本文選題：缺血再灌注損傷 + 膽堿能抗炎通路��； 參考：《中南大學(xué)》2009年博士論文

【摘要】： 背景:心肌缺血再灌注(ischemia/reperfusion I/R)損傷是一種多因素參與的復(fù)雜病理過程,涉及到多個(gè)環(huán)節(jié)。目前認(rèn)為,心肌I/R損傷是炎癥反應(yīng)過度表達(dá),系統(tǒng)炎癥反應(yīng)和局部炎癥反應(yīng)共同參與的結(jié)果,由白細(xì)胞(中性粒細(xì)胞為主)和細(xì)胞因子、趨化因子激活為主的炎癥反應(yīng)在心肌缺血階段即被激活,再灌注則顯著加劇心肌的炎癥級聯(lián)反應(yīng)。因此防治心肌I/R損傷的有效辦法除了盡快恢復(fù)心肌血流的再灌注外,如何拮抗炎癥的過度表達(dá)在心肌中的損傷作用,適時(shí)保護(hù)心肌是我們目前研究的主要方向。研究表明,刺激外周迷走神經(jīng)及應(yīng)用膽堿能遞質(zhì)乙酰膽堿或擬膽堿藥物煙堿能抑制內(nèi)毒素血癥導(dǎo)致的全身炎癥反應(yīng)綜合征,顯著降低細(xì)胞因子如TNF-α、IL-1β、I L-6、IL-8等的釋放,同時(shí)不影響抑炎因子IL-10的釋放,并將之命名為“膽堿能抗炎通路”(the cholinergic anti-inflammatorypathway,CAP)。心肌缺血再灌注損傷與內(nèi)毒素血癥、低血容量失血性休克均有相同病理機(jī)制,即由許多炎癥介質(zhì)參與、機(jī)體失控性過度的炎癥反應(yīng)的病理過程,同時(shí)基于傳出迷走神經(jīng)電刺激對上述動(dòng)物模型均具有抗過度炎癥以及器官保護(hù)的研究結(jié)果,我們假設(shè)采取任何措施興奮迷走神經(jīng)傳出纖維,都有可能激活該通路達(dá)到治療心肌I/R損傷。現(xiàn)有資料表明,針刺足三里能夠興奮迷走神經(jīng)中樞,并增加其傳出纖維的電活動(dòng),而有關(guān)電針足三里對大鼠缺血再灌注損傷心肌的影響和機(jī)制研究目前尚未有人報(bào)道,因此本實(shí)驗(yàn)擬通過電針足三里,研究該方法對I/R損傷心肌是否也具有保護(hù)作用。 目的:通過運(yùn)用電針足三里刺激或迷走神經(jīng)電刺激研究興奮膽堿能抗炎通路對大鼠缺血再灌注損傷心肌的保護(hù)作用,探討其作用機(jī)制。 方法:雄性SD大鼠100只,隨機(jī)分為5組:①手術(shù)對照組(SHAM組)②心肌缺血再灌注組(IR組)③迷走神經(jīng)刺激組(STM組)④電針足三里組(ZSL組)⑤非經(jīng)非穴組(FJFX組),除SHAM組外,各組均行心肌缺血30min再灌注,STM組于再灌前后各10min,共20min,以5 V、2ms、1HZ強(qiáng)度持續(xù)刺激左頸迷走神經(jīng),ZSL組于再灌前后各15min,共30min,行電針足三里刺激,FJFX組于再灌前后各15min,共30min,行電針非經(jīng)非穴刺激。記錄心律失常發(fā)生率,心率(heart rate,HR),平均動(dòng)脈壓(mean arterial pressure,MAP)變化;再灌注120min后,處死動(dòng)物,采集標(biāo)本。常規(guī)方法石蠟切片行蘇木素一伊紅(Hematoxylin and Eosin Staining,HE)染色,光學(xué)顯微鏡下觀察心肌的炎癥改變;伊文思藍(lán)/氯化三苯基四唑(Evan's Blue/2,3,5-Triphenyl-2H-Tetrazoliumchloride,EB/TTC)測定心肌梗死面積與免疫比濁法測定血漿肌鈣蛋白Ⅰ濃度;酶聯(lián)免疫吸附試驗(yàn)(enzyme-linkedimmunosorbent assay,ELISA)檢測心肌和血漿中腫瘤壞死因子(tumornecrosis factor,TNF)含量和白介素-6(interleukin-6,IL-6)含量;分光光度法測定心肌組織髓過氧化物酶(Myeloperoxidas MPO)活性;硫代巴比妥酸法測定心肌組織丙二醛(Malondialdehyde MDA)含量;免疫組化分析心肌核轉(zhuǎn)錄因子p65(nulear factor-kappa Bp65 NF-κBp65)表達(dá);原位末端標(biāo)記法(TUNEL)檢測心肌細(xì)胞凋亡。 結(jié)果:1.血流動(dòng)力學(xué)和心律失常觀察:除SHAM組外,再灌120min后各組心率,平均動(dòng)脈壓較缺血前均有下降(p＜0.05),各組之間MAP差異無統(tǒng)計(jì)學(xué)意義(p＞0.05)。STM組、ZSL組心率在刺激階段有下降,STM組下降較ZSL組更明顯,但波動(dòng)幅度仍在10%以內(nèi),刺激停止后,心率回升。STM組、ZSL組再灌后心律失常發(fā)生率明顯低于IR組,差異具有統(tǒng)計(jì)學(xué)意義(p＜0.05); 2、組織形態(tài)學(xué)觀察:光鏡下發(fā)現(xiàn)SHAM組心肌結(jié)構(gòu)正常,細(xì)胞排列緊密、界限清楚,無水腫,無炎性細(xì)胞浸潤;IR組、FJFX組心肌排列稀疏,不規(guī)則,細(xì)胞水腫,部分細(xì)胞出現(xiàn)明顯空泡變性、心肌纖維斷裂,細(xì)胞間隙明顯增寬,大量炎性細(xì)胞浸潤;STM組、ZSL組心肌細(xì)胞排列較規(guī)則,細(xì)胞輕度水腫,細(xì)胞間隙增寬,炎性細(xì)胞散在浸潤; 3、心肌損傷程度觀察:各組心肌行EB/TTC染色顯示,各組間缺血范圍差異無統(tǒng)計(jì)學(xué)意義(P＞0.05),與IR組相比,STM組和ZSL組心肌梗死范圍差異有統(tǒng)計(jì)學(xué)意義(p＜0.05),心肌梗死范圍減小,而ZSL組和STM組比較,無統(tǒng)計(jì)學(xué)差異(P＞0.05),FJFX組和IR組比較,無統(tǒng)計(jì)學(xué)差異(P＞0.05),各組心肌肌鈣蛋白(cTnI)在心肌缺血前無明顯差別,再灌注120min后,除SHAM組外,其他各組cTnI較缺血前均有不同程度的升高(P＜0.05),STM組和ZSL組cTnI濃度低于IR組(P＜0.05),STM組和ZSL組比較,無統(tǒng)計(jì)學(xué)差異(P＞0.05); 4、炎性標(biāo)志物和氧化損害程度觀察:與SHAM組相比,各組MPO活性和MDA含量顯著增高,以IR組升高更為顯著(P＜0.01),與ZSL組和STM組相比,差異也有統(tǒng)計(jì)學(xué)意義(P＜0.05),心肌組織和血漿中的TNF及IL-6的濃度在IR組、STM組、ZSL組、FJFX組明顯升高,與SHAM組相比差異均有統(tǒng)計(jì)學(xué)意義(P＜0.05);其中IR組升高更為顯著,與ZSL組相比,差異也有統(tǒng)計(jì)學(xué)意義(P＜0.05);STM組與ZSL組相比差異無統(tǒng)計(jì)學(xué)意義(P＞0.05)。免疫組化檢測NF-κBp65表達(dá),IR組大部分心肌細(xì)胞中,細(xì)胞漿染色呈陽性,細(xì)胞核染色呈陽性,ZSL組和STM組心肌細(xì)胞的胞漿呈弱陽性或陽性,細(xì)胞核染色呈弱陽性,核移位細(xì)胞數(shù)目減少。平均光密度值比較發(fā)現(xiàn)STM組、ZSL組NF-κBp65表達(dá)低于IR組(P＜0.05); 5、細(xì)胞凋亡觀察:TUNEL法檢測細(xì)胞凋亡發(fā)現(xiàn),在SHAM組中,未發(fā)現(xiàn)TUNEL陽性細(xì)胞,相反,在IR組中,呈現(xiàn)大量TUNEL陽性細(xì)胞核呈棕黃色反應(yīng),核內(nèi)染色質(zhì)濃縮,形成密集顆粒位于核膜下,有的胞核被降解,胞漿不著色。ZSL組、STM組可見散在的陽性細(xì)胞。凋亡指數(shù)(apoptosis index AI)比較發(fā)現(xiàn),STM組、ZSL組AI值低于IR組(P＜0.05)。 結(jié)論:電針足三里能夠抑制心肌缺血再灌注損傷引發(fā)的炎癥反應(yīng),對心肌起到保護(hù)作用;其機(jī)制可能是通過興奮膽堿能抗炎通路,削弱N F-κB表達(dá),減少TNF、IL-6產(chǎn)生,抑制細(xì)胞凋亡有關(guān)。
[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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R245

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