电针“内关”预处理对心肌缺血再灌注大鼠p38MAPK信号通路的影响及机制研究

发布时间：2017-12-28 06:41

本文关键词：电针“内关”预处理对心肌缺血再灌注大鼠p38MAPK信号通路的影响及机制研究　出处：《湖北中医药大学》2017年博士论文　论文类型：学位论文

【摘要】：目的利用电针的低创伤、易操作和可控性强的优势,结合针灸经典理论“心胸内关谋”,并借助已有的关于内关-心脏相关性的研究基础,观察电针“内关”预处理对心肌缺血再灌注大鼠早期心功能参数、心室重构、心肌梗死情况,血清及心肌相关因子、线粒体呼吸功能的影响,并借用p38MAPK通路阻滞剂SB203580从p38MAPK信号通路的角度分析其作用机制,为临床各型I/R的防治方法提供新的理论探讨和实验基础。方法选用SPF级雄性Wistar大鼠200只,取材前体重180g-220g,采用随机数字表法,将大鼠随机分为五组:假手术组(Shame operation group,S组)、模型组(Model group,M组)、电针预处理组(electroacupuncture pretreatment group,EA组)、抑制剂预处理组(SB203580 group,SB组)和电针+抑制剂预处理组(electroacupuncture pretreatment+SB203580 group,EA+SB组)。假手术组:常规饲养,捆绑1次/天,共7天,第8天手术过程中,只开胸穿线,不结扎左冠状动脉前降支;模型组:捆绑1次/天,共7天,第8天通过采用推管法行左冠状动脉缺血再灌注术,制备在体心肌缺血再灌注模型;抑制剂预处理组:捆绑1次/天,共7天,第8天将p38MAPK抑制剂SB203580溶于二甲基亚砜成20%溶液,在造模前给予0.3mg/kg皮下注射,后造模过程同模型组;电针预处理组:捆绑1次/天。电针双侧内关穴操作,直刺深度约5mm,采用HANS-200型电针治疗仪,选用连续波,频率2Hz,强度1mA,电针预处理1次/天,通电治疗20min。共7天,后造模过程同模型组;电针+SB203580预处理组:捆绑1次/天,共7天,电针预处理过程同电针预处理组,第8天造模前给予抑制剂SB203580皮下注射,过程同抑制剂预处理组,后造模过程同模型组。复灌即刻,利用生物信号及压力测试系统(BL-Newcentury 410)测定大鼠左心室心功能指标(舒张末期压力、左室收缩期平均压、短轴缩短率和射血分数),后处死大鼠,取血清待检,剪取心脏,称取梗死区重量和全心重量,分别计算心肌梗死区重量和全心重量的比值。ttc染色法分析心肌梗死面积,采用全自动生化分析仪检测血清肌酸激酶(ck)、乳酸脱氢酶(ldh)、肌钙蛋白i(ctni),采用比色法检测ros含量,采用黄嘌呤氧化法检测心肌超氧化物歧化酶(sod)活性,硫代巴比妥酸法检测心肌丙二醛(mda)含量,高效液相色谱法计算心肌组织中atp的含量。利用激光共聚焦显微镜、he染色和透射电镜观察大鼠心肌细胞尤其是线粒体的形态变化。结果(1)m组的梗死面积和梗死程度的比值,与s组相比较,均显著升高(p0.05),sb组、ea组和ea+sb组的梗死面积和梗死程度的比值均较m组有降低,差异有统计学意义(p0.05),ea组、ea+sb组和sb组之间无显著性差异(p0.05)。(2)m组的lvedp与s组相比较,显著升高(p0.05),sb组、ea组和ea+sb组的lvedp均较m组显著降低,差异有统计学意义(p0.05),ea组、ea+sb组和sb组之间无显著性差异(p0.05)。m组的lvsp、fs、ef值与s组相比较,显著升高(p0.05),sb组、ea组和ea+sb组的lvsp、fs、ef值均较m组显著升高,差异有统计学意义(p0.05),ea组、ea+sb组和sb组之间无显著性差异(p0.05)。(3)m组的ldh、ck及ctni与s组相比较,显著升高(p0.05),sb组、ea组和ea+sb组的ldh、ck及ctni均较m组显著降低,差异有统计学意义(p0.05),ea组的ldh、ck与ea+sb组和sb组之间无显著性差异(p0.05)。ea组和ea+sb组的ctni与sb组比较,有显著降低,差异有统计学意义(p0.05)。(4)m组的tnf-α、il-1β和il-6与s组相比较,显著升高(p0.05),sb组、ea组和ea+sb组的tnf-α、il-1β和il-6均较m组显著降低,差异有统计学意义(p0.05),ea组的tnf-α、il-1β和il-6与ea+sb组和sb组比较,显著降低,差异有统计学意义(p0.05)。m组的il-4和il-10与s组比较,差异无统计学意义(p0.05)。sb组、ea组和ea+sb组的il-4和il-10与m组比较,有显著性差异(p0.05)。各治疗组之间差异性无统计学意义(p0.05)。(5)m组的icam-1和vcam-1与s组相比较,显著升高(p0.05),sb组、ea组和ea+sb组的icam-1和vcam-1均较m组显著降低,差异有统计学意义(p0.05),ea组的icam-1和vcam-1明显低于ea+sb组和sb组,差异有统计学意义(p0.05)。(6)m组的ros和mda与s组相比较,显著升高(p0.05),sb组、ea组和ea+sb组的ros和mda均较m组显著降低,差异有统计学意义(p0.05),ea组的ros和mda明显低于ea+sb组和sb组,差异有统计学意义(p0.05)。m组的sod和atp与s组相比较,显著降低(p0.05),sb组、ea组和ea+sb组的sod和atp均较m组显著升高,差异有统计学意义(p0.05),ea组的ros和mda明显高于ea+sb组和sb组,差异有统计学意义(p0.05)。(7)与s组比较,m组、ea组、sb组及ea+sb组大鼠心肌细胞na+-k+-atpase和ca2+-atpase的活性明显下降(p0.05),ea组、sb组及ea+sb组大鼠心肌细胞na+-k+-atpase和ca2+-atpase的活性较m组明显提高(p0.05),ea组中na+-k+-atpase和ca2+-atpase的改善明显优于sb组及ea+sb组,差异具有统计学意义(p0.05)。(8)m组的mkk3/6、p38及p-p38蛋白表达与s组相比较,显著升高(p0.05),sb组、ea组和ea+sb组的mkk3/6、p38及p-p38蛋白表达均较m组显著降低,差异有统计学意义(p0.05),ea组的mkk3/6、p38及p-p38蛋白表达数值低于ea+sb组和sb组,但差异无统计学意义(p0.05)。(9)s组正常心肌纤维排列规则,细胞结构完整,细胞核及线粒体形态和数量正常,线粒体嵴清晰,心肌间未见异常。m组心肌肌纤维出现溶解断裂甚至坏死,间质增大,肿胀明显。部分线粒体固缩变小,部分线粒体肿胀、或外膜破损。线粒体嵴不规则、断裂成絮状或缺失。EA组、SB组及EA+SB组肌纤维间隙轻度增宽,坏死灶减轻,心肌细胞轻微肿胀,细胞核及线粒体结构基本完整,小部分线粒体仍存在空泡样病变。结论1.电针“内关”预处理可使I/R模型大鼠心肌梗死面积和梗死程度明显下降,可减轻线粒体肿胀和坏死程度,减轻心肌细胞间炎症因子浸润,病理形态学检测提示电针可显著改善受损心肌组织的病理损害程度。2.电针“内关”预处理可使I/R模型大鼠LVEDP降低,LVSP、FS和EF升高,可降低血清中LDH、CK及cTnI的含量,保护大鼠心肌组织,改善大鼠心功能。3.电针“内关”预处理可以抑制TNF-a、IL-1β、IL-6等炎性因子的表达,增加IL-4和IL-10等抗炎因子的表达,可以下调心肌粘附分子ICAM-1和VCAM-1的蛋白表达,通过调控炎症反应相关因子保护受损心肌组织。4.电针“内关”预处理可以减少I/R模型大鼠心肌细胞ROS及MDA的含量,提高的ATP含量,激活SOD和ATP酶的活性,改善RCR,通过保护线粒体结构和功能的完整性来减轻I/R中心肌损伤。5.MKK3/6/p38MAPK信号传导通路参与在体I/R模型大鼠的病理过程,I/R中大鼠MKK3/6和p38MAPK表达上调,电针“内关”预处理可以明显抑制I/R中MKK3/6、p38MAPK的蛋白表达,抑制p38MAPK的磷酸化,对心肌细胞p38信号转导通路的调节可能只是电针“内关”预处理保护I/R,发挥其抗心肌缺血的作用机制之一。6.电针组与p38抑制剂组和电针+p38抑制剂组相比较,对I/R大鼠模型相关指标结果的调控上可见明显差异,但p38抑制剂组和电针+p38抑制剂组之间并未出现明显叠加效应,说明电针内关预处理对I/R的保护机制可能是多层次、多途径的综合效应。
[Abstract]:Objective to use electroacupuncture low trauma, easy operation and high controllability advantages, combined with the classical theory of "mind in Guan acupuncture plan, and with existing on the acupoint Neiguan-heart correlation research foundation, to observe the effect of electroacupuncture at Neiguan on myocardial ischemia reperfusion in rats with early heart function, ventricular remodeling, myocardial infarction the situation, related factors, effects of serum and myocardial mitochondrial respiratory function, and p38MAPK pathway blocker SB203580 to analyze the mechanism of p38MAPK signaling pathway from the angle, to provide theoretical and experimental basis for the new control methods of various types of clinical I/R. Methods male Wistar SPF rats were taken before 200, weight 180g-220g, were randomly, the rats were randomly divided into five groups: sham operation group (Shame operation, group, S group), model group (Model group, M group), electroacupuncture pretreatment group (electroacupuncture pretreatment, group, EA group), preconditioning group (SB203580 inhibitor group, SB group) and electroacupuncture + inhibitor pretreatment group (electroacupuncture pretreatment+SB203580, group, EA+SB group). Sham operation group: conventional breeding, tied 1 times / day, 7 days, eighth days during the surgery, only thoracotomy threading, without ligating the left anterior descending coronary artery; model group: bind 1 times / day, 7 days, eighth days by pushing pipe for the left coronary artery ischemia perfusion, preparation of myocardial ischemia reperfusion model; inhibitor pretreatment group: bind 1 times / day, a total of 7 days, the p38MAPK inhibitor SB203580 dissolved in two dimethyl sulfoxide into 20% solution for eighth days, given subcutaneous injection of 0.3mg/kg before modeling, after modeling process is the same as the model group; Electroacupuncture pretreatment group: bind 1 times / day. Electroacupuncture at Neiguan point operation, puncture depth of about 5mm, using HANS-200 type electric acupuncture apparatus, using continuous wave, frequency of 2Hz and intensity 1mA, electroacupuncture pretreatment 1 times / day, electricity treatment 20min. A total of 7 days later, the modeling process was the same as the model group. The electroacupuncture +SB203580 pretreatment group was bound for 1 days / day for 7 days, the EA pretreatment process was combined with the electro acupuncture pretreatment group, and eighth days before the injection, the inhibitor SB203580 was injected subcutaneously, the process was the same as the inhibitor pretreatment group, and the modeling process was the same as the model group. Reperfusion immediately, using biological signal and pressure test system (BL-Newcentury 410) to measure the cardiac function indexes of rats (left ventricular end diastolic pressure, left ventricular systolic pressure, average shortening and ejection fraction), after the rats were killed, serum to be detected, their heart, weigh the infarction area and heart weight the weight ratio were calculated infarct weight and heart weight. Analysis of myocardial infarction area by TTC staining, serum creatine kinase detection automatic biochemical analyzer (CK), lactate dehydrogenase (LDH), troponin I (cTnI), the ratio of ROS content detection method to detect myocardial superoxide dismutase by xanthine oxidation (SOD) activity, detection of myocardial malondialdehyde thiobarbituric acid method (MDA) content, calculate the contents of myocardial ATP by hplc. The morphological changes of rat cardiac myocytes, especially mitochondria, were observed by laser confocal microscopy, he staining and transmission electron microscopy. Results (1) ratio of M group, infarct size and degree of infarction, compared with the s group, were significantly increased (P0.05), the ratio of sb group, EA group and ea+sb group the infarct size and degree of infarction were compared with the M group decreased, the difference was statistically significant (P0.05), no significant difference between EA group, ea+sb group and Sb group (P0.05). (2) LVEDP in group M increased significantly compared with group s (P0.05), LVEDP in sb group, EA group and ea+sb group was significantly lower than that in M group, the difference was statistically significant (P0.05), but there was no significant difference between the EA group, the M group and the control group. The values of LVSP, FS and EF in group M were significantly higher than those in s group (P0.05), and the values of LVSP, FS and FS in sb group, EA group and ea+sb group were significantly higher than those in group A. There was no significant difference between them. (3) LDH, CK and cTnI in group M were significantly higher than those in s group (P0.05), and the LDH, CK and LDH in sb group, EA group and ea+sb group were significantly lower than those in group A. There was no significant difference between them. The cTnI and Sb in group EA and group ea+sb were significantly lower than those in group sb, and the difference was statistically significant (P0.05). (4) M group tnf- alpha, IL-1 beta and IL-6 compared with the s group, was significantly increased (P0.05), Sb group, EA group and ea+sb group of tnf- alpha, IL-1 beta and IL-6 were significantly lower than those in M group, the difference was statistically significant (P0.05), EA group, tnf- alpha, IL-1 beta and IL-6 compared with ea+sb group and Sb group significantly decreased, the difference was statistically significant (P0.05). There was no significant difference in IL-4 and IL-10 between group M and group s (P0.05). There were significant differences in IL-4 and IL-10 between group sb, group EA and group ea+sb compared with that of group M (P0.05). There was no significant difference between the treatment groups (P0.05). (5) ICAM-1 and VCAM-1 in group M increased significantly compared with group s (P0.05), and ICAM-1 and VCAM-1 in sb group, EA group and ea+sb group were significantly lower than those in group C. The difference was statistically significant (P0.05). (6) ROS and MDA in group M increased significantly compared with group s (P0.05), and ROS and MDA in sb group, EA group and ea+sb group were significantly lower than those in group C. The difference was statistically significant (P0.05). The SOD and ATP in group M were significantly lower than those in group s (P0.05). The SOD and ATP in sb group, EA group and ea+sb group were significantly higher than those in group C. The difference was statistically significant (P0.05). (7) compared with group s, the activities of na+-k+-atpase and ca2+-atpase in M group, EA group, Sb group and ea+sb group were significantly decreased (P0.05), and the activities of myocardial cells in EA group, Sb group and ea+sb group were significantly higher than those in group A.
【学位授予单位】：湖北中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R245

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