【作者】 黄日红；

【导师】 曲鹏；

【作者基本信息】 大连医科大学 ， 内科学， 2010， 博士

【摘要】 目的：脓毒症早期即存在心血管功能异常,尤其是严重脓毒症和脓毒症休克患者,有50%存在左室收缩／舒张功能异常。关于脓毒症继发心功能障碍的机制复杂多样,主要与细菌内毒素(主要成分为脂多糖LPS)激活血管内皮、中性粒细胞、单核巨噬细胞系统,进而激活凝血、补体激肽系统等,释放多种炎症介质和细胞因子如肿瘤坏死因子-α(TNF-α)、白细胞介素-1 p(IL-1 p)、溶酶体、一氧化氮(NO)等有关,这些心肌抑制因子(MDS)可以直接损伤心肌细胞,同时亦影响心肌线粒体、内质网代谢功能。此外脓毒症时心肌细胞钙循环障碍以及TNF-α诱导的心肌细胞凋亡等也促进了心功能障碍的发生。涉及面甚广,难以从单一环节阻断。Toll样受体(TLR)能够识别病原体,并在病原体入侵机体的早期启动天然免疫,促进免疫细胞成熟分化及调节免疫应答,LPS激活TLR4的信号转导通路后导致NF-K B激活,使炎症因子大量表达,导致全身炎症反应和多器官功能衰竭的发生。A20作为一种NF-κB依赖性表达的胞质蛋白,对NF-κB活性有负反馈调控作用,参与了体内炎症反应的调节和凋亡抑制。所以通过屏蔽TLR4或下调TLR4表达以阻断感染的源头;同时促进A20表达,抑制LPS信号转导,成为近年来防治感染继发心功能障碍研究的热点。谷胺酰胺(Glutamine, Gln)作为体内最丰富的游离氨基酸,能够通过诱导GSH合成及热休克蛋白70表达以增强抗氧化物质活性、抑制NO的产生,是一种多靶点的心肌保护药物。因此,Gln已经作为心肌缺血再灌注损伤保护剂及危重症患者肠粘膜免疫屏障保护剂应用于临床,甚至可以改善Sepsis患者的预后,证实该药是安全可靠的。鉴于已有研究证实：Gln可以下调sepsis大鼠肠道粘膜TLR4信号通路表达,从而减轻肠粘膜损伤；Gin还可以减少I-κB的分解,抑制NF-κB的激活使促炎性细胞因子水平降低。简言之,Gln可以在上游TLR4表达和下游NF-κB激活双环节抑制TLR4信号传导,从而减轻炎性损伤。同时戚仁斌等应用Gin进行大鼠离体心脏主动脉逆灌流,对LPS损伤引起的心肌细胞单相动作电位幅度减小、MAPD及MAPD20延长、心肌张力降低、心率变慢等变化有明显的改善作用,说明Gin对LPS所致心肌损伤有一定保护作用。因此我们通过应用LPS血症大鼠模型,观察作为抗氧化、免疫营养调理制剂应用的Gin能否通过抑制TLR4-NF-κB信号通路,抑制LPS信号转导,减轻心肌损伤及心肌凋亡而保护心功能,以期为防治感染继发心功能障碍提供有效辅助治疗药物。方法：1.动物模型制备和实验分组：健康清洁级SD大鼠90只,雄性,体质量(350±25)g随机分为为3组：对照组10只：生理盐水5ml／只；LPS组40只：LPS10mg/、kg(以生理盐水稀释至5m1):LPS+Gln组40只：LPS 1 Omg/kg+Gln 1 g/kg(以生理盐水稀释至5ml)腹腔注射。于注药后6、24、48、72h处死动物。2.腹腔注药后6、24、48、72h后右颈总动脉插管至左心室,RM6240BD多功能生理记录仪测定平均血压(MAP)、左室收缩末期压力(LVESP).左室压力上升及下降最大变化速率(士dp/dtmax)等心功能指标。3.心尖组织固定、脱水、包埋,HE染色,观察心肌病理学变化。4.RT-PCR检测心肌TLR4.A20.TNF-αmRNA表达,Western-blotting法检测TLR4.A20.NF-κB p65蛋白表达。5.RT-PCR检测心肌Bax.Bcl-2 mRNA表达,免疫组化方法测定心肌Bax.Bcl-2蛋白表达;TUNEL检测心肌细胞凋亡水平；比色法测定Caspase-3活性。6.ELISA测定心肌TNF-α、血清CTnI.NT-ProBNP含量。结果：1.大鼠毒血症表现：大鼠注射LPS 6h后即出现倦怠、嗜睡,进食进水明显减少,呼吸急促,松毛,眼鼻血性分泌物,腹泻,体重下降。解剖见心脏扩大、双肺淤血。2.心功能测定：与对照组比较,LPS注射6h后即先后出现大鼠心脏+dp/dtmax.-dp/dtmax下降,且呈进行性下降趋势,至72h回升。Gln可使不同时间点LVESP.+dp/dtmax.一dp/dtmax升高(P<0.05)。3.病理检查：LPS组大鼠心肌细胞水肿、变性坏死,Gln治疗组心肌细胞损伤减轻。5.LPS组心肌组织TLR4.A20.TNF-αmRNA及TLR4.A20.NF-κBp65蛋白表达升高,LPS+Gln24h组较LPS24h组有下降趋势,差异显著(P<0.05)。6.LPS导致心肌Bcl-2/Bax下降；Caspase一3活性及凋亡率明显升高；Gln可提升心肌Bcl-2／Bax,降低Caspase-3活性及凋亡率(P<0.05)。7.LPS组心肌TNF-α、血清CTnI.BNP浓度显著升高, Gln能降低TNF-α.CTnI.NT-proBNP浓度,使TNF-α提前降至正常。(P<0.05)。结论：1.LPS通过TLR4信号转导通路诱导炎症介质表达和心肌细胞凋亡等,导致心肌损伤及心功能障碍。2. Gln能抑制TLR4-NF-κB信号转导通路,对LPS致心肌损伤具有一定保护作用。3. Gln可部分抑制LPS致心肌凋亡作用,并部分改善心功能。4. A20基因表达上调系NF-κB自身调节所致,Gln无上调A20基因表达作用。更多还原

【Abstract】 Objective:Cardiovascular dysfunction was found at the early stage of sepsis.50% of patients with severe sepsis and septic shock had left ventricular systolic/diastolic dysfunction. Mechanisms of cardiac dysfunction secon-dary to sepsis were complex and diverse. The primary mechanism was the activation of vascular endothehial cells, neutrophils, monocyte-macrophage system, as well as blood coagulation, complement and kinin systems by bacterial endotoxin(major component was lipopolysaccharide, LPS), proceeded with the release of various inflammatory mediators and cytokines (TNF-a, IL-β,lysosome, NO etc). these myocardial depressant factors (MDS) directly damaged myocardial cells and the metabolic functions of myocardial mitchondria and endoplasmic reticulum. In addition, calcium circulatory disorder of myocardial cells in sepsis and TNF-a-induced myocardial apoptosis promotes the occurrence of cardiac dysfunction. Factors involved in the mechanisms were so complex and various to find one single part to block. Toll-like receptors (TLRs) Could recognize the pathogen initiate innate immune response at the early stage of pathogen invasion, promote maturation and classification of immune cells, regulate immune response, and trigger inflammatory response. LPS could activate the TLR4 signaling pathways resulting in the activation of NF-κB, and then large amounts of inflammatory factors would be expressed, Leading to the occurrence of systemic inflammatory response and multiple organ failure. A20,a NF-KB-dependant cytosolic protein, Could block NF-κB activation via a negative feedback loop, and participate in the regulation of inflammatory response in vivo. Therefore, screening and downregulation of TLR4 to inhibit the source of infection, together with upregulation of the A20 expression and inhibition of LPS signaling transduction to protect cardiac function has become the hot spot in recent years.Gin, as the body’s most abundant free amino acids, was a multi-target drug for myocardial protection, which could enhance the capacity of antioxidant activity and inhibition of nitric oxide production through the synthesis of GSH and induction of heat shock protein 70 expression.Therefore, Gin had been used in clinical as its protection for myocardial ischemia and reperfusion injury, as well as protection for intestinal mucosa immunological barrier. It could improve the prognosis of sepsis and had been proved safe. Studies had confirmed that Gin could downregulate the expression of TLR4 signaling pathway in sepsis to reduce the mucosal injury. Gin could also reduce the decomposition of I-KB and inhibit the activation of NF-κB to reduce the level of pro-inflammatory cytokines. In short, gln could inhibit TLR4 signaling by influnce the expression of TLR4 in upstream and activation of NF-κB in downstream, and thereby reduced inflammatory injury, meanwhile Gin had been used in isolated rat heart against aortic perfusion, and was proved protective effects on LPS induced myocardial injury as follows, improving the LPS-induced myocardial injury in monophosic activating amplitude, decreasing the extention of MAPD and MAPD 20,and reducing myocardial tension and heart rate. In our study, we constructed a LPS induced septic model, to find the protective effects of Gin on myocardial injury through the inhibition of TLR4-NF-κB signalling and LPS signalling. We hope to find an effective adjuvant treatment to prevent cardiac dysfunction secondary to sepsis.METHOEDS:1 Animal model and experimental groups:healthy and clean SD rat 90,male, body weight (350±25)g were randomly divided into 3groups: control group 40:saline 5ml/only;LPS group 40:LPS 10mg/Kg (in normal saline diluted to 5ml) LPS+Gln group 40:LPS 10mg/Kg+Glnlg/Kg in normal saline diluted to 5ml, intraperitoneally we sacrificed 6,24,48,72h after the animals injected.2 Intubate a catheter through the right carotid artery to the left ventricle at 6,24,48 and 72h after intraperitoneal injection.RM6240 BD multifunctional physiological recorders,was used to record the average blood pressure (MAP),left ventricular end systolic pressure,pressure rise and fall of left the maximum rate of change (±dp/dtmax) and other cardcial function.3 Apical tissues were fixed, dehydrated, embeded, HE stained, and we oberseved the changes of cardiac pathology.4 RT-PCR was used to detect myocardial TLR4,A20 and TNF-a mRNA expression,and Western-blotting was used to detect the expression of TLR4, A20,NF-κB P65 protein.5 RT-PCR was used to detect cardiac Bax,Bcl-2 mRNA expression, immunohistochemistry was used to measure myocardial Bax Bcl-2 protein expression TUNEL was used to examine the level of cardiomyocyte apoptosis,and colorimetric technique was used to determine caspase-3 activity.6 ELISA was used to measure the content of myocardial TNF-a serum CTnl,NT-proBNP.Results:1 Toxemia performance in rats:6h after injection of LPS, rats appeared fatigue sleepiness, reduction in the consumption of water,shortness of breath,increasing of eyes and nose’s bloody dischange,diarrhea and weight loss. Heart enlargement and lung congestion were observed anatomically.2 Cardiac functional measurements:compared with controlled group, LVESP,+dp/dtmax,-dp/dtmax decreased 6h after LPS injection, and progressively decline and then rebounded till 72h. Gln increased +dp/dtmax,+dp/dtmax at different time points (p<0.05).3 Pathology:LPS included myocardical cell edema, degeneration and necrosis. Gin treatment group reduced myocardial cell injury.4 LPS group:myocardial TLR4, A20 and TNF-a mRNA and TLR4,A20, NF-κB 65protein expression increased; compared with LPS group, LPS+Glngroup decreased.The difference was significant (p<0.05).5 LPS led to decline in myocardial Bcl-2/Bax,caspase-3 activity and apoptosis rate Gln could significantly improve Bcl-2/Bax,and reduce the caspase-3 activity and apoptosis rat (p<0.05).6 In LPS group, myocardial TNF-a,serum cTnI,BNP concentration increased significantly.Gln could decrease TNF-a, CTnl,NT-ProBNP concentration (p<0.05).Conclusion:1 LPS signal pathway could induce expression of inflammatory mediators and myocardial apoptosis through TLR4, leading to myocardial injury.2 Gln could inhibit the TLR4/NF-κB signal pathway, and had a protective effect on the LPS induced myocardial injury.3 Gln could partially inhibit LPS-induced myocardial apoptosis, and partially improve cardiac function.4 Expression of the A20 was self regulation of NF-kappaB. Gln couldn’t upregulate the expression of A20.更多还原