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

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0.3 μmol·L^-1，舒芬太尼组（SF组）加入舒芬太尼至终浓度0.000 3 μmol·L^-1，PKC激动剂PMA+吗啡组（PMA+MF组）加入PMA至终浓度0.02 μmol·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5 μmol·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0.02 μmol·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5 μmol·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，这种心肌细胞保护作用可能与PKC信号通路的激活有关。

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

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 0.3 μmol·L^-1, SF group with sufentanyl 0.000 3 μmol·L^-1, PMA + MF group with phorbol and 10 minutes later with morphine at 0.3 μmol·L^-1, ROT + MF group with rottlerin and 10 minutes later with morphine at 0.000 3 μmol·L^-1, PMA + SF group with phorbol and 10 minutes later with sufentanyl at 0.000 3 μmol·L^-1, ROT + SF group with rottlerin and 10 minutes later with sufentanyl at 0.000 3 μmol·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 of Cx43, the total proteins of Cx43 in SF group were significantly decreased (P<0.05), but the P-Cx43 protein was increased (P<0.05), cell proliferation, the AOD of Cx43, the total proteins of Cx43 and P-Cx43 in PMA + MF group were significantly increased (P<0.05). Compared with SF group, cell proliferation, the AOD of Cx43, the total proteins of Cx43 and P-Cx43 in ROT+SF group were significantly decreased (P<0.05), cell proliferation, the AOD of Cx43, the total proteins of Cx43 and P-Cx43 in PMA+MF group were significantly increased (P<0.05). CONCLUSION Preconditioning with morphine and sufentanil can reduce myocardial hypoxia/reoxygenation injury. And this protective effect of morphine for myocardial cells is stronger than sufentanil, which may be related to activated PKC signaling pathways.