There is no safe and effective antidote for cardiotoxicity induced by bupivacaine, an extensively used anaesthetic. Our previous research shows that acupoint treatment may partially improve bupivacaine-induced cardiotoxicity, but the mechanism of action needs to be further studied.

To observe the effect of acupoint injection of puerarin on mitochondrial energy metabolism in myocardia in bupivacaine-poisoned rats, and to explore its mechanism of action.

This experiment was conducted from January to December 2019. Forty 6-month-old male SPF Wistar rats were selected and equally randomized into a blank group (group C) , a model group (group B) , a treatment group (group T) , and a prevention group (group P) . Interventions were given to the groups as follows: Group P received: 0.1 ml puerarin injection injected into bilateral Neiguan acupoints. Then 5 minutes later, bupivacaine-poisoned model was established for three groups (except for group C) using intravenous infusion of 0.5% bupivacaine (10 mg/kg) via femoral vein within 2 minutes. Group C received 0.1 ml of 0.9% sodium chloride solution injected into bilateral Neiguan acpoints. : Immediately after successful modeling, 0.1 ml of 0.9% sodium chloride solution was injected into bilateral Neiguan acupoints for group B, and: 0.1 ml puerarin injection was injected into bilateral Neiguan points for C. The rats in group C were sacrificed at the 8th minute of intravenous infusion of 0.9% sodium chloride solution, and those in groups B, T, and P were sacrificed at the 8th minute of intravenous infusion of bupivacaine, then the left ventricular myocardium of all rats were taken out and cardiomyocyte mitochondria were isolated, the structure of which was observed under the transmission electron microscope. The semi-quantitative analysis of mitochondrial structure was evaluated by Flameng classification system. The activity of mitochondrial adenosine triphosphate synthase (ATPase) was measured by ELISA. The content of adenosine triphosphate (ATP) was measured by colorimetric assay. The mitochondrial membrane potential (MMP) was detected by JC-1 fluorescence staining and colorimetry. The protein levels of myocardial mitochondrial peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) , nuclear respiratory factor 1 (NRF1) , and mitochondrial transcription factor A (mtTFA) were measured by Western blotting.

Group C had lower Flameng score than did other three groups (P<0.05) . The Flameng score in group P was lower than that of groups B and T (P<0.05) . Group C had lower activity of ATPase than did groups B and T (P<0.05) . The activity of ATPase in group P was higher than that in groups B and T (P<0.05) . Group C had higher ATP content and MMP, and protein levels of PGC-1α, NRF1 and mtTFA than did other three groups (P<0.05) . Group B had lower ATP content, MMP, and PGC-1α protein level than did groups T and P (P<0.05) . Group B had lower protein level of mtTFA than did groups T and P (P<0.05) . The protein level of mtTFA in group P was higher than that in group T (P<0.05) .

Acupoint injection of puerarin could reduce the damage of myocardial mitochondria caused by bupivacaine, and its mechanism of action may be related to puerarin's protection of mitochondrial energy metabolism by regulating the content of ATP synthesis-related proteins PGC-1α, NRF1 and mtTFA in mitochondria.

布比卡因是临床中广泛使用的麻醉药物，但其具有心脏毒性，目前尚没有安全有效的解毒方法。本课题组前期实验结果显示穴位处理用于治疗布比卡因所引起的心脏毒性有一定作用，但其具体的作用机制还有待进一步研究。

观察穴位注射葛根素对布比卡因中毒大鼠心肌线粒体能量代谢的影响，并探究其作用机制。

实验时间为2019年1—12月，选取6月龄SPF级Wistar雄性大鼠40只，依据随机数字表法将大鼠分为空白组（C组）、模型组（B组）、治疗组（T组）、预防组（P组），每组10只。P组：双侧内关穴分别注入0.1 ml的葛根素注射液。5 min后，开始建立布比卡因中毒模型（C组除外），建模具体方法为经股静脉2 min内静脉滴注0.5%布比卡因10 mg/kg。C组不建模，于双侧内关穴分别注入0.1 ml 0.9%氯化钠溶液。B组：建模成功后即刻取双侧内关穴分别注入0.1 ml 0.9%氯化钠溶液。T组：建模成功后即刻取双侧内关穴分别注入0.1 ml葛根素注射液。C组在静脉滴注0.9%氯化钠溶液的第8分钟，B组、T组、P组分别在静脉滴注利多卡因的第8分钟处死大鼠，提取所有大鼠左室心肌，分离心肌细胞线粒体。采用透射电镜观察线粒体结构，Flameng评分法评价线粒体半定量结构，酶联免疫吸附实验（ELISA）法测定心肌线粒体三磷酸腺苷合成酶（ATPase）活性，比色法测定三磷酸腺苷（ATP）含量，荧光比色法测定线粒体膜电位（MMP），Western blotting法测定心肌线粒体过氧化物酶体增殖物激活受体γ共激活因子1α（PGC-1α）、核呼吸因子（NRF1）、线粒体转录因子A（mtTFA）蛋白水平。

B组、T组、P组大鼠Flameng评分法得分高于C组，P组大鼠Flameng评分法得分低于B组、T组（P<0.05）。B组、T组大鼠ATPase活性低于C组，P组大鼠ATPase活性高于B组、T组（P<0.05）。B组、T组、P组大鼠ATP含量、MMP及PGC-1α、NRF1、mtTFA蛋白水平低于C组（P<0.05）。T组、P组大鼠ATP含量、MMP、PGC-1蛋白水平高于B组（P<0.05）。P组大鼠NRF1蛋白水平高于B组、T组（P<0.05）。T组、P组大鼠mtTFA蛋白水平高于B组，P组大鼠mtTFA蛋白水平高于T组（P<0.05）。

穴位注射葛根素可减轻布比卡因致心肌线粒体的损伤，其作用机制可能为调节线粒体内ATP合成相关蛋白PGC-1α、NRF1、mtTFA含量来保护线粒体能量代谢。