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目的:研究地黄多糖(rehmannia polysaccharide)对H_2O_2诱导乳鼠心肌细胞损伤的保护作用及其机制研究。方法:无菌环境下分离并培养SD大鼠乳鼠心肌细胞72h后随机分为:空白对照组、H_2O_2干预组、地黄多糖(10、20和40μg/ml)+H_2O_2干预组和舒血宁注射液(SXN,100μg/ml)+H_2O_2干预组(阳性对照组),每组设10个复孔。各组细胞经药物干预6h后,通过光学纤维镜观察细胞形态并采用MTT法检测细胞存活率;检测培养液中心肌酶:谷草转氨酶(AST)、磷酸肌酸激酶(CPK)、乳酸脱氢酶(LDH)活性;测定细胞中抗氧化酶:超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性和丙二醛(MDA)含量;通过酶联免疫(ELISA)法测定培养液中炎症因子:肿瘤坏死因子-α(TNF-α)、白细胞介素-1(IL-1)、白细胞介素-6(IL-6)含量水平;通过Flow Cytometry检测细胞凋亡状况并计算凋亡率,通过RT-PCR检测细胞中AKT mRNA、bcl-2 mRNA、Bax mRNA表达并计算bcl-2/Bax表达比值。结果:较H_2O_2干预组,地黄多糖(20、40μg/ml)+H_2O_2干预组乳鼠心肌细胞形态明显好转,细胞存活率显著升高,培养液中AST、CPK、LDH活性显著降低,细胞中SOD、CAT活性显著升高且MDA含量显著降低,培养液中TNF-α、IL-1、IL-6含量显著降低,细胞凋亡状况明显改善、凋亡率显著降低,bcl-2 mRNA表达显著上调且Bax mRNA表达显著下调、bcl-2/Bax表达比值显著升高,其中地黄多糖40μg/ml+H_2O_2干预组AKT mRNA表达显著上调。结论:地黄多糖能够有效改善H_2O_2诱导损伤的乳鼠心肌细胞生存状态、提高细胞存活率、降低细胞凋亡率,提示地黄多糖对H_2O_2诱导损伤的乳鼠心肌细胞具有保护作用;其作用机制可能与地黄多糖能够有效改善抗氧化酶活性、抑制氧化应激损伤,调节凋亡相关基因表达,抑制炎症反应相关。
Objective: To study the protective effect of rehmannia polysaccharide on cardiomyocyte injury induced by H_2O_2 and its mechanism. Methods: The isolated and cultured SD rat neonatal rat cardiomyocytes were randomly divided into control group, H 2 O 2 intervention group, Rehmannia glutinosa polysaccharide (10, 20 and 40 μg / ml) + H 2 O 2 intervention group and Shuxuening injection Liquid (SXN, 100μg / ml) + H 2 O 2 intervention group (positive control group). The cells in each group were subjected to drug treatment for 6 hours. The cell morphology was observed by optical fiber microscope and the cell viability was detected by MTT assay. The levels of myocardial enzymes such as aspartate aminotransferase (AST), creatine phosphokinase (CK), lactate dehydrogenase (LDH) activity in the culture medium were measured. The activities of superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) in the cells were measured. The inflammation in the culture medium was determined by enzyme-linked immunosorbent assay The levels of tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1) and interleukin-6 (IL-6) were measured by flow cytometry. The expressions of AKT mRNA, bcl-2 mRNA and Bax mRNA were detected by RT-PCR and the ratio of bcl-2 / Bax expression was calculated. Results: Compared with H 2 O 2 intervention group, the morphology of neonatal rat cardiomyocytes in the treatment of Rehmannia glutinosa polysaccharide (20,40μg / ml) + H 2 O 2 significantly improved, the cell viability increased significantly, the activities of AST, CPK and LDH in the culture fluid decreased significantly, , CAT activity increased significantly and the content of MDA decreased significantly. The content of TNF-α, IL-1 and IL-6 in the culture medium decreased significantly, the apoptosis status was significantly improved, the apoptosis rate was significantly decreased, and the expression of bcl- And the expression of Bax mRNA was significantly down-regulated and the ratio of bcl-2 / Bax expression was significantly increased. The AKT mRNA expression was significantly up-regulated by Rehmannia glutinosa polysaccharide 40μg / ml + H 2 O 2 intervention group. Conclusion: Rehmannia glutinosa polysaccharide can effectively improve the survival of neonatal rat cardiomyocytes induced by H 2 O 2, improve the cell survival rate and reduce the apoptosis rate, suggesting that Rehmannia glutinosa polysaccharide has a protective effect on neonatal rat cardiomyocytes induced by H 2 O 2; its mechanism may be related to Rehmannia polysaccharide can effectively improve antioxidant enzyme activity, inhibit oxidative stress injury, regulate apoptosis-related gene expression and inhibit the inflammatory response.