葛根素穴位注射预处理对布比卡因诱发的心肌损伤影响及机制探讨

Puerarin point injection pre-treatment alleviates myocardial injury through regulating mitochondrial function

目的探讨葛根素穴位注射预处理对布比卡因所致大鼠心肌线粒体损伤的保护作用及可能机制。方法采用随机数字表法,将40只雄性Wistar大鼠分为空白组、布比卡因组、葛根素治疗组、葛根素预防组,每组10只。空白组在双侧内关穴注入生理盐水各0.1 mL;布比卡因组经股静脉输注0.5%布比卡因10 mg/kg,出现心律失常后立即在双侧内关穴注入生理盐水各0.1 mL;葛根素治疗组经股静脉输注0.5%布比卡因10 mg/kg,出现心律失常后立即在双侧内关穴注入葛根素注射液各0.1 mL;葛根素预防组在双侧内关穴注入葛根素注射液各0.1 mL后5 min,输注0.5%布比卡因10 mg/kg。空白组在注入生理盐水的第8 min、其余组在注入利多卡因的第8 min处死大鼠。取左室心肌组织,分离心肌细胞线粒体。采用ELISA法测定细胞色素C氧化酶(COX)、单胺氧化酶(MAO)活性,Western blot法测定电压依赖性阴离子通道1(VDAC1)、解偶联蛋白2(UCP2)、腺嘌呤核甘酸转运子1(ANT1)、B细胞淋巴瘤-2(Bcl-2)蛋白表达情况,RT-PCR法测定VDAC1、UCP2、ANT1、Bcl-2 mRNA表达情况。结果与空白组比较,布比卡因组心肌细胞线粒体中MAO活性和VDAC1、UCP2、ANT1蛋白及mRNA表达量均明显升高(P均<0.05),COX活性、Bcl-2蛋白及mRNA表达量均明显降低(P均<0.05)。与布比卡因组比较,葛根素预防组心肌细胞线粒体中MAO活性和VDAC1、UCP2、ANT1蛋白及mRNA表达量均明显降低(P均<0.05),COX活性、Bcl-2蛋白及mRNA表达量均明显升高(P均<0.05)。结论葛根素穴位注射预处理可能通过调节线粒体COX、MAO活性和VDAC1、UCP2、ANT1、Bcl-2表达来减轻布比卡因中毒大鼠心肌损伤。

Objective It is to investigate the protective effect of puerarin point injection pre-treatment on the damage of myocardial mitochondria in bupivacaine intoxicated rats.Methods A total of 40 male Wistar rats were divided into 4 groups by random number table method:blank group,bupivacaine group,puerarin treatment group and puerarin pre-treatment group,with 10 rats in each group.The blank was injected with 0.9%normal saline 0.1 mL via bilateral PC6 points;the bupivacaine group was infused with 0.5%bupivacaine 10 mg/kg via femoral vein and 0.9%normal saline 0.1 mL via bilateral PC6 points immediately after arrhythmia;the puerarin treatment group was infused with 0.5%bupivacaine 10 mg/kg via femoral vein,and puerarin point injection 0.1 mL via bilateral PC6 points immediately after arrhythmia occurred;the puerarin pre-treatment group was injected with puerarin point 0.1 mL via bilateral PC6 points and 0.5%bupivacaine(10 mg/kg)via femoral vein 5 min later.The rats in the blank group were sacrificed 8 minutes after the injection of normal saline,and the rats in the other groups were sacrificed 8 minutes after the infusion of bupivacaine.Left ventricular myocardial tissue was taken and mitochondria of myocardial cells were isolated.The activities of cytochrome C oxidase(COX)and monoamine oxidase(MAO)were measured by ELISA method.The protein expressions of uncoupling protein 2(UCP2),voltage-dependent anion channel 1(VDAC1),adenine ribonucleic acid transporter 1(ANT1)and B cell lymphoma-2(Bcl-2)were measured by Western blot.The expression of UCP2,VDAC1,ANT1 and Bcl-2 mRNA was measured by RT-PCR.Results Compared with the blank group,the activity of MAO and the expression of UCP2,VDAC1,ANT1 protein and mRNA in bupivacaine group were significantly increased(all P<0.05),while the activity of COX,the expression of Bcl-2 protein and mRNA were significantly decreased(all P<0.05).Compared with bupivacaine group,the activity of MAO and the expression of UCP2,VDAC1,ANT1 protein and mRNA in pueratin pretreatment group were significantly decreased(all P<0.05),while the activity of COX,the expression of Bcl-2 protein and mRNA were significantly increased(all P<0.05).Conclusion Puerarin acupoint injection pre-treatment may alleviate bupivacaine-induced myocardial damage by regulating the activities of mitochondrial COX and MAO,and the expression of UCP2,ANT1,VDAC1,Bcl-2.