电针内关穴对氟哌利多处理大鼠QTc间期和心肌Cx43表达的影响

发布时间：2018-06-27 23:52

  本文选题：电针 + 内关穴　； 参考：《南京中医药大学》2014年博士论文

【摘要】：背景：氟哌利多是临床麻醉中常用的丁酰苯类药物,主要对中枢神经边缘系统、下丘脑、黑质和纹状体等部位的多巴胺受体产生阻断作用,因而具有强大的神经安定和镇吐作用,可与芬太尼合用于神经安定麻醉或小剂量单独用于恶心呕吐的防治。但美国食品药品监督管理局(Food and Drug Administration, FDA)于2001年,基于多起使用氟哌利多出现尖端扭转性室速猝死的病例,对其提出黑框警告,限制了氟哌利多的临床使用,但很多麻醉学者认为长期的临床麻醉实践了证实小剂量氟哌利多的安全性,其优良的止吐效果和低廉的价格优势使得目前尚无最佳的可替代药物,而FDA这一行为有失公允,在麻醉学界产生了广泛的争议。电针麻醉曾广泛用于临床实践并取得良好的效果,也是现代麻醉学对中华传统医学的重要应用和发展。近年来,电针广泛用于防治麻醉相关的不良反应,如恶心呕吐、缺血再灌注损伤和脏器功能不全等,在国际麻醉学研究中也有所报道,并越来越受重视。尤其是电针内关穴能产生较好的止吐效果,有研究还提示电针内关具有心血管保护和抗心律失常作用,而电针内关穴是否能减轻氟哌利多诱发的QT间期延长,尚缺乏相关研究。通过研究相关机制可以为临床麻醉使用电针提供理论支持,也有助于研究电针对相关药物的心脏毒性保护作用并推动电针在麻醉学中的应用,有重要的科研价值和社会意义,并有助于节约和降低医疗费用。缝隙连接蛋白是存在于相邻心肌细胞间的一种重要的膜通道蛋白,它参与了心肌细胞的电和化学信号的传导,并通过调控离子交换影响心肌细胞间的电偶联和机械收缩偶联。缝隙连接蛋白是由两个对称的连接子组成,每个连接子由连接蛋白的六聚体结构形成。心肌中最为重要的连接蛋白是缝隙连接蛋白43(connexin 43, Cx43),主要位于心肌细胞间的闰盘部位。相关文献认为Cx43密切参与了QT间期的形成,并对QT间期延长产生重要影响。心肌中Cx43蛋白的数量和分布变化会改变心肌细胞的电偶联状态,Cx43蛋白减少会降低心肌细胞电传导的均质性,心肌去极化和复极化时间延长,可能是QT间期延长的重要病理生理机制；而增加Cx43蛋白的含量和功能则可能改善心肌细胞间电传导的异质化,减弱QT间期的延长。目的：(1)观察不同剂量的氟哌利多和电针内关穴预处理对大鼠QT间期的影响。(2)研究不同剂量的氟哌利多和电针内关穴预处理对大鼠Cx43mRNA和蛋白表达的影响。方法：(1)动物分组：将雌性SD大鼠随机分成正常对照组C组(8只)、小剂量氟哌利多组DL组(8只)、大剂量氟哌利多组DH组(8只)、电针对照组EC组(8只)、小剂量氟哌利多电针预处理组EDL组(8只)和大剂量氟哌利多电针预处理组EDH组(8只)。C组经股静脉给予等量生理盐水,DL组经股静脉给予氟哌利多0.13mg/kg, DH组经股静脉给予氟哌利多1.3mg/kg, EC组预先电针左侧内关穴30min后经股静脉给予等量生理盐水,EDL组预先电针左侧内关穴30min后经股静脉给予氟哌利多0.13mg/kg,EDH组预先电针左侧内关穴30min后经股静脉给予氟哌利多1.3mg/kg。(2)使用Ⅱ导联监测并记录给药前0min、给药后5min、10min、15min、30min、60min和120min心电图,根据Bazett's公式QTc-B=QT/(?) (RR)计算大鼠QTc值。(3)RT-PCR法检测心室肌组织中Cx43mRNA表达情况,凝胶成像分析系统拍摄电泳条带,BandScan4.3软件进行灰度分析,将Cx43mRNA和内参基因GAPDHmRNA的光密度比值作为Cx43mRNA的表达水平。(4)Western blotting法检测心室肌组织中Cx43蛋白的表达,并采用放射自显影法记录影像,凝胶成像系统拍摄后,使用image Pro Plus6.0软件进行灰度分析,计算Cx43和内参蛋白GAPDH的光密度比值,作为Cx43蛋白的表达水平。(5)免疫组化法研究心室肌中Cx43的分布表达,心室肌组织经固定、切片、脱蜡、抗原-抗体反应和染色等步骤后,在光学显微镜40倍下观察切片中Cx43蛋白的表达情况,取3个高表达区域400倍下拍照保存,并使用Image Pro Plus6.0软件进行平均光密度分析。结果：(1)电针内关穴对氟哌利多处理大鼠QTc间期的影响：①与正常对照组对比,小剂量氟哌利多组在5min、10min、15min和30min各时点QTc间期显著延长,P0.01差异有显著性；大剂量氟哌利多组与正常对照组对比,在5min、10min、15min、30min、60min和120min等时点QTc间期均显著延长,P0.01差异有显著性。②电针预处理后,EDL组在给药后5min和15minQTc值均显著低于DL组,P0.01差异有显著性；10min和30min各时点的QTc间期值均显著低于DL组,P0.05差异有统计学意义。③电针预处理后,EDH组在给药后5min时QTc值显著低于DH组,P0.05差异有统计学意义；30min和60min时点的QTc值均显著低于DH组,P0.01差异有显著性。④电针内关穴与生理盐水注射对照组比,QTc间期无显著统计学差异。(2)电针内关对Cx43mRNA表达影响,Cx43mRNA表达水平EC组(0.92±0.03)和C组(0.84±0.02)比较,P0.05差异有统计学意义；EDL组(0.91±0.03)和DL组(0.84±0.02)相比较,P0.05差异有统计学意义； EDH组(0.73±0.02)和DH组(0.64±0.03)相比较,P0.05差异有统计学意义。(3) Western blotting法检测Cx43蛋白表达水平,EC组(1.46±0.15)和C组(1.09±0.08)比较,P0.01差异有显著性；EDL组(1.57±0.16)和DL组(1.09±0.10)相比较,P0.01,差异有显著性；EDH组(1.15±0.05)和DH组(0.96±0.08)相比较,P0.01差异有显著性。(4)免疫组织化学染色法检测心肌Cx43蛋白分布和表达：①C组Cx43阳性部分呈深棕色密集颗粒状分布于心肌细胞交界和闰盘处；DL组与C组类似；DH组Cx43蛋白呈淡棕黄色细颗粒状表达,呈散在点状分布于心肌细胞交界和闰盘处；EC组中Cx43蛋白呈深棕黄色粗线状广泛分布于心肌细胞交界和闰盘处；EDL组Cx43呈深棕黄色颗粒状分布于心肌细胞交界和闰盘处；EDH组心室肌中Cx43呈棕黄色颗粒状分布于心肌细胞交界和闰盘处。②Cx43蛋白平均光密度值在EC组(0.189±0.008),C组(0.168±0.009),二者比较,P0.01差异有显著性； Cx43蛋白平均光密度值在EDL组(0.202±0.009),DL组(0.160±0.009),二者比较,P0.01差异有显著性； Cx43蛋白平均光密度值在EDH组(0.198±0.009),DH组(0.153±0.007),二者比较,P0.01差异有显著性。结论：1.氟哌利多呈剂量依赖性导致大鼠QTc间期延长。2.电针内关穴可减轻氟哌利多诱发的大鼠QTc间期延长。3.电针内关穴可上调心肌Cx43mRNA和蛋白的表达,从而减少氟哌利多造成的心肌细胞复极过程中动作电位的异质性,这可能是电针改善氟哌利多诱发QTc间期延长的机制之一。
[Abstract]:Background: droperidol is a common butanyl drug in clinical anesthesia. It is mainly used to block the dopamine receptors in the central nervous system, the hypothalamus, the substantia nigra, and the striatum, and thus has a strong neurostability and antiemetic effect. It can be combined with fentanyl in neurodiazepam anesthesia or small dose alone for nausea nausea. The prevention and treatment of spitting. But in 2001, the Food and Drug Administration (FDA), based on multiple use of droperidol's case of sudden sudden ventricular tachycardia death, proposed a black frame warning, limiting the clinical use of droperidol, but many anesthesiologists believe that long-term clinical anesthesia has proven small doses. The safety of droperidol, its excellent antiemetic effect and low price advantage make no best alternative drugs at present, and the behavior of FDA is unfair and has produced widespread controversy in the anesthesiology. Electroacupuncture anesthesia has been widely used in clinical practice and has achieved good results. It is also a modern anesthesiology for Chinese traditional medicine. Important applications and development. In recent years, electroacupuncture has been widely used to prevent and cure anaesthesia related adverse reactions, such as nausea, vomiting, ischemia reperfusion injury and organ dysfunction. It has also been reported in international Anesthesiology, and is becoming more and more important. Especially, electroacupuncture Neiguan acupoints can produce better antiemetic effect. It has the effect of cardiovascular protection and antiarrhythmia, and whether the electroacupuncture Neiguan acupoint can relieve the prolonged QT interval induced by droperidol is still lack of related research. The application of drunken learning has important scientific and social significance and helps to save and reduce medical costs. Gap junctional protein is an important membrane channel protein that exists between adjacent myocardial cells. It participates in the conduction of electrical and chemical signals of cardiac myocytes and through regulating ion exchange to influence the coupling of electric couples among cardiac myocytes. The connexin is composed of two symmetrical connexions, each of which is formed by the six polymer structure of the connexin. The most important connexin in the myocardium is the gap connexin 43 (connexin 43, Cx43), mainly located in the intercalated disc between the cardiac myocytes. The relevant literature considers that Cx43 is closely involved in the QT interval. The formation of QT has an important effect on the prolongation of the interval of interphase. The changes in the number and distribution of Cx43 protein in the myocardium will change the electric couple state of the cardiac myocytes. The decrease of Cx43 protein will reduce the homogeneity of the electrical conduction of the cardiac myocytes, the depolarization and repolarization time of the myocardium, which may be the important pathophysiological mechanism of the prolongation of the interphase of QT; and the increase of Cx43 The protein content and function may improve the heterogeneity of the intercellular electrical conduction and the prolongation of the QT interval. Objective: (1) observe the effect of different doses of droperidol and electroacupuncture Neiguan point on the QT interval in rats. (2) the expression of Cx43mRNA and protein in rats with different doses of droperidol and Electroacupuncture Neiguan point preconditioning Methods: (1) animal groups: the female SD rats were randomly divided into the normal control group C group (8 rats), the small dose droperidol group DL group (8), the large dose droperidol group DH group (8 rats), the electroacupuncture control group EC group (8), the small dose droperidol Electroacupuncture group EDL group (8 rats) and the large dose droperidol electroacupuncture pretreatment group EDH group (8).C group. The same amount of physiological saline was given via femoral vein, group DL was given droperidol 0.13mg/kg through femoral vein, group DH was given droperidol 1.3mg/kg through femoral vein, group EC was given to the left Neiguan point 30min after 30min in group EC, and EDL group was given left Neiguan point after 30min to be given droperidol 0.13mg/kg in the femoral vein, and the EDH group was beforehand. The electroacupuncture at the left Neiguan point 30min was given to droperidol 1.3mg/kg. (2) via the femoral vein to monitor and record 0min before administration by the lead II. 5min, 10min, 15min, 30min, 60min and 120min electrocardiogram were calculated after the administration. (3) the expression of the rat ventricular muscle was detected by the Bazett's formula QTc-B=QT/ (RR). The system photographed electrophoresis strip, BandScan4.3 software carried out gray analysis, and the ratio of light density of Cx43mRNA and internal reference gene GAPDHmRNA was used as the expression level of Cx43mRNA. (4) Western blotting method was used to detect the expression of Cx43 protein in ventricular muscle, and the autoradiography was recorded by autoradiography, and image Pro Pl was used after the gel imaging system was taken. Us6.0 software was used for gray analysis to calculate the light density ratio of Cx43 and GAPDH as the expression level of Cx43 protein. (5) the distribution and expression of Cx43 in ventricular muscle were studied by immunohistochemistry. After fixed, slicing, dewaxing, antigen antibody reaction and staining, the Cx43 protein in the slice was observed under the optical microscope at 40 times of the optical microscope. The expression of 3 high expression regions was taken at 400 times, and the average light density analysis was performed using Image Pro Plus6.0 software. The results were as follows: (1) the effect of the electroacupuncture Neiguan point on the QTc interval of droperidol treatment in rats: (1) the QTc interval of the small dose of droperidol group was significant at 5min, 10min, 15min and 30min at each time point. The difference in P0.01 was significant, and the time points of QTc interval in 5min, 10min, 15min, 30min, 60min and 120min were significantly longer in the large dose droperidol group than in the normal control group, and the difference of P0.01 was significant. 2. After the electroacupuncture treatment, the 5min and 15minQTc values of the EDL group were significantly lower than those in the group. The QTc interval values of each time point were significantly lower than that of the DL group, and the difference in P0.05 was statistically significant. After the electroacupuncture treatment, the QTc value of the EDH group was significantly lower than that of the DH group after 5min, and the P0.05 difference was statistically significant. The QTc values of 30min and 60min points were significantly lower than those of the DH group. There was no significant difference in QTc interval. (2) the effect of electroacupuncture Neiguan on Cx43mRNA expression, the level of Cx43mRNA expression in EC group (0.92 + 0.03) and C group (0.84 + 0.02), the difference between P0.05 and EDL group (0.91 + 0.03) and DL group (0.84 + 0.02) was statistically significant; EDH group (0.73 + 0.02) and DH group (0.64 + 0.03) were compared. The difference of P0.05 was statistically significant. (3) the expression level of Cx43 protein was detected by Western blotting method, the difference in P0.01 was significant in EC group (1.46 + 0.15) and C group (1.09 + 0.08), and in EDL group (1.57 + 0.16) and DL group (1.09 + 0.10), P0.01, the difference was significant; EDH group (1.15 + 0.05) was compared with DH group (0.96 + 0.08). (4) immunohistochemical staining was used to detect the distribution and expression of Cx43 protein in myocardium: (1) the Cx43 positive part of group C was distributed in the junction of cardiac myocytes and intercalated discs; the DL group was similar to that in the C group; the Cx43 protein in the DH group was pale brown and fine granular, distributed in the point like distribution at the junction of cardiac myocytes and intercalated discs, and in EC group. The Cx43 protein was widely distributed in the junction of cardiac myocytes and intercalated discs at the junction of cardiac myocytes and intercalated discs. In group EDL, Cx43 was distributed in the junction of cardiac myocytes and intercalated discs, and in group EDH, Cx43 was brown and yellow granules distributed at the junction of cardiac myocytes and intercalated discs. The mean optical density of Cx43 egg white was in group EC (0.189 + 0.008), C Group (0.168 + 0.009), compared with the two, the difference in P0.01 was significant, the mean light density of Cx43 protein was in group EDL (0.202 + 0.009), DL group (0.160 + 0.009), and the difference of P0.01 was significant in the two group; the average optical density of Cx43 protein was in group EDH (0.198 + 0.009), DH group (0.153 + 0.007), and P0.01 difference was significant. Conclusion: fluoro piperli Dose dependence can lead to prolongation of.2. electroacupuncture Neiguan point in QTc interval in rats. The prolongation of droperidol induced QTc interval in rats by prolonging.3. electroacupuncture Neiguan point can increase the expression of Cx43mRNA and protein in myocardium, thus reducing the heterogeneity of action potential during the repolarization process of droperidol. This may be the improvement of droperidol by electroacupuncture. One of the mechanisms that induce the prolongation of QTc interval.
【学位授予单位】：南京中医药大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R614

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