PKC在缺氧预处理和去甲肾上腺素预处理减轻乳鼠心肌细胞缺氧复氧损伤中的作用

Role of protein kinase C in reduction of hypoxia-reoxygenation injury by hypoxic preconditioning or norepinephrine preconditioning in cultured neonatal rat cardiomyocytes

目的 评价蛋白激酶C（PKC）在缺氧预处理和去甲肾上腺素预处理减轻乳鼠心肌细胞缺氧复氧损伤中的作用.方法 原代培养乳鼠心肌细胞,随机分为6组（n=25）：对照组（Ⅰ组）常规培养;缺氧复氧组（Ⅱ组）细胞缺氧3 h,复氧1 h;缺氧预处理组（Ⅲ组）缺氧20 min,复氧20 min后制备缺氧复氧模型;去甲肾上腺素预处理组（Ⅳ组）细胞经终浓度为10-7 mol/L去甲肾上腺素孵育30 min后,去除去甲肾上腺素,再行缺氧复氧;H7＋缺氧预处理组（Ⅴ组）细胞经终浓度为5×10-5 mol/L的H7孵育10 min后,去除H7,其余操作同Ⅲ组;H7＋去甲肾上腺素预处理组（Ⅵ组）细胞经终浓度为5×10-5 mol/L的H7（PKC活性抑制剂）孵育10 min后,去除H7,其余操作同Ⅳ组.复氧结束后,测定心肌细胞存活率、培养液乳酸脱氢酶（LDH）、肌酸激酶（CK）活性和心肌细胞MDA含量和SOD活性.结果 与Ⅰ组比较,Ⅱ组细胞存活率和SOD活性降低,LDH、CK的活性及MDA含量升高（P＜0.01）.与Ⅱ组比较,Ⅲ组和Ⅳ组细胞存活率和SOD活性升高,LDH、CK活性及MDA含量降低（P＜0.01）.与Ⅲ组比较,Ⅴ组细胞存活率和SOD活性降低,LDH、CK活性及MDA含量升高（P＜0.01）.与Ⅳ组比较,Ⅵ组细胞存活率和SOD活性降低,LDH、CK活性及MDA含量升高（P＜0.05）.结论 PKC激活参与了缺氧预处理与去甲肾上腺素预处理减轻乳鼠心肌细胞缺氧复氧损伤.

Objective To evaluate the role of protein kinase C （PKC） in reduction of hypoxia-reoxygenation （H/R） injury by hypoxic preconditioning or norepinephrine preconditioning in cultured neonatal rat cardiomyocytes. Methods Primary cultured neonatal rat cardiomyocytes were randomly divided into 6 groups （n = 25 each）： control group （group Ⅰ）, H/R group （group Ⅱ）, hypoxia preconditioning group （group Ⅲ）, norepinephrine preconditioning group （group Ⅳ）, H7 ＋ hypoxia preconditioning group （group Ⅴ） and H7 ＋ norepinephrine preconditioning group （group Ⅵ）. In group Ⅱ , the cardiomyocytes were exposed to 3 h of hypoxia followed by 1 h of reoxygenation. In group Ⅲ, the cells were subjected to 20 min of hypoxia followed by 20 min of reoxygenation before H/R. Norepinephrine was added to the culture medium with a final concentration of 10- 7 mol/L,and then the cells were cultured for 30 min before H/R in group Ⅳ. H7 was added to the culture medium with a final concentration of 5 × 10-5 mol/L, the cells were then cultured for 10 min, and the following procedures before H/R were the same as thase described in group Ⅴ . H7 was added to the culture medium with a final concentration of 5 × 10-5 mol/L, the cells were then cultured for 10 min, and the following procedures were the same as those described in group Ⅵ. The cell survival rate, the activities of LDH and CK in the supernatant, and the content of MDA and activity of SOD in cardiomyocytes were determined. Results The cell survival rate and activity of SOD were significantly lower, while the LDH and CK activities and MDA content higher in group Ⅱ than in group Ⅰ ,in group Ⅴ than in group Ⅲ, and in group Ⅵ than in group Ⅳ （P 〈 0.01）. The cell survival rate and activity of SOD were significantly increased, while the LDH and CK activities and MDA content decreased in group Ⅲ and Ⅳ compared with group Ⅱ （P〈0.01）.Conclusion The activiation of PKC is involved in the reduction of H/R injury by hypoxic preconditioning or norepinephrine preconditioning in cultured neonatal rat cardiomyocytes.