【作者】 高好考；

【导师】 王海昌；

【作者基本信息】 第四军医大学 ， 内科学， 2009， 博士

【摘要】 研究背景:缺血性心脏病(IHD)是当今世界威胁人类健康的首敌。随着冠状动脉内溶栓及冠脉搭桥等内外科治疗手段的广泛应用,继之出现的心肌细胞再灌注损伤日益受到心脏病学家的高度重视。来自动物实验和临床观察的证据表明,心脏缺血再灌注(MI/R)后局部组织过度的炎症反应是造成再灌注损伤的主要原因之一,也是心脏缺血继发性损伤的重要病理生理机制。多项研究证实,p38丝裂原活化蛋白激酶(p38-MAPK)和核转录因子-κB(NF-κB)是各器官I/R炎症损伤中密切相关的两个转录因子,并对炎症介质如TNF-α、IL-1β、IL-6和黏附分子(ICAM-1、VCAM-1)等的表达及凋亡细胞的调控发挥着重要作用。因此,药物预处理下调p38MAPK和NF-κB的活性,有望成为控制TNF-α等表达的策略,从而减轻MI/R炎性损伤。研究认为急性期MI/R损伤是固有免疫应答的一部分,而导致这种非感染刺激炎症应答的机制部分是通过Toll样受体(TLRs)介导的信号。TLRs在心肌细胞的表达不但对心肌细胞诱导炎症应答带来新的认识,同时也提供降低MI/R损伤的靶点。TLRs在缺血性心脏损伤中的作用研究才刚刚起步,目前和心脏缺血关系比较明确是TLR2和TLR4。TLR2和TLR4均可以识别缺血中损伤释放的内源性“危险信号”,这为TLRs和心脏缺血的关系研究提供了理论依据。MI/R增加TLR4 mRNA和蛋白表达,TLR4激活下游MAPK、NF-κB等活性,随后炎症因子瀑布表达,引起心肌损伤。但在MI/R中TLR2表达情况仍不清楚,药物是否能降低心脏TLR2信号通路减轻炎性损伤的研究也未见报道。目前,再灌注治疗显著促进急性心肌梗死(AMI)患者的预后,然而,将近25%患者再灌注治疗不能提供足够的存活心肌,而导致严重心力衰竭,故寻找安全有效的抗心肌缺血再灌注损伤药物和方法是十分必要的。研究证实许多具有心肌保护的药物通过多种信号通路最后聚集在糖原合酶激酶(GSK)-3β这一靶点上,即增加GSK-3β位点9丝氨酸(Ser9)的磷酸化,抑制其活性降低线粒体通透性转换孔(mPTP)开放保护心肌,然而GSK-3β是否通过其他机制影响再灌注损伤仍不清楚。最新研究证实GSK-3β也调节炎症如增强NF-κB活性,因此GSK-3β成为治疗I/R和炎症性疾病的新靶点。但是,在MI/R过程中,应用GSK-3β抑制剂TDZD-8抑制其活性是否具有抗炎和免疫作用及其涉及的机制,尚需进一步深入研究。因此,本研究的实验目的:1.明确GSK-3β抑制剂TDZD-8治疗能否抑制MI/R中炎性因子的产生,减轻MI/R病理损伤过程中的炎症反应,从而发挥抗炎作用。2.如果是,进一步探讨GSK-3β抑制剂TDZD-8作用的具体信号转导机制,尤其是p38MAPK和NF-κB分子在其中的作用。3.确定GSK-3β抑制剂TDZD-8能否下调再灌注激活的TLR2/NF-κB信号通路活性。4.研究GSK-3β抑制剂的上述抗炎和抑制免疫效应是否最终有助于减少I/R导致的细胞凋亡和心梗范围,从而发挥显著的心脏保护作用。实验方法1.雄性SD大鼠,腹腔麻醉后,开胸选择性结扎冠状动脉左室支,制备心肌I/R大鼠模型。分离股动脉并插管记录动脉压,分离颈静脉建立静脉通路。缺血30 min,再灌注6 h,再灌注开始后封闭胸腔,恢复动物自主呼吸。再灌注前5min颈静脉推注不同浓度TDZD-8(0.1,1mg/kg)。I/R术中通过八道生理记录仪持续记录大鼠心电图、心率、动脉压及左室内压等血流动力学指标;分别于缺血前、缺血30 min、再灌注2h、6 h由尾静脉取血,测血糖水平;再灌注6 h后由颈动脉取血2 ml,分光光度法检测血清肌酸激酶(CK)和乳酸脱氢酶(LDH)活性。2.动物模型的制作同第一部分。再灌注前5min分别静脉推注二甲基亚砜(DMSO)或TDZD-8(1mg/kg)。再灌注6h后采用DAPI细胞核标记和末端脱氧核苷基转移酶介导的dUTP原位平端标记法(TUNEL)复染定性和定量检测心肌细胞凋亡指数(AI)。AI以TUNEL阳性细胞数占总细胞计数的百分比表示;采用伊文蓝-TTC双染法测定心肌梗死范围(IS),IS以心脏梗死范围(INF)占缺血区面积(AAR)的百分比表示(INF/AAR×100%);留取心肌组织,用ELISA试剂盒检测组织TNF-α和IL-6的含量;分光光度法检测心肌髓过氧化物酶(MPO)活性;再灌注30min后采用Western Blot方法测定缺血心肌组织NF-κBp65, p38MAPK,GSK-3βSer9磷酸化蛋白含量,以检测其活性。3.动物模型的制作同第一部分,应用实时荧光定量(RT)-PCR观察再灌注不同时间点(1h-24h)TLR2mRNA;免疫组织化学检测再灌注12h后TLR2在心脏组织中定位情况。于再灌注前5min静脉推注TDZD-8(1mg/kg),观察再灌注1h后TLR2mRNA和下游炎症因子TNF-α和IL-6mRNA变化及两者相关性;Western Blot方法测定缺血心肌组织NF-κBp65磷酸化蛋白含量及与TLR2mRNA相关性;采用伊文蓝-TTC双染法测定心肌梗死范围(IS)。实验结果1.各组间心率在缺血期和再灌注期均不存在显著性差异,各组动脉压在缺血期间较假手术组下降(P<0.05),再灌注期间无差异。I/R组室内收缩压(LVSP)及其微分(±dP/dtmax)均出现降低,左室舒张末压(LVEDP)升高,有显著差异(P<0.01)。TDZD-8小剂量LVSP未见变化。与I/R组相比,TDZD-8大剂量组LVSP明显增高(P<0.05),LVEDP及±dP/dtmax均明显改善(P<0.05)。CK和LDH活性反映心肌损伤程度。二者在大剂量TDZD-8组显著降低(P<0.05)。提示再灌注时给予大剂量TDZD-8不影响血糖水平的改变而减轻再灌注心肌损伤并且增强心功能。2.再灌注期给予TDZD-8发挥显著的抗心肌细胞凋亡作用,细胞凋亡率从22%降到12%(P<0.05);MI/R大鼠心肌梗死面积明显增加,再灌注时给予TDZD-8心肌梗死面积从58%降到34%(P<0.05)。TDZD-8单独应用并未影响心肌梗死面积和细胞凋亡。3.与假手术组相比,缺血30 min再灌注6 h导致心肌组织中TNF-α,IL-6生成及MPO(反映中性粒细胞的浸润水平)大量增加。与MI/R组相比,给予TDZD-8显著减少心肌TNF-α,IL-6生成,分别从165.5pg/mg protein,44.7pg/mg protein降到107.4pg/mg protein和29.28 pg/mg protein(P<0.05),同时TDZD-8降低心肌MPO活性,从36.40 U/100 mg tissue降到21.80 U/100 mg tissue(P<0.05)。这些结果提示再灌注时给予TDZD-8可抑制减轻心肌组织局部的炎症反应。4.再灌注30min使缺血心肌的NF-κBp65磷酸化增加3.4倍,p38MAPK磷酸化增加4倍,GSK-3βSer9磷酸化水平下降,而TDZD-8抑制MI/R诱导的NF-κBp65、p38MAPK磷酸化阻断其活化(P<0.05),增加GSK-3βSer9磷酸化(P<0.05)。5. TLR2mRNA随着再灌注时间(1h-24h)的延长逐渐升高。1, 6, 12和24h TLR2mRNA分别是较假手术组TLR2mRNA的2.1-, 2.9- , 4.7-和3.5-倍(P<0.001),12hTLR2mRNA达到最高点;免疫组织化学检测发现再灌注12h后缺血区相邻的2个或多个心肌细胞膜TLR2呈团簇样表达,提示再灌注激活固有免疫系统。6.再灌注1h后相关性分析TLR2mRNA表达与NF-κBp65活化显著相关(r=0.89, P<0.001),与TNF-α,IL-6呈正相关(分别为: r=0.65; r=0.68, P<0.05)。TDZD-8处理显著降低TLR2mRNA和TNF-α、IL-6mRNA(分别从2.1-,4.5-,4.3-倍降到1.5-,3.0-,2.7-倍, P<0.05);NF-κBp65磷酸化蛋白含量较对照组明显下降(P<0.05);心肌梗死面积较MI/R组下降约33% (P<0.05)。结论1. TDZD-8通过增加GSK-3βSer9磷酸化而降低NF-κB和p38MAPK活性,减少再灌注心肌TNF-α、IL-6生成,抑制心肌中性粒细胞的浸润,减轻心肌损伤(心肌梗死面积和细胞凋亡),改善左室功能,保护心肌。2.再灌注引起固有免疫系统TLR2功能性表达,TDZD-8降低再灌注后TLR2表达及其信号通路而保护心肌。3.上述结果提示GSK-3β抑制剂TDZD-8心血管保护的抗炎和免疫的新机制,为GSK-3β抑制剂更有效、合理应用于治疗缺血再灌注损伤其他炎症相关性疾病提供了理论依据和参考资料。更多还原

【Abstract】 BackgroundGrowing evidence from both animal experiments and clinical observations indicates that inflammatory response and cytokine production are particularly active after myocardial infarction and contribute to cardiac function and eventual host outcome. Cytokines such as TNF-αreleased after myocardial ischemic injury can acutely regulate myocyte survival or deaths and trigger subsequent cellular inflammatory response. Many studies indicated that p38MAPK and NF-κB are more important translation signaling, they induced the inflammation cytokines. So the drugs which decrease the active of p38MAPK and NF-κB signaling can protects the heart from acute ischemia-reperfusion injury via inhibition of inflammation and apoptosis.Recently, the acute phase of I-R has been viewed as part of the innate immune response, with a lack of vascular perfusion and oxygenation. These non-infectious stimuli contribute to the inflammatory response, in part by signaling via Toll-like receptors (TLRs)-mediated pathways. The identification of TLRs on cardiomyocytes not only has brought new insights on the inflammatory response initiated by cardiomyocytes themselves, but also offered potential targets to reduce I-R injury. Till date, there are no other studies on the role of TLR2 in MI-R injury documented. It has not been reported until now whether drug administration could modulate TLR2 /NF-κB signaling following MI-R injury in rats.Introduction of reperfusion therapy has markedly improved prognosis of patients with acute myocardial infarction (AMI). However, current reperfusion therapy cannot afford sufficient myocardial salvage in approximately 25% of patients, who subsequently develop severe heart failure. Thus, there is a clinical need for novel therapy to protect cardiomyocytes from ischemia/ reperfusion-induced necrosis. Many drugs which lead to myocardial protection from infarction induce Ser 9 -phosphorylation of GSK-3βand regulate of mPTP opening. Several reports have shown that chemical inhibitors of GSK-3βcan reduce the levels of pro-inflammatory cytokines in acute systemic inflammation. However, it is not known whether the GSK-3βinhibitor TDZD-8-elicited anti-inflammatory property contributes to the cardioprotective and prosurvival effects, if so, what the molecular mechanism is.Objectives1. To determine whether treatment with TDZD-8 may inhibit inflammation cytokines induction and reduce acute inflammatory response in MI/R.2. If so, to investigate the possible mechanism(s) involved in TDZD-8-induced inflammation cytokines induction in MI/R.3. To determine the modulatory role of TDZD-8 on TLR2/NF-κB signaling following MI-R injury in rats.4. To elucidate whether the anti-inflammatory and anti-apoptosis effect afforded by TDZD-8 may contribute to its cardio-protection in MI/R.Methods1. Rats were anesthetized and myocardial ischemia was produced by exteriorizing the heart through a left thoracic incision and placing a 6-0 silk and making a slipknot around the left anterior descending coronary artery. After 30 minutes of ischemia, the slipknot was released and the myocardium was reperfused for 6 hours. The administration of different concentration TDZD-8 (from 0.1 mg/kg to 1 mg/kg intravenous bolus) was gave 5 min prior to surgical procedures. Hemodynamic data were continuously monitored on a polygraph (RM-6200C) and simultaneously digitized by using a computer interfaced with an analog-to-digital converter. Blood samples were drawn from caudal vein before ischemia, 30 minutes after ischemia and 2/6 hours after reperfusion respectively to measure blood glucose levels. Plasma creatine kinase (CK) and lactate dehydrogenase (LDH) activities were measured spectrophotometrically (Beckman DU 640, USA) at 6 hours after reperfusion.2. The animal model was the same as that in the first part. Rats were received DMSO or TDZD-8 (1 mg/kg intravenous bolus) 5 min prior to surgical procedures. After 6h reperfusion, the myocardial infarct size was determined by means of a double-staining technique and was analyzed by digital imaging systems. Evan’s blue stained area, TTC stained area and TTC stained negative area were measured digitally using Image Pro Plus software. Myocardial apoptotic index was analyzed by TUNEL assay. A double-staining technique was used, i.e., TUNEL staining for apoptotic cell nuclei and DAPI staining for all myocardial cell nuclei. Myocardial TNF-αand IL-6 were detected using the enzyme-linked immunosorbent assay (ELISA) kits. Myocardial myeloperoxidase (MPO) was also deteced at the end of reperfusion. NF-κBp65, p38MAPK and GSK-3βSer9 phosphorylation were determined by western blot at the end of 30 min reperfusion.3. The animal model was the same as that in the first part. Real-time reverse transcriptase- polymerase chain reaction (RT-PCR), and immunohistochemist -ry (IHC) were used to analyze the presence and quantity of TLR2 mRNA and protein. Rats were received TDZD-8 (1 mg/kg) 5 min prior to surgical procedures. TNF-αmRNA and IL-6 mRNA were analyzed by RT-PCR. The activation of NF-κB was detected by western blot. The relation of TLR2 with NF-κB and its downstream cytokines was analysised. Myocardial infarct size was assessed by Evans blue-TTC staining.Results1. Low and high concentration of TDZD-8 did not change the level of blood glucose and heart rate, blood pressure before and after I-R in all study groups.±dp/dtmax decreased in MI/R group and Low concentration of TDZD-8 after 2h and 6h of reperfusion compared with sham group (P<0.05). High concentration of TDZD-8 increased±dp/dtmax compared with MI/R group (P<0.05). 2. MI/R resulted in a significant increase in TUNEL-positive nuclei (AI: 22±2% vs. sham groups, p <0.05) Administration of TDZD-8 caused a significant reduction in AI compared with the MI-R group (12±1%, p < 0.05). The infarct size (IS)/AAR ratio was about 58.64±9.45% in MI-R group. However, the IS/AAR ratio was reduced in TDZD-8 animals (34.42±8.65% of AAR) by nearly 43% compared with MI-R animals (p < 0.05). Administration of TDZD-8 alone did not affect the apoptosis and infarct size.3. Following 6h reperfusion, the activity of MPO was significantly elevated after MI-R when compared to the sham groups (p < 0.05). In the MI-R group, reperfusion resulted in a noticeable increase in cardiac TNF-αcontent compared with the sham groups (165.5±9.6 pg/mg protein, p < 0.05). The administration of TDZD-8 caused a significant attenuation of cardiac TNF-αcontent (107.4±7.3pg/mg protein vs. MI-R group, p <0.05). The same trend was observed in the levels of IL-6. Compared with the sham groups, MI-R-mediated injury significantly increased IL-6 levels (44.7±7.5 pg/mg protein, p <0.05), while TDZD-8 administration significantly decreased (29.28±6.3 pg/mg protein vs. MI-R group, p <0.05).4. Consistent with the levels of p-NF-κB p65 and p-p38MAPK protein resulted in 3.4-fold and 4-fold increase, but decrease in GSK-3βSer9 phosphorylation in myocardium in I/R rats. Treatment with TDZD-8 resulted in the decrease of p-NF-κB p65 and p-p38MAPK protein, and increased the p-GSK-3βSer9.5. After reperfusion, TLR2 mRNA expression was greater and increased over time. At 1, 6, 12 and 24h reperfusion, expression of the TLR2 gene was 2.1-, 2.9- , 4.7- and 3.5-fold higher compared to the sham group (p<0.001), respectively. IHC staining confirmed the expression of TLR2 occurred mainly in cardiac myocytes. In cardiac sections adjacent to the site of ischemic injury, enhanced and predominantly sarcolemmal staining of TLR2 was observed. Scattered foci of intense TLR2 staining involving 2 or more contiguous myocytes were noted in the I-R tissue.6. To investigate whether the functional effects of TLR2 are associated with pro-inflammatory cytokine production in MI-R, the levels of tissue TNF-αand IL-6 mRNA were measured after 1 h reperfusion. These mRNA levels were significantly increased after 1h reperfusion (TLR2: 2.1±0.1-fold higher than sham; TNF-α: 4.5±0.4-fold higher than sham, and IL-6: 4.3±0.4-fold higher than sham, p<0.05). The mRNA levels of TNF-αand IL-6 were positively correlated with TLR2 mRNA levels after 1 h reperfusion. (TLR2 vs. TNF-αmRNA: r=0.65, p<0.05; TLR2 vs. IL-6 mRNA: r=0.68, p<0.05). Treatment with TDZD-8 had a significant effect on the mRNA levels of TLR2, TNF-αand IL-6 compared with MI-R (2.1±0.1 to 1.5±0.1; 4.5±0.4 to 3.0±0.5; 4.3±0.4 to 2.7±0.3; respectively, p<0.05). There was a significant correlation between TLR2 mRNA levels and the levels of phospho- NF-κB p65 protein after 1h reperfusion. (TLR2 mRNA vs. p- NF-κB p65: r=0.89, p<0.001).The levels of phospho-NF-κB p65 protein were reduced in the nuclear fractions of the hearts of animals that received TDZD-8 (p < 0.05 vs MI-R group). The ratio of the infarct size (IS)/AAR was reduced in the animals treated with TDZD-8 by nearly 33% compared with the MI-R animals (p<0.05). Conclusions1. We have demonstrated that GSK-3βinhibitor TDZD-8 reduced myocardial infarct size, decreased neutrophil infiltration and suppressed NF-κB and p38 MAPK activation and associated proinflammatory cytokine expression, and reduced myocardial apoptosis in a rat model of MI-R through increasing the phospho- GSK-3βSer9 in MI/R.2. The functional effects of TLR2 are associated with pro-inflammatory cytokine production in MI-R and GSK-3βinhibitor reduced TLR2/ NF-κB signaling following MI/R.3. The anti-inflammatory property elicited by GSK-3βinhibitor may contribute to its cardioprotective effects in I/R injury and inflammatory related illness.更多还原