线粒体通透性转换孔在脂肪乳逆转布比卡因大鼠心肌毒性中的作用

Role of mitochondrial permeability transition pore in lipid emulsion-induced inversion of bupivacaine myocardiotoxicity in rats

目的 评价线粒体通透性转运孔在脂肪乳逆转布比卡因大鼠心肌毒性中的作用.方法 培养H9c2心肌细胞,以105个/ml的密度接种于6孔板,采用随机数字表法分为4组,每组6孔,每孔2 ml：对照组（C组）加入PBS 100μl;布比卡因组（B组）加入布比卡因,终浓度为1 mmol/L;脂肪乳＋布比卡因组（LB组）同时加入脂肪乳和布比卡因,终浓度分别为1％和1 mmol/L;脂肪乳＋布比卡因＋苍术苷组（LBA组）同时加入脂肪乳、布比卡因和线粒体通透性转换孔开放剂苍术苷,终浓度分别为1％、1 mmol/L和30 μmol/L,均孵育24 h.孵育结束后,采用Western blot法测定Bcl-2、Bax、磷酸化Bad （p-Bad）、caspase-3、活化的caspase-3、caspase-9、活化的caspase-9和细胞色素c（Cyt c）的表达水平,采用RT-PCR法检测Bcl-2 mRNA、Bax mRNA、Bad mRNA、caspase-9 mRNA和Cyt c mRNA的表达水平.结果 与C组比较,B组Bax/Bcl-2比值、活化的caspase-3/caspase-3比值、活化的caspase-9/caspase-9比值和Bax mRNA/Bcl-2 mRNA比值升高,p-Bad表达下调,Cyt c、Bad mRNA、caspase-9mRNA和Cyt c mRNA的表达上调（P＜0.05）,LB组上述各指标差异无统计学意义（P＞0.05）;与B组比较,LB组和LBA组Bax/Bcl-2比值、活化的caspase-3/caspase-3比值和活化、caspase-9/caspase-9比值和Bax mRNA/Bcl-2 mRNA比值降低,p-Bad表达上调,Cyt c、Bad mRNA、caspase-9 mRNA和Cyt cmRNA的表达下调（P＜0.05）;与LB组比较,LBA组Bax/Bcl-2比值、活化的caspase-3/caspase-3比值、活化的caspase-9/caspase-9比值和Bax mRNA/Bcl-2 mRNA比值升高,p-Bad表达下调,Cyt c、BadmRNA、caspase-9 mRNA和Cyt c mRNA的表达上调（P＜0.05）.结论 脂肪乳逆转布比卡因大鼠心肌毒性作用的机制与抑制线粒体通透性转换孔的开放有关.

Objective To evaluate the effect of mitochondrial permeability transition pore （mPTP） in lipid emulsion-induced inversion of bupivacaine myocardiotoxicity in rats.Methods H9c2 cells were inoculated in 6-well plates at a density of 105 cells/ml, and were randomly divided into 4 groups （6 wells in each group, 2 ml/well） using a random number table： control group （group C） , bupivacaine group （group B） , lipid emusion ＋ bupivacaine group （group LB） , and lipid emusion ＋ bupivacaine ＋ atractyloside group （group LBA）.Phosphate buffer solution 100 μl was added to the culture medium in group C.In group B, bupivacaine was added to the culture medium with the final concentration of 1 mmol/L.In group LB, lipid emusion and bupivacaine were added to the culture medium with the final concentrations of 1% and 1 mmol/L, respectively.In group LBA, lipid emusion, bupivacaine and atractyloside （an mPTP opener） were added to the culture medium with the final concentrations of 1%, 1 mmol/L and 30 μmol/L, respectively.All the cells were incubated for 24 h.After the end of incubation, the expression of Bcl-2, Bax, phosphorylated Bad （p-Bad） , caspase-3, activated caspase-3, caspase-9,activated caspase-9 and cytochrome c （Cyt c） was detected using Western blot.The expression of Bcl-2 mRNA, Bax mRNA, Bad mRNA, caspase-9 mRNA and Cyt c mRNA was detected using real-time reverse transcriptase polymerase chain reaction.The ratios of Bax/Bcl-2, activated caspase-3/caspase-3, activated caspase-9/caspase-9, and Bax mRNA/Bcl-2 mRNA were calculated.Results Compared with group C,the ratios of Bax/Bcl-2, activated caspase-3/caspase-3, activated caspase-9/caspase-9, and Bax mRNA/ Bcl-2 mRNA were significantly increased, the expression of p-Bad was down-regulated, and the expression of Cyt c, Bad mRNA, caspase-9 mRNA and Cyt c mRNA was up-regulated in group B （P〈0.05） , and no significant change was found in the parameters mentioned above in group LB （P〉0.05）.Compared with group B, the ratios of Bax/Bcl-2, activated caspase-3/caspase-3, activated caspase-9/caspase-9, and Bax mRNA/Bcl-2 mRNA were significantly decreased, the expression of p-Bad was up-regulated, and the expression of Cyt c, Bad mRNA, caspase-9 mRNA and Cyt c mRNA was down-regulated in LB and LBA groups （P〈 0.05）.Compared with group LB, the ratios of Bax/Bcl-2, activated caspase-3/caspase-3, activated caspase-9/caspase-9, and Bax mRNA/Bcl-2 mRNA were significantly increased, the expression of p-Bad was down-regulated, and the expression of Cyt c, Bad mRNA, caspase-9 mRNA and Cyt c mRNA was up-regulated in group LBA （P 〈 0.05）.Conclusion The mechanism underlying lipid emulsioninduced inversion of bupivacaine myocardiotoxicity is related to inhibited mPTP opening in rats.