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

发布时间：2018-04-22 18:40

本文选题：PKC + 阿片药物　；参考：《中国医院药学杂志》2017年16期

【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(P0.05);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(P0.05);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(P0.05),SF组心肌细胞存活率、Cx43 AOD值和Cx43总蛋白降低(P0.05),而P-Cx43表达增高(P0.05),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(P0.05);ROT+SF组较SF组心肌细胞Cx43 AOD值、Cx43总蛋白和P-Cx43表达低(P0.05),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(P0.05)。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,这种心肌细胞保护作用可能与PKC信号通路的激活有关。
[Abstract]:Aim: to investigate the role of protein kinase C kinase (PKC) signaling pathway and cardiac gap junction protein (Cx43) in opioid preconditioning. Methods: primary cardiomyocytes were isolated and cultured. Cardiomyocytes cultured for 5 days were divided into 8 groups. Normal control group (group C) is not given any treatment, The model of hypoxic / reoxygenation injury of cultured neonatal rat cardiomyocytes was established. I / R group, morphine group (MF group)) were added morphine to the final concentration of 0. 3 渭 mol / L, and sufentanil group (SF group) were added sufentanil to the final concentration of 0.000 渭 mol / L PMAMF). The final concentration of PMA was 0. 02 渭 mol / L ~ (-1), and then the morphine preconditioning was performed with Rottlerin, the inhibitor of PKC. The rot MF group was added with Rottlerin to the final concentration of 5 渭 mol L ~ (-1). Then the morphine pretreatment with PMA sufentanil agonist PMA sufentanil was used to add PMA to the final concentration of 0. 02 渭 mol L ~ (-1), and then to do sufentanil (0. 02 渭 mol L- 1). Rottlerin sufentanil group was pretreated with sufentanil (5 渭 mol L ~ (-1)) and then sufentanil was pretreated with sufentanil (5 渭 mol 路L ~ (-1)). The cell survival rate was measured after the above treatment. The average optical density of gap junction protein (Connexin 43 Cx43) was detected by immunofluorescence confocal technique, and the expression of Cx43 total protein and its phosphorylation level (P-Cx43) was detected by Western-blot. Results: compared with group C, the survival rate of cardiac myocytes in other groups was determined by Cx43 AOD. Compared with the I / R group, the total protein expression of Cx43 and the expression of P-Cx43 in cardiomyocytes were decreased (P0.05); compared with the I / R group, the expression of Cx43 AOD and the expression of Cx43 total protein and P-Cx43 were increased in the myocytes of the MF group compared with that of the group of MF, and the myocardial fine cells in the group of row MF were compared with those in the group of MF, and the expression of Cx43 total protein and P-Cx43 was increased in the group of PMA-SF than in the group of MF. The expression of Cx43 total protein and P-Cx43 decreased in P0.05 / SF group, while the expression of Cx43 AOD and P-Cx43 in P0.05 / PMA-SF group was significantly higher than that in P0.05 / SF group. The expression of Cx43 total protein and Cx43 protein in SF group were significantly higher than that in P0.05 group (P < 0.05), while the expression of Cx43 AOD and Cx43 total protein and P-Cx43 in SF group were significantly higher than those in P0.05 group (P < 0.05). The expression of Cx43 total protein and P-Cx43 in SF group was significantly higher than that in P0.05 group. Compared with SF group, the expression of Cx43 total protein and P-Cx43 in SF group was lower than that in SF group, but the survival rate of PMA SF group was higher than that of SF group. The expression of Cx43 total protein and P-Cx43 in cardiomyocytes was high (P 0.05). Conclusion: morphine and sufentanil preconditioning can attenuate hypoxia / reoxygenation injury of cardiomyocytes and the protective effect of morphine on cardiomyocytes is stronger than sufentanil which may be related to the activation of PKC signaling pathway.
【作者单位】：北京市海淀妇幼保健院麻醉科;宁夏医科大学总医院麻醉科;宁夏医科大学;
【基金】：国家自然科学基金(编号:81260029)
【分类号】：R614

【相似文献】