摘要
目的 评价阿片受体、磷脂酰肌醇-3-激酶/丝氨酸-苏氨酸蛋白激酶（PI3K/Akt）和细胞外信号调节激酶（ERK）信号通路在瑞芬太尼预处理（RPC）减轻大鼠心肌细胞缺氧/复氧（H/R）损伤中的作用.方法 健康成年雄性SD大鼠,处死后取心室肌组织,原代培养心肌细胞,调整细胞浓度为2×104个/ml后接种于24孔细胞培养板（500 μl/孔）中,采用随机数字表法,将其中108个培养孔分为12组（n=9）：正常对照组（C组）；H/R损伤组（H/R组）采用缺氧90 min/复氧120 min的方法制备心肌细胞H/R损伤模型；缺氧预处理组（HPC组）在H/R前,培养孔经缺氧10 min/复氧30 min；瑞芬太尼预处理组（RPC组）在H/R前,以终浓度1μmol/L瑞芬太尼孵育心肌细胞10 min,再常规培养30 min;δ受体拮抗剂纳曲吲哚＋ RPC组（NTD＋ RPC组）、κ受体拮抗剂nor-binahorphimine（nor-BNI）＋ RPC组（BNI＋RPC组）、PI3K/Akt信号通路阻断剂渥曼青霉素＋RPC组（W＋ RPC组）、ERK信号通路阻断剂PD98059＋ RPC组（PD＋ RPC组）在RPC前10 min分别维持培养孔终浓度5μmol/L纳曲吲哚和nor-BNI、0.1μmol/L渥曼青霉素和30 μmol/L PD98059,然后与瑞芬太尼共孵育10 min,再常规培养30 min,随后行H/R；试剂对照组分别为NTD组、BNI组、W组和PD组.于复氧结束时,测定心肌细胞活力、细胞凋亡率和培养液乳酸脱氢酶（LDH）活性.结果 与C组比较,H/R组心肌细胞活力降低,细胞凋亡率和培养液LDH活性升高（P＜0.05）；与H/R组比较,HPC组、RPC组和BNI＋ RPC组心肌细胞活力升高,细胞凋亡率和培养液LDH活性降低（P＜0.05）,NTD＋ RPC组、W＋ RPC组、PD＋ RPC组、NTD组、BNI组、W组和PD组上述指标差异无统计学意义（P＞0.05）；与RPC组比较,NTD＋ RPC组、BNI＋ RPC组、W＋ RPC组和PD＋ RPC组心肌细胞活力降低,细胞凋亡率和培养液LDH活性升高（P＜0.05）.结论 瑞芬太尼预处理可能通过激活δ受体,进而激活PI3K/Akt和ERK信号通路,从而减轻大鼠心肌细胞H/R损伤.
Objective To evaluate the role of opioid receptors and phosphatidylinositol 3-kinase/proteinserine-threonine kinases （PI3K/Akt） and extracellular signal-regulated kinase （ERK） signaling pathways in reduction of hypoxia/reoxygenation （H/R）-induced injury to cardiomyocytes by remifentanil preconditioning in rats.Methods Primary cardiomyocytes were obtained from adult male Sprague-Dawley rats and cultured in DMEM culture medium.The cells were seeded in 48-well plates （density 2 × 104 cells/ml,500 μl/well） and randomly divided into 12 groups （n =9 each）：control group （group C）,group H/R,hypoxia preconditioning group （group HPC）,remifentanil preconditioning （RPC） group,naltrindole （δ receptor antagonist） ＋ RPC group,nor-binaltorphimine （κ receptor antagonist） ＋ RPC group （BNI ＋ RPC group）,wortmannin （PI3K inhibitor） ＋ RPC group （W＋ RPC group）,PD98059 （ERK inhibitor） ＋ RPC group （PD ＋ RPC group）,NTD group,BNI group,W group and PD group.In group H/R,the cardiomyocytes were exposed to 90 min of hypoxia,followed by 120 min of reoxygenation.In group HPC,the cardiomyocytes were exposed to 10 min of hypoxia,followed by 30 min of reoxygenation before H/R.In group RPC,the cardiomyocytes were preconditioned with remifentanil with the final concentration of 1 μmol/L for 10 min,followed by 30 min routine culture before H/R.In NTD ＋ RPC,BNI ＋ RPC,W ＋ RPC and PD ＋ RPC groups,naltrindole 5μmol/L （final concentration）,nor-binaltorphimine 5 μmol/L （final concentration）,wortmannin 0.1 μmol/L （final concentration） and PD98059 30μmol/L （final concentration）were added,respectively,and then the cells were coincubated with remifentanil for 10 min,followed by 30 min routine culture before H/R.The viability of cardiomyocytes,cell apoptosis and activity of lactate dehydrogenase （LDH） in the culture medium were detected.The apoptosis rate （AR） was calculated.Results Compared with group C,the viability of cardiomyocytes,AR and activity of LDH in the culture medium were significantly increased in group H/R （P 〈 0.05）.Compared with group H/R,the viability of cardiomyocytes,AR and activity of LDH in the culture medium were significantly decreased in HPC,RPC and BNI ＋ RPC groups （P 〈 0.05）,and no significant changes were found in the parameters mentioned above in NTD ＋ RPC,W ＋ RPC,PD ＋ RPC,NTD,BNI,W,and PD groups （P 〉 0.05）.The viability of cardiomyocytes was significantly lower,and the AR and activity of LDH in the culture medium were higher in NTD ＋ RPC,BNI ＋ RPC,W ＋ RPC,and PD ＋ RPC groups than in RPC group （P 〈 0.05）.Conclusion Remifentanil preconditioning activates PI3K/Akt and ERK signaling pathways possibly through activating δ opioid receptors thus attenuating H/R-induced injury to cardiomyocytes in rats.
出处
《中华麻醉学杂志》
CAS
CSCD
北大核心
2013年第9期1117-1120,共4页
Chinese Journal of Anesthesiology
基金
国家自然科学基金青年科学基金（81200171）
