PKC及缝隙连接蛋白Cx43改变在吗啡和舒芬太尼预处理缺氧复氧损伤心肌中的作用  

Role of protein kinase C and Connexin43 alteration during morphine and sufentanil preconditioning in hypoxic myocardium

作　　者：高小茹 张冬梅[2] 王萌[3] 宋华丽[3] 

机构地区：[1]北京市海淀妇幼保健院麻醉科,北京100080 [2]宁夏医科大学总医院麻醉科,宁夏银川750004 [3]宁夏医科大学,宁夏银川750004

出　　处：《中国医院药学杂志》2017年第16期1570-1575,共6页Chinese Journal of Hospital Pharmacy

基　　金：国家自然科学基金(编号:81260029)

摘　　要：目的:探讨蛋白激酶C(PKC)信号通路及心肌缝隙连接蛋白Cx43改变在阿片药物预处理中的作用。方法:原代心肌细胞分离培养。将培养5 d的心肌细胞分成8组。正常对照组(C组)不给予任何处理,建立培养乳鼠心肌细胞缺氧/复氧损伤模型(I/R组),吗啡组(MF组)加入吗啡至终浓度0.3μmol·L^(-1),舒芬太尼组(SF组)加入舒芬太尼至终浓度0.000 3μmol·L^(-1),PKC激动剂PMA+吗啡组(PMA+MF组)加入PMA至终浓度0.02μmol·L^(-1),再进行吗啡预处理;PKC抑制剂Rottlerin+吗啡组(ROT+MF组)加入Rottlerin至终浓度5μmol·L^(-1),再进行吗啡预处理;PKC激动剂PMA+舒芬太尼组(PMA+SF组)加入PMA至终浓度0.02μmol·L^(-1),再进行舒芬太尼预处理;PKC抑制剂Rottlerin+舒芬太尼组(ROT+SF组)加入Rottlerin至终浓度5μmol·L^(-1),再进行舒芬太尼预处理。各组做上述相应处理后取材检测细胞存活率。免疫荧光共聚焦技术检测缝隙连接蛋白Connexin 43(Cx43)的平均光密度(AOD),用Western-blot检测细胞Cx43总蛋白及其磷酸化水平(P-Cx43)的表达量。结果:与C组比较,其余各组心肌细胞存活率、Cx43 AOD值、心肌细胞Cx43总蛋白表达及P-Cx43表达降低(P<0.05);与I/R组比较,MF组、SF组、ROT+MF组、ROT+SF组、PMA+MF组、PMA+SF组心肌细胞存活率、Cx43 AOD值、Cx43总蛋白和P-Cx43表达增高(P<0.05);与MF组比较,ROT+MF组心肌细胞存活率、Cx43 AOD值、Cx43总蛋白及P-Cx43表达降低(P<0.05),SF组心肌细胞存活率、Cx43 AOD值和Cx43总蛋白降低(P<0.05),而P-Cx43表达增高(P<0.05),PMA+MF组心肌细胞存活率、Cx43 AOD值、Cx43总蛋白和P-Cx43表达均增高(P<0.05);ROT+SF组较SF组心肌细胞Cx43 AOD值、Cx43总蛋白和P-Cx43表达低(P<0.05),而PMA+SF组较SF组心肌细胞存活率、心肌细胞Cx43 AOD值、Cx43总蛋白和P-Cx43表达高(P<0.05)。结论:吗啡和舒芬太尼预处理可减轻心肌细胞缺氧/复氧损伤,且吗啡对心肌细胞的保护作用较舒芬太尼强,这种心肌OBJECTIVE To investigate the role of the signaling pathway of protein kinase C and alteration of connexin43 with opioid preconditioning.METHODS Primary myocardial cells were isolated and cultured,and 5 days later cells were allocated to eight groups：control（C）group,hypoxia/reoxygenation（I/R）injury group,morphine preconditioning（MF）group,sufentanil preconditioning（SF）group,phorbol＋ morphine preconditioning（PMA＋MF）group,Rottlerin ＋ morphine preconditioning（ROT ＋ MF）group,phorbol＋sufentanyl preconditioning（PMA＋SF）group,rottlerin＋sufentanyl preconditioning（ROT＋SF）group.The control group was not given any intervention.Myocardial cells I/R injury models were established with neonatal rats.MF group were preconditioned with morphine at the final concentration of 0.3μmol·L^-1,SF group with sufentanyl 0.000 3μmol·L^-1,PMA ＋ MF group with phorbol and 10 minutes later with morphine at 0.3μmol·L^-1,ROT ＋ MF group with rottlerin and 10 minutes later with morphine at 0.000 3μmol·L^-1,PMA ＋ SF group with phorbol and 10 minutes later with sufentanyl at 0.000 3μmol·L^-1,ROT ＋ SF group with rottlerin and 10 minutes later with sufentanyl at 0.000 3μmol·L^-1.After the intervention,cell survival rate was measured.The average optical density（AOD）of the Cx43 protein was observed by immunofluorescence confocal technology,and Western-blot was used to measure the total proteins of Cx43 and P-Cx43.RESULTS Compared with C group,the cell proliferation,the AOD of Cx43,the total proteins of Cx43 and P-Cx43 in each group were significantly decreased（P〈0.05）.Compared with I/R group,cell proliferation,the AOD of Cx43,the total proteins of Cx43 and P-Cx43 in MF group,SF group,ROT ＋ MF group,ROT ＋ SF group,PMA＋ MF group and PMA ＋ SF group were significantly increased（P〈0.05）.Compared with MF group,cell proliferation,the AOD of Cx43,the total proteins of Cx43 and P-Cx43 in ROT ＋ MF group were significantly decreased（P〈0.05）,cell proliferation,the AOD

关 键 词：PKC 阿片药物 缺氧/复氧损伤 Cx43 

分 类 号：R978.6[医药卫生—药品]