糖原合成酶激酶3β对布比卡因所致大鼠心肌细胞凋亡的影响

Effects of Glycogen Synthase Kinase 3β on Bupivacaine-induced Cardiomyocyte Apoptosis in Rats

目的证实糖原合成酶激酶3β(GSK3β)通过影响促凋亡因子、线粒体通透性转运孔(mPTP)、细胞色素C(CytC)表达参与脂肪乳救治布比卡因所致的大鼠心肌细胞凋亡。方法采用RNA干扰(RNAi)腺病毒法,H9C2大鼠心肌细胞复苏培养后,以1×10^5个/mL细胞密度接种于96孔板上,采用随机数字表法分为8个组:空白对照组(C组)、布比卡因组(B组)、脂肪乳组(LE组)、布比卡因+脂肪乳组(B+LE组)、空白对照组+GSK3βRNAi腺病毒(GSK3βi)组(C+GSK3βi组)、布比卡因+GSK3βi组(B+GSK3βi组)、脂肪乳+GSK3βi组(LE+GSK3βi组)、布比卡因+脂肪乳+GSK3βi组(B+LE+GSK3βi组)。B组、LE组、B+LE组分别用终浓度为1 mmol·L^-1布比卡因、1%脂肪乳、1 mmol·L^-1布比卡因+1%脂肪乳的培养基孵育。C+GSK3βi组、B+GSK3βi组、LE+GSK3βi组、B+LE+GSK3βi组经GSK3βi腺病毒感染后第2 d给予以上药物孵育。药物孵育24 h后采用Western blot法检测Bad和CytC蛋白表达,采用流式细胞仪检测细胞凋亡率,采用激光共聚焦显微镜观察钙黄绿素荧光强度值(mPTP开放情况)。结果与C组比较,B组Bad、CytC蛋白表达水平和细胞凋亡率均增加,钙黄绿素荧光强度值下降(P均<0.05);与B组比较,LE组、B+LE组Bad、CytC蛋白表达水平和细胞凋亡率均下降,钙黄绿素荧光强度值增加(P均<0.05);与B+LE组比较,B+LE+GSK3βi组Bad、CytC蛋白表达水平和细胞凋亡率增加,钙黄绿素荧光强度值下降(P均<0.05);与B+GSK3βi组比较,C+GSK3βi组、LE+GSK3βi组Bad、CytC蛋白表达水平和细胞凋亡率下降,钙黄绿素荧光强度值增加(P均<0.05),B+LE+GSK3βi组Bad蛋白表达水平差异无统计学意义(P>0.05),CytC蛋白表达水平下降,细胞凋亡率增加,钙黄绿素荧光强度值下降(P均<0.05)。结论脂肪乳抑制布比卡因所致大鼠心肌细胞凋亡的机制可能与其通过GSK3β减少促凋亡因子Bad的表达,减少mPTP开放,减少CytC表达有关。

Objective To prove glycogen synthase kinase 3β(GSK3β)is involved in the treatment of rat myocardial cell apoptosis caused by bupivacaine through the influence of pro-apoptotic factor,mitochondrial permeability transport pore(m PTP)and cytochrome C(CytC)expression.Methods Using RNA interference(RNAi)adenovirus method,H9C2 cells were recoveried and cultured,then transferred into 96-well cell plates with a density of 105 cells/m L,and were randomly divided into eight groups using a random number table:control group(group C),bupivacaine group(group B),lipid emulsion group(group LE),and bupivacaine+lipid emulsion group(group B+LE),control group+GSK3βi(group C+GSK3βi),bupivacaine+GSK3βi group(group B+GSK3βi),lipid emulsion+GSK3βi group(group LE+GSK3βi),and bupivacaine+lipid emulsion+GSK3βi group(group B+LE+GSK3βi).In groups B,LE and B+LE,the cells were incubated with culture medium containing 1 mmol·L^-1 bupivacaine,1%lipid emulsion,1 mmol·L^-1 bupivacaine and 1%lipid emulsion,respectively.In groups C+GSK3βi,B+GSK3βi,LE+GSKin3βi,B+LE+GSK3βi,the cells were incubated by different drugs as above after infected by adenovirus for two days.After incubation for 24 h,the expressions of Bad and CytC were measured by Western blot,the fluorescence intensity of calcein was observed by laser confocal microscopy(opening of m PTP),and the apoptosis rate was detected by flow cytometry.Results Compared with group C,the expressions of Bad and CytC,apoptosis rate were increased,the fluorescence intensity of calcein was decreased in group B(P all<0.05).Compared with group B,the expressions of Bad and CytC,apoptosis rate were decreased,the fluorescence intensity of calcein was increased in groups LE and B+LE(P all<0.05).In group B+LE,the expressions of Bad and CytC,apoptosis rate were increased,the fluorescence intensity of calcein was decreased compared with group B+LE+GSK3βi(P all<0.05).Compared with group B+GSK3βi,the expressions of Bad and CytC,apoptosis rate were decreased,the fluorescence intensity of calcein were increased in group C+GSK3βi and group LE+GSK3βi(P all<0.05),but in group B+LE+GSK3βi no significant change was found in Bad expression(P>0.05),but the expression of CytC and apoptosis rate were decreased,the fl uorescence intensity of calcein was increased(P all<0.05).Conclusion The mechanism of lipid emulsion inhibited the apoptosis of rat cardiomyocytes induced by bupivacaine is related to GSK3βacting on m PTP by reducing the expression of the pro-apoptotic factor Bad and CytC.