【摘要】 目的探讨右美托咪定(DEX)对小鼠心脏手术疼痛以及心肌保护的作用及其有关机制。方法取24只8周龄无特定病原体(SPF)雄性C57BL/6小鼠用于构建心肌缺血模型,并根据随机数字表法分为3组:右美托咪定低剂量组(25μg/kg)、高剂量组(50μg/kg)和模型组(注射等量生理盐水)。术后3 d,对小鼠进行疼痛评估。然后分离模型小鼠心肌细胞,根据实验目的分为:对照组:等体积磷酸盐缓冲溶液干预;渥曼青霉素(Wm)组:PI3K抑制剂渥曼青霉素(1 mg/mL)干预; DEX组:右美托咪定(10μmol/L)干预; DEX+Wm组:右美托咪定(10μmol/L)和渥曼青霉素(1 mg/m L)干预。培养72 h后,采用CCK-8检测细胞增殖、TUNEL检测凋亡情况;免疫印迹检测小鼠心肌细胞中磷脂酰肌醇-3-激酶(PI3K)、丝磷酸化苏氨酸蛋白激酶(p-AKT)、Caspase-3和Bcl-2的蛋白表达情况。结果与模型组比较,右美托咪定低剂量组和高剂量组可显著减轻心脏搭桥手术小鼠MGS得分(P <0.05),其中以右美托咪定高剂量组作用更明显(P <0.05);右美托咪定低剂量组和高剂量组小鼠的心率和平均动脉压在心脏搭桥手术后恢复显著(P <0.05)。右美托咪定干预12～72 h,与对照组相比,Wm组和DEX组心肌细胞活力显著降低(P <0.05),而DEX+Wm组与对照组相比则差异无统计学意义(P> 0.05);与Wm组相比,DEX组和DEX+Wm组心肌细胞活力显著上升,且DEX+Wm组也显著高于DEX组(P <0.05)。与对照组相比,右美托咪定处理组小鼠心肌细胞凋亡率降低(P <0.05),而该作用被PI3K抑制剂渥曼青霉素阻断(P <0.05)。在右美托咪定处理的小鼠心肌细胞中,Bcl-2的表达水平上调,而Caspase-3的表达水平显著下调(P <0.05);上述作用同样被PI3K抑制剂渥曼青霉素阻断(P <0.05)。与对照组相比,DEX处理小鼠的心肌细胞内PI3K和AKT的表达水平明显升高,且AKT磷酸化也显著上调(P <0.05)。PI3K抑制剂Wm可明显拮抗右美托咪定的心肌细胞抗凋亡作用(P <0.05)。结论右美托咪定在心脏手术麻醉中可通过活化PI3K/AKT信号通路抑制小鼠心肌细胞凋亡,有利于减轻疼痛、发挥心肌保护作用。更多还原

【Abstract】 Objective To investigate the effect of dexmedetomidine(DEX) on the pain and myocardial protection of mouse cardiac surgery and its related mechanisms. Methods Twenty-four 8-week-old male C57 BL/6 mice with Specific Pathogen Free( SPF) were used to construct the myocardial ischemia model and were divided into 3 groups according to the random number table method: Dexmedetomidine low-dose group( 25 μg/kg),high-dose group( 50 μg/kg) and model group( injected equal amount of saline). Three days after surgery,the mice were evaluated for pain. Then the model mouse cardiomyocytes were isolated. According to the purpose of the experiment,they were divided into:control group: equal volume PBS intervention; wortmannin(Wm) group: PI3K inhibitor wortmannin( 1 mg/m L) intervention; DEX group:dexmedetomidine( 10 μmol/L) intervention; DEX + Wm group: dexmedetomidine(10 μmol/L) and wortmannin(1 mg/m L) intervention. After72 hours of culture,CCK-8 was used to detect cell proliferation and TUNEL was used to detect apoptosis. The protein expressions of phosphatei-dylinositol 3 kinase( PI3K),phosphorylation serine protein kinase( p-Akt),Caspase-3 and Bcl-2 in mouse cardiomyocytes were detected by Western blotting. Results Pretreatment with different doses of dexmedetomidine could significantly reduce the MGS scores of mice undergoing cardiac bypass surgery( P < 0. 05),and the effect of high-dose dexmedetomidine group was more obvious( P < 0. 05). The heart rate and mean arterial pressure of mice in the low-dose and high-dose groups of dexmedetomidine recovered significantly after cardiac bypass surgery( P <0. 05). After dexmedetomidine intervention for 12 to 72 h,compared with the control group,the vitality of myocardial cells in the Wm group and the DEX group was significantly reduced( P < 0. 05),while the DEX + Wm group had no significant difference compared with the control group( P > 0. 05). Compared with the Wm group,the vitality of myocardial cells in the DEX group and the DEX + Wm group increased significantly,and the DEX + Wm group was also significantly higher than the DEX group( P < 0. 05). Compared with the control group,the apoptotic rate of myocardial cells in the dexmedetomidine-treated mice was reduced( P < 0. 05),and this effect was blocked by the PI3K inhibitor wortmannin( P < 0. 05). In dexmedetomidine-treated mouse cardiomyocytes,the expression level of Bcl-2 was up-regulated,while the expression level of Caspase-3 was significantly down-regulated( P < 0. 05); the above effects were also blocked by the PI3K inhibitor wortmannin( P < 0. 05).Compared with the control group,the expression levels of PI3K and AKT in the cardiomyocytes of DEX-treated mice were significantly increased,and AKT phosphorylation was also significantly up-regulated( P < 0. 05). PI3K inhibitor Wm could significantly antagonize the anti-apoptotic effect of dexmedetomidine in cardiomyocytes( P < 0. 05). Conclusion Dexmedetomidine can inhibit mouse myocardial cell apoptosis by activating PI3K/AKT signaling pathway during cardiac surgery anesthesia,which is beneficial to alleviate pain and exert myocardial protection.更多还原