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及其机制研究

doi:10.12114/j.issn.1007-9572.2021.02.143

中国全科医学 ›› 2022, Vol. 25 ›› Issue (18): 2280-2285.DOI: 10.12114/j.issn.1007-9572.2021.02.143

所属专题：心血管最新文章合集

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及其机制研究

于妍¹, 王春爱²^,^*(), 秦晓宇³, 李玉兰⁴, 梁曦², 温晓辉⁵, 刘敏⁶

1.730050　甘肃省兰州市，甘肃省中医院心内科
2.730050　甘肃省兰州市，甘肃省中医院麻醉科
3.730000　甘肃省兰州市，甘肃中医药大学第一临床医学院
4.730013　甘肃省兰州市，兰州大学第一医院麻醉科
5.730000　甘肃省兰州市，甘肃中医药大学免疫教研室
6.730099　甘肃省兰州市，甘肃中医药大学附属医院针灸科

收稿日期:2021-10-20 修回日期:2021-12-29 出版日期:2022-06-20 发布日期:2022-01-13
通讯作者: 王春爱
于妍，王春爱，秦晓宇，等.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及其机制研究[J].中国全科医学，2022，25（18）：2280-2285. [www.chinagp.net]
作者贡献：于妍、王春爱、李玉兰提出研究的构思与设计，负责研究的实施推进与可行性分析，并进行结果的分析与解释；于妍负责论文起草；秦晓宇、梁曦、温晓辉、刘敏负责动物模型的制备、实验指标的检测；梁曦、刘敏进行数据收集、整理；温晓辉进行统计分析；秦晓宇绘制图表；于妍、王春爱负责最终版本修订，对论文负责。所有作者确认论文终稿。
基金资助:
国家自然科学基金资助项目(81760892)

Effect and Mechanism of Action of Acupoint Injection of Puerarin on Myocardial Mitochondria Energy Metabolism in Bupivacaine-poisoned Rats

Yan YU¹, Chunai WANG²^,^*(), Xiaoyu QIN³, Yulan LI⁴, Xi LIANG², Xiaohui WEN⁵, Min LIU⁶

1. Department of Cardiology, Gansu Provincial Hospital of TCM, Lanzhou 730050, China
2. Department of Anesthesiology, Gansu Provincial Hospital of TCM, Lanzhou 730050, China
3. First School of Clinical Medical, Gansu University of Chinese Medicine, Lanzhou 730000, China
4. Department of Anesthesiology, the First Hospital of Lanzhou University, Lanzhou 730013, China
5. Department of Immunology, Gansu University of Chinese Medicine, Lanzhou 730000, China
6. Department of Acupuncture and Moxibustion, Affiliated Hospital of Gansu University of Chinese Medicine, Lanzhou 730099, China

Received:2021-10-20 Revised:2021-12-29 Published:2022-06-20 Online:2022-01-13
Contact: Chunai WANG
About author:
YU Y, WANG C A, QIN X Y, et al. Effect and mechanism of action of acupoint injection of puerarin on myocardial mitochondria energy metabolism in bupivacaine-poisoned rats[J]. Chinese General Practice, 2022, 25 (18) : 2280-2285.

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摘要/Abstract

摘要： 背景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。目的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。方法实验时间为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。结果 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）。结论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。

关键词: 心脏毒性, 心肌, 线粒体, 葛根素, 水针疗法, 布比卡因, 能量代谢, 药理作用分子作用机制（中药）

Abstract:

Background

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.

Objective

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.

Methods

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.

Results

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) .

Conclusion

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.

Key words: Cardiotoxicity, Myocardium, Mitochondria, Puerarin, Hydro acupuncture therapy, Bupivacaine, Energy metabolism, Molecular mechanisms of pharmacological action (TCD)

于妍,王春爱,秦晓宇,等. 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及其机制研究[J]. 中国全科医学, 2022, 25(18): 2280-2285. DOI: 10.12114/j.issn.1007-9572.2021.02.143.
Yan YU,Chunai WANG,Xiaoyu QIN, et al. Effect and Mechanism of Action of Acupoint Injection of Puerarin on Myocardial Mitochondria Energy Metabolism in Bupivacaine-poisoned Rats[J]. Chinese General Practice, 2022, 25(18): 2280-2285. DOI: 10.12114/j.issn.1007-9572.2021.02.143.

图/表 5

图1 四组大鼠心肌线粒体结构电镜图（3%醋酸铀-枸橼酸铅双染色，×20 000）注：C组为空白组，B组为模型组，T组为治疗组，P组为预防组

Figure 1 Transmission electron micrographs of myocardial mitochondria in four groups of rats（double staining with 3% uranyl acetate and lead citrate，×20 000）

表1 四组大鼠Flameng评分法得分比较（±s，分）

Table 1 Comparison of the Flameng scores of four groups of rats

分组	只数	Flameng评分
C组	10	0.29±0.02
B组	10	2.54±0.09^a
T组	10	2.24±0.15^a
P组	10	1.55±0.22^abc
F值		139.37
P值		<0.05

表2 四组大鼠ATPase活性、ATP含量、MMP比较（±s）

Table 2 Comparison of ATPase activity，ATP content and MMP of four groups of rats

分组	只数	ATPase（pg/ml）	ATP（μmol/g）	MMP
C组	10	338.79±22.38	1 132.58±140.11	0.38±0.02
B组	10	261.61±21.83^a	629.90±135.81^a	0.14±0.04^a
T组	10	281.85±25.00^a	773.06±104.54^ab	0.21±0.02^ab
P组	10	300.89±27.38^bc	786.29±71.79^ab	0.23±0.05^ab
F值		37.06	66.49	75.63
P值		<0.05	<0.05	<0.05

表3 四组大鼠PGC-1α、NRF1、mtTFA蛋白水平比较（±s）

Table 3 Comparison of the protein levels of PGC-1α，NRF1 and mtTFA in four groups of rats

分组	只数	PGC-1α	NRF1	mtTFA
C组	10	1.00±0.04	1.00±0.04	1.00±0.06
B组	10	0.66±0.04^a	0.55±0.02^a	0.74±0.07^a
T组	10	0.75±0.04^ab	0.59±0.03^a	0.81±0.07^ab
P组	10	0.77±0.04^ab	0.70±0.02^abc	0.89±0.07^abc
F值		175.17	166.84	212.46
P值		<0.05	<0.05	<0.05

图2 不同处理方式对心肌线粒体PGC-1α、NRF1、mtTFA蛋白影响的电泳图注：PGC-1α=过氧化物酶体增殖物激活受体γ共激活因子1α，NRF1=核呼吸因子，mtTFA=线粒体转录因子A

Figure 2 Electrophoresis diagram of the effects of different treatments on myocardial mitochondrial PGC-1α，NRF1，and mtTFA protein

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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及其机制研究

Effect and Mechanism of Action of Acupoint Injection of Puerarin on Myocardial Mitochondria Energy Metabolism in Bupivacaine-poisoned Rats

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