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目的探讨加味丹参饮治疗心肌缺血再灌注损伤的可能作用机制。方法将体外培养的H9C2心肌细胞随机分为6组,每组6个复孔。空白血清组在含15%空白血清培养液中常规培养29 h;缺氧预处理组更换缺氧培养液缺氧20 min,复氧20 min,再缺氧20 min后换为空白血清培养液常规培养24 h,缺氧2 h,再给氧3 h;缺氧/复氧组在含15%空白血清培养液中常规培养24 h,更换缺氧培养液,缺氧2 h,再给氧3 h;含药血清组在含15%药物血清中常规培养24 h,缺氧2 h,再给氧3 h;含药血清+自噬抑制剂组用3-甲基腺嘌呤10 mmol/L预处理30 min,余同含药血清组;含药血清+自噬激动剂组用雷帕霉素100 nmol/L预处理30 min,余同含药血清组。倒置显微镜观察各组心肌细胞生长及形态变化,比色法检测定各组细胞培养上清液中乳酸脱氢酶(LDH)漏出率,透射电子显微镜观察心肌细胞自噬体形态及数量变化,Western blot法检测各组自噬相关蛋白LC3Ⅱ及Beclin1表达。结果与空白血清组比较,缺氧/复氧组镜下心肌细胞变性坏死程度增加,电镜下有少量自噬体,培养液中LDH漏出率增加,Beclin1蛋白水平增加(P<0.05或P<0.01);与缺氧/复氧组比较,含药血清组及含药血清+自噬激动剂组镜下心肌细胞变性坏死程度减轻,电镜下自噬体数量增多,培养液中LDH漏出率显著降低,LC3Ⅱ及Beclin1蛋白表达上升(P<0.05)。结论加味丹参饮可能通过上调自噬相关蛋白LC3Ⅱ及Beclin1表达,增强细胞自噬,从而保护损伤的心肌细胞。
Objective To explore the possible mechanism of modified Danshen Decoction on myocardial ischemia-reperfusion injury. Methods H9C2 cardiomyocytes cultured in vitro were randomly divided into 6 groups with 6 replicate wells. The blank serum group was routinely cultured for 29 h in medium containing 15% blank serum. In hypoxic preconditioning group, hypoxia medium was replaced by hypoxia for 20 min and reoxygenated for 20 min, and then replaced by blank serum medium The cells were cultured for 24 h in hypoxia for 2 h and then in oxygen for 3 h. Hypoxia / reoxygenation group was cultured in 15% blank serum medium for 24 h, h; serum-containing group was routinely cultured in serum containing 15% for 24 h, hypoxia for 2 h, then oxygen for 3 h; serum-containing + autophagy inhibitor group was treated with 10 mmol / L 3-methyladenine Treatment with 30 min, more than the same drug-containing serum group; drug-containing serum + autophagy agonist group with rapamycin 100 nmol / L pretreatment for 30 min, Yu the same drug-containing serum group. The growth and morphological changes of cardiomyocytes in each group were observed by inverted microscope. The leakage rate of lactate dehydrogenase (LDH) in the cell culture supernatant of each group was determined by colorimetric assay. The morphological and quantitative changes of autophagosomes were observed by transmission electron microscopy. blot was used to detect the expression of autophagy-related proteins LC3Ⅱ and Beclin1 in each group. Results Compared with blank serum group, the degree of degeneration and necrosis of cardiomyocytes in hypoxia / reoxygenation group was increased, and a small amount of autophagosome was observed under electron microscope. The leakage rate of LDH increased and Beclin1 protein level increased (P <0.05 or P <0.01) ). Compared with the hypoxia / reoxygenation group, the degree of degeneration and necrosis of myocardial cells in the drug-containing serum group and the drug-containing serum + autophagy agonist group was reduced as compared with the hypoxia / reoxygenation group. The number of the autophagosome increased under the electron microscope and the LDH leakage rate in the culture medium was significantly decreased , LC3Ⅱ and Beclin1 protein expression increased (P <0.05). Conclusion Modified Salvia miltiorrhiza drink may protect the damaged cardiomyocytes by up-regulating the expression of autophagy-related proteins LC3 Ⅱ and Beclin1 and enhancing autophagy.