓度分别为10mg/ml、1mg/ml、0.1mg/ml的培养液中常规培养6h。流式细胞仪测定心肌细胞线粒体凋亡率和线粒体膜电位,荧光定量PCR测定Bcl-2mRNA和MnSODmRNA的表达量,黄嘌呤氧化酶法检测细胞内MnSOD的活性。结果:与缺氧/复氧组相比较,予10mg/ml浓度的附子多糖后处理可以有效保护线粒体膜电位的稳定,促进Bcl-2mRNA的表达,抑制心肌细胞凋亡的发生。附子多糖可有效促进MnSOD基因表达和增加MnSOD的活性,并呈一定的浓度依赖性。结论:附子多糖后处理对缺氧/复氧后心肌细胞具有保护作用,其机制可能与附子多糖促进锰超氧化物歧化酶的表达合成,保护线粒体,抑制细胞凋亡有关。
OBJECTIVE: To observe the protection of aconite polysaccharide aftertreatment on hypoxia-reoxygenation cardiomyocytes and investigate its mechanism of action by using manganese superoxide dismutase (MnSOD) as the focus. Methods: The hypoxia / reoxygenation model was established in neonatal rat cardiomyocytes. Cardiomyocytes were divided into normal control group, hypoxia / reoxygenation group, post-hypoxia adaptation group, monkshood polysaccharide (0.1mg / ml, 10 mg / ml) post-treatment group. In hypoxia / reoxygenation group, cardiomyocytes were reoxygenated for 6 hours after hypoxia for 3 hours. After hypoxia for 3 hours, cells in hypoxia group were given hypoxia for 5 min, reoxygenation / 5 min before reoxygenation, 6h; the aconite polysaccharide aftertreatment group 3h after hypoxia, the exchange of myocardial cells containing monkshood polysaccharides were 10mg / ml, 1mg / ml, 0.1mg / ml of culture medium routinely cultured 6h. Mitochondrial apoptotic rate and mitochondrial membrane potential were measured by flow cytometry. The expression of Bcl-2 mRNA and MnSOD mRNA were detected by fluorescence quantitative PCR. The activity of MnSOD in cells was detected by xanthine oxidase method. Results: Compared with the hypoxia / reoxygenation group, postconditioning with monkshood polysaccharide at a concentration of 10 mg / ml could effectively protect the mitochondrial membrane potential, promote the expression of Bcl-2 mRNA and inhibit the apoptosis of cardiomyocytes. Monkshood polysaccharide can effectively promote MnSOD gene expression and increase MnSOD activity, and showed a certain concentration-dependent. CONCLUSION: Aconite polysaccharide postconditioning can protect cardiomyocytes against hypoxia / reoxygenation. The mechanism may be that monkshood polysaccharide can promote the expression of manganese superoxide dismutase, protect mitochondria and inhibit apoptosis.