电针足三里对大鼠缺血再灌注损伤心肌的保护作用

发布时间：2018-05-28 22:53

本文选题：缺血再灌注损伤 + 胆碱能抗炎通路　；参考：《中南大学》2009年博士论文

【摘要】： 背景:心肌缺血再灌注(ischemia/reperfusion I/R)损伤是一种多因素参与的复杂病理过程,涉及到多个环节。目前认为,心肌I/R损伤是炎症反应过度表达,系统炎症反应和局部炎症反应共同参与的结果,由白细胞(中性粒细胞为主)和细胞因子、趋化因子激活为主的炎症反应在心肌缺血阶段即被激活,再灌注则显著加剧心肌的炎症级联反应。因此防治心肌I/R损伤的有效办法除了尽快恢复心肌血流的再灌注外,如何拮抗炎症的过度表达在心肌中的损伤作用,适时保护心肌是我们目前研究的主要方向。研究表明,刺激外周迷走神经及应用胆碱能递质乙酰胆碱或拟胆碱药物烟碱能抑制内毒素血症导致的全身炎症反应综合征,显著降低细胞因子如TNF-α、IL-1β、I L-6、IL-8等的释放,同时不影响抑炎因子IL-10的释放,并将之命名为“胆碱能抗炎通路”(the cholinergic anti-inflammatorypathway,CAP)。心肌缺血再灌注损伤与内毒素血症、低血容量失血性休克均有相同病理机制,即由许多炎症介质参与、机体失控性过度的炎症反应的病理过程,同时基于传出迷走神经电刺激对上述动物模型均具有抗过度炎症以及器官保护的研究结果,我们假设采取任何措施兴奋迷走神经传出纤维,都有可能激活该通路达到治疗心肌I/R损伤。现有资料表明,针刺足三里能够兴奋迷走神经中枢,并增加其传出纤维的电活动,而有关电针足三里对大鼠缺血再灌注损伤心肌的影响和机制研究目前尚未有人报道,因此本实验拟通过电针足三里,研究该方法对I/R损伤心肌是否也具有保护作用。目的:通过运用电针足三里刺激或迷走神经电刺激研究兴奋胆碱能抗炎通路对大鼠缺血再灌注损伤心肌的保护作用,探讨其作用机制。方法:雄性SD大鼠100只,随机分为5组:①手术对照组(SHAM组)②心肌缺血再灌注组(IR组)③迷走神经刺激组(STM组)④电针足三里组(ZSL组)⑤非经非穴组(FJFX组),除SHAM组外,各组均行心肌缺血30min再灌注,STM组于再灌前后各10min,共20min,以5 V、2ms、1HZ强度持续刺激左颈迷走神经,ZSL组于再灌前后各15min,共30min,行电针足三里刺激,FJFX组于再灌前后各15min,共30min,行电针非经非穴刺激。记录心律失常发生率,心率(heart rate,HR),平均动脉压(mean arterial pressure,MAP)变化;再灌注120min后,处死动物,采集标本。常规方法石蜡切片行苏木素一伊红(Hematoxylin and Eosin Staining,HE)染色,光学显微镜下观察心肌的炎症改变;伊文思蓝/氯化三苯基四唑(Evan's Blue/2,3,5-Triphenyl-2H-Tetrazoliumchloride,EB/TTC)测定心肌梗死面积与免疫比浊法测定血浆肌钙蛋白Ⅰ浓度;酶联免疫吸附试验(enzyme-linkedimmunosorbent assay,ELISA)检测心肌和血浆中肿瘤坏死因子(tumornecrosis factor,TNF)含量和白介素-6(interleukin-6,IL-6)含量;分光光度法测定心肌组织髓过氧化物酶(Myeloperoxidas MPO)活性;硫代巴比妥酸法测定心肌组织丙二醛(Malondialdehyde MDA)含量;免疫组化分析心肌核转录因子p65(nulear factor-kappa Bp65 NF-κBp65)表达;原位末端标记法(TUNEL)检测心肌细胞凋亡。结果:1.血流动力学和心律失常观察:除SHAM组外,再灌120min后各组心率,平均动脉压较缺血前均有下降(p＜0.05),各组之间MAP差异无统计学意义(p＞0.05)。STM组、ZSL组心率在刺激阶段有下降,STM组下降较ZSL组更明显,但波动幅度仍在10%以内,刺激停止后,心率回升。STM组、ZSL组再灌后心律失常发生率明显低于IR组,差异具有统计学意义(p＜0.05); 2、组织形态学观察:光镜下发现SHAM组心肌结构正常,细胞排列紧密、界限清楚,无水肿,无炎性细胞浸润;IR组、FJFX组心肌排列稀疏,不规则,细胞水肿,部分细胞出现明显空泡变性、心肌纤维断裂,细胞间隙明显增宽,大量炎性细胞浸润;STM组、ZSL组心肌细胞排列较规则,细胞轻度水肿,细胞间隙增宽,炎性细胞散在浸润; 3、心肌损伤程度观察:各组心肌行EB/TTC染色显示,各组间缺血范围差异无统计学意义(P＞0.05),与IR组相比,STM组和ZSL组心肌梗死范围差异有统计学意义(p＜0.05),心肌梗死范围减小,而ZSL组和STM组比较,无统计学差异(P＞0.05),FJFX组和IR组比较,无统计学差异(P＞0.05),各组心肌肌钙蛋白(cTnI)在心肌缺血前无明显差别,再灌注120min后,除SHAM组外,其他各组cTnI较缺血前均有不同程度的升高(P＜0.05),STM组和ZSL组cTnI浓度低于IR组(P＜0.05),STM组和ZSL组比较,无统计学差异(P＞0.05); 4、炎性标志物和氧化损害程度观察:与SHAM组相比,各组MPO活性和MDA含量显著增高,以IR组升高更为显著(P＜0.01),与ZSL组和STM组相比,差异也有统计学意义(P＜0.05),心肌组织和血浆中的TNF及IL-6的浓度在IR组、STM组、ZSL组、FJFX组明显升高,与SHAM组相比差异均有统计学意义(P＜0.05);其中IR组升高更为显著,与ZSL组相比,差异也有统计学意义(P＜0.05);STM组与ZSL组相比差异无统计学意义(P＞0.05)。免疫组化检测NF-κBp65表达,IR组大部分心肌细胞中,细胞浆染色呈阳性,细胞核染色呈阳性,ZSL组和STM组心肌细胞的胞浆呈弱阳性或阳性,细胞核染色呈弱阳性,核移位细胞数目减少。平均光密度值比较发现STM组、ZSL组NF-κBp65表达低于IR组(P＜0.05); 5、细胞凋亡观察:TUNEL法检测细胞凋亡发现,在SHAM组中,未发现TUNEL阳性细胞,相反,在IR组中,呈现大量TUNEL阳性细胞核呈棕黄色反应,核内染色质浓缩,形成密集颗粒位于核膜下,有的胞核被降解,胞浆不着色。ZSL组、STM组可见散在的阳性细胞。凋亡指数(apoptosis index AI)比较发现,STM组、ZSL组AI值低于IR组(P＜0.05)。结论:电针足三里能够抑制心肌缺血再灌注损伤引发的炎症反应,对心肌起到保护作用;其机制可能是通过兴奋胆碱能抗炎通路,削弱N F-κB表达,减少TNF、IL-6产生,抑制细胞凋亡有关。
[Abstract]:Background: myocardial ischemia reperfusion (ischemia/reperfusion I/R) injury is a complex pathological process involving multiple factors involved in multiple links. It is currently considered that myocardial I/R damage is a result of excessive expression of inflammatory reaction, systemic inflammatory reaction and local inflammatory reaction, which are derived from leukocytes (neutrophil dominated) and cytokines. The inflammatory response mainly activated by the activation of chemical factor is activated in the phase of myocardial ischemia, and reperfusion can significantly increase the inflammatory cascade reaction of the myocardium. Therefore, the effective way to prevent and cure I/R injury is how to antagonize the damage effect of overexpression of inflammation in the cardiac muscle as soon as possible, and protect the myocardium at the right time. The research shows that stimulation of the peripheral vagus nerve and the use of cholinergic acetylcholine or choline nicotine can inhibit the systemic inflammatory response syndrome caused by endotoxemia, significantly reducing the release of cytokines such as TNF- alpha, IL-1 beta, I L-6, IL-8 and so on, without affecting the release of anti inflammatory factor IL-10, and will It is named "the cholinergic anti-inflammatorypathway, CAP". Myocardial ischemia reperfusion injury and endotoxemia, hypovolemic hemorrhagic shock has the same pathological mechanism, that is, many inflammatory mediators are involved in the pathological process of excessive inflammation of the body, and the vagus nerve is based on the efferent vagus nerve. The electrical stimulation results in the above animal models against excessive inflammation and organ protection. We assume that any action to excite the vagus nerve efferent fibers may activate the pathway to treat the I/R injury of the myocardium. The study of the effect and mechanism of electroacupuncture at Zusanli on myocardial ischemia reperfusion injury in rats has not been reported. Therefore, this experiment is to use electroacupuncture at Zusanli to study the protective effect of this method on I/R injured myocardium.
Objective: To study the protective effect of the excitatory cholinergic anti-inflammatory pathway on the myocardial ischemia reperfusion injury in rats by using electroacupuncture at Zusanli or the electric stimulation of the vagus nerve, and to explore the mechanism of its action.
Methods: 100 male SD rats were randomly divided into 5 groups: (group SHAM): myocardial ischemia reperfusion group (group IR) (group STM) (group STM): Electroacupuncture Zusanli group (group ZSL) (group ZSL) without non acupoint group (group FJFX), except group SHAM, cardiac ischemia 30min reperfusion, STM group before and after reperfusion, 10min, 20min, 5 V, 1, 1 HZ intensity continued to stimulate left cervical vagus nerve, group ZSL was 15min before and after reperfusion, a total of 30min, electroacupuncture was stimulated by Zusanli, FJFX group was 15min before and after reperfusion, and 30min was performed without non acupoint stimulation. The incidence of arrhythmia was recorded, heart rate (heart rate, HR), mean arterial pressure (mean arterial). The routine method of paraffin section was stained with Hematoxylin and Eosin Staining (HE) and the inflammatory changes of the myocardium were observed under the optical microscope; Evans blue / chlorinated three phenyl four azole (Evan's Blue/2,3,5-Triphenyl-2H-Tetrazoliumchloride, EB/ TTC) was used to determine the area of myocardial infarction and immunoturbidimetry for the determination of plasma The concentration of troponin I; enzyme-linkedimmunosorbent assay (ELISA), the content of tumor necrosis factor (TumorNecrosis factor, TNF) in myocardium and plasma and the content of IL -6 (interleukin-6, IL-6); Spectrophotometric Determination of myocardium myeloperoxidase (Myeloperoxidas MPO) activity; thiobarbituric acid. The content of malondialdehyde (Malondialdehyde MDA) in myocardial tissue was measured and the expression of myocardial nuclear transcription factor p65 (nulear factor-kappa Bp65 NF- kappa Bp65) was analyzed by immunohistochemistry, and apoptosis of cardiac myocytes was detected by in situ end labeling (TUNEL).
Results: 1. hemodynamics and arrhythmia observation: except SHAM group, the heart rate of each group was decreased after reperfusion after 120min (P < 0.05). There was no significant difference in MAP between each group (P > 0.05).STM group. The heart rate of ZSL group decreased in the stimulation stage, and the decrease of STM group was more obvious than that in the ZSL group, but the amplitude of the fluctuation was still within 10%. After cardiac arrest, heart rate increased in group.STM, and the incidence of arrhythmia after reperfusion in group ZSL was significantly lower than that in group IR (P < 0.05).
2, histomorphological observation: under the light microscope, the myocardial structure of SHAM group was normal, the cells were arranged closely, the boundaries were clear, no edema, no inflammatory cell infiltration. In group IR, the myocardium of group FJFX was sparse, irregular, cell edema, obvious vacuolar degeneration in some cells, fibrous fracture of myocardium, obvious widening of intercellular space and a large number of inflammatory cells; STM group, ZS In group L, the myocardial cells were arranged in a regular way, the cells were slightly edema, the cell gap widened, and the inflammatory cells were scattered.
3, the degree of myocardial injury: EB/TTC staining showed that there was no significant difference in the range of ischemic range between each group (P > 0.05). Compared with group IR, there was a significant difference in the range of myocardial infarction in group STM and ZSL group (P < 0.05), and the range of myocardial infarction decreased, but there was no statistical difference between the ZSL group and the STM group (P > 0.05), and the ratio of FJFX and IR groups. There was no statistical difference (P > 0.05). There was no significant difference in myocardial troponin (cTnI) before ischemia in each group. After 120min reperfusion, all cTnI in other groups were higher than before SHAM (P < 0.05), and cTnI concentration in STM group and ZSL group was lower than that of IR group (P < 0.05).
4, inflammatory markers and oxidative damage degree observation: compared with group SHAM, MPO activity and MDA content increased significantly in group IR (P < 0.01). Compared with group ZSL and STM group, the difference was statistically significant (P < 0.05). The concentration of TNF and IL-6 in myocardial tissue and plasma was in IR group, STM group, group, and IL-6 were significantly higher. The difference in group IR was statistically significant (P < 0.05), and the increase in group IR was more significant than that in group ZSL (P < 0.05), and there was no statistical difference between group STM and ZSL group (P > 0.05). Immunohistochemistry was used to detect NF- kappa Bp65 expression, and the cytoplasm staining was positive in the large part of the musculocutaneous cells in the IR group, and the nucleus staining was positive. The cytoplasm of myocardial cells in group ZSL and group STM was weak positive or positive, the nucleus staining was weak positive and the number of nuclear displaced cells decreased. The average density of light density was compared to group STM, and the expression of NF- kappa Bp65 in group ZSL was lower than that in group IR (P < 0.05).
5, apoptosis observation: TUNEL detection of apoptosis found that in group SHAM, no TUNEL positive cells were found. On the contrary, in group IR, a large number of TUNEL positive nuclei showed brown yellow reaction, chromatin concentration in the nucleus was concentrated, dense particles were located under the nuclear membrane, some nuclei were degraded, cytoplasm was not stained with.ZSL group, and the positive cells scattered in the STM group were visible. Apoptotic index (apoptosis index AI) found that in group STM, AI value in group ZSL was lower than that in IR group (P < 0.05).
Conclusion: the Electroacupuncture of Zusanli can inhibit the inflammatory response induced by myocardial ischemia reperfusion injury and protect the myocardium. The mechanism may be related to the inhibition of the expression of N F- kappa B by stimulating the cholinergic anti-inflammatory pathway, reducing the production of TNF, IL-6, and inhibiting the apoptosis.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R245

【参考文献】