高迁移率族蛋白1在大鼠心肌缺血再灌注损伤中的作用及丙酮酸乙酯的疗效
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摘要
再灌注治疗虽然使缺血部位心肌血供得到恢复,但也可导致再灌注损伤。心肌缺血-再灌注损伤(IRI)的存在限制了再灌注治疗效果。炎症反应、氧化应激、钙超载均为心肌IRI的重要机制,但其始发因素及信号转导机制尚未彻底阐明。为此,深入探讨心肌IRI的分子调控机制及有效防治对策必将具有重要的临床及理论价值。本研究利用大鼠心肌缺血再灌注损伤模型,着重探讨心肌缺血-再灌注后高迁移率族蛋白1(HMGB1)的表达及其与心肌IRI的关系,同时观察丙酮酸乙酯干预对心肌IRI的作用及分子机制,研究主要由4部分组成:①心肌缺血-再灌注后高迁移率族蛋白1的表达及其作用,②丙酮酸乙酯干预心肌缺血-再灌注损伤的剂量及时机,③高迁移率族蛋白1介导炎性缺血-再灌注损伤的机制及丙酮酸乙酯的作用,④丙酮酸乙酯对心肌缺血-再灌注后氧自由基及线粒体的影响。
一、心肌缺血再灌注后高迁移率族蛋白1的表达及其作用
目的探讨高迁移率族蛋白1(high mobility group box 1,HMGB1)在心肌缺血-再灌注后的时空表达特点;观察外源性HMGB1对心肌缺血再灌注损伤的影响及其可能机制。
方法第I部分:清洁级健康雄性SD大鼠分为5组,每组6只,分别于心肌缺血前、缺血30分钟、再灌注30分钟、再灌注24小时、再灌注48小时处死大鼠,留取标本检测HMGB1的表达。第II部分:清洁级健康雄性SD大鼠分为4组,每组6只,均给予缺血30分钟,再灌注48小时:对照组(Control),HMGB1-1μg /kg组(HMGB1-1μg),HMGB1-10μg /kg组(HMGB1-10μg),HMGB1-100μg /kg组(HMGB1-100μg),后3组在缺血再灌注后注射相应剂量rhHMGB1。采用有创血流动力学检测大鼠左心功能变化,TTC染色计算心肌梗死面积,免疫组化、Real-time PCR、Western Blot检测HMGB1及其受体RAGE、TLR-2、TLR-4的表达。
结果第I部分:在正常心肌组织中,HMGB1蛋白定位于心肌细胞核内;心肌缺血再灌注后,HMGB1不仅在心肌细胞核内表达,而且大量表达于梗死区域内的炎性细胞;HMGB1在大鼠心肌缺血30分钟后显著增高,并持续至再灌注48小时。第II部分:与Control组相比,HMGB1-1μg组、HMGB1-10μg组心肌梗死面积、±dp/dt max、RAGE、TLR-2、TLR-4无显著差异(P >0.05);但与Control组相比,HMGB1-100μg组心肌梗死面积显著增大(56.4±5.1% vs. 44.6±3.7%,P<0.05),±dp/dt max显著恶化(2199±185 mmHg/s vs. 2890±217mmHg/s; 1536±122mmHg/s vs.1979±190 mmHg/s;p<0.05);HMGB1、TNF-α及IL-6水平显著增高,与Control组相比,HMGB1-100μg组RAGE、TLR-2、TLR-4水平明显增高,其中以RAGE升高幅度最为明显。
结论心肌缺血再灌注后心肌及炎症细胞胞核浆HMGB1表达显著增高,外源性HMGB1进一步放大心肌炎症反应、增大梗死面积;HMGB1主要通过RAGE受体介导加重炎症反应及心肌损伤。
二、丙酮酸乙酯干预心肌缺血再灌注损伤的剂量及时机
目的观察不同剂量丙酮酸乙酯(Ethyl pyruvate, EP)对缺血-再灌注心肌的保护作用即量效关系研究;在合适的剂量下,探索其有效的治疗窗口即时效关系研究。
方法量效关系部分:清洁级健康雄性SD大鼠分为6组,每组6只,给予缺血30分钟,再灌注48小时,具体分组如下:Control组、10mg/kg组、20mg/kg组、40mg/kg组、80mg/kg组、160mg/kg组。Control组给予乳酸林格斯液静脉注射,其余各组均于缺血前2分钟给予相应剂量的EP静脉注射。时效关系部分:清洁级健康雄性SD大鼠分为4个组,每组6只,给予缺血30分钟,再灌注48小时,具体分组如下:Control组(缺血前2分钟给予乳酸林格斯液注射),缺血前2分钟给药组(Is),再灌注前2分钟给药组(R),再灌注后30分钟给药组(R30)。除Control组外,其余各组均给予EP(40mg/kg)于相应的时间点静脉注射。采用有创血流动力学检测大鼠左心功能变化,TTC染色计算心肌梗死面积。
结果在量效关系部分,与Control组相比,心梗面积在20mg/kg组就显著减小(36.1±1.6% vs. 45.9±4.2%, P<0.05),心功能显著改善,当剂量增加到40mg/kg时,心梗面积进一步减小(28.7±1.5% vs. 45.9±4.2%, P<0.05),心功能进一步改善,当给药剂量进一步增加到80mg/kg、160mg/kg时,心梗面积未见进一步减小。在时效关系部分,与Control组相比,Is组、R组均可显著减少心肌梗死面积(29.0±2.5%, 35.3±2.8 vs. 46.4±3.8%, P<0.05),改善心功能;R30组心梗面积有减小趋势,但无统计学差异(42.7±3.4% vs. 46.4±3.8%, P>0.05)。
结论40mg/kg的EP是治疗大鼠心肌缺血-再灌注损伤的最佳剂量;缺血前预先应用EP的疗效优于再灌注即刻给药,当给药时间延迟到再灌注后30分钟则EP的心肌保护作用消失;缺血前2分钟至再灌注前2分钟是EP保护心肌缺血-再灌注损伤的最佳时间窗。
三、HMGB1介导炎性缺血-再灌注损伤的机制及EP的作用
目的观察丙酮酸乙酯(EP)干预对炎症因子表达及炎症性心肌缺血-再灌注损伤的影响;研究EP干预对心肌缺血-再灌注后炎症因子表达变化的调控机制。
方法清洁级健康雄性SD大鼠分为4组,每组12只,均给予缺血30分钟,再灌注48小时:1.假手术组(Sham),只穿线不结扎;2.对照组(Control),于再灌注前给予PBS 0.5ml+RLS 0.5ml静脉注射;3. EP治疗组(EP),再灌注前2分钟给予静脉注射EP(40mg/kg);4. EP+HMGB1组(EP+HMGB1):缺血再灌注前2分钟静脉注射EP(40mg/kg)+重组HMGB1(100μg /kg)。采用有创血流动力学检测大鼠左心功能变化,TTC染色计算心肌梗死面积,ELISA法检测血清HMGB1、TNF-α、IL-6,Western Blot检测心肌磷酸化P38的表达。
结果与Sham组相比,Control组心肌梗死面积显著增加,心功能显著恶化,血清HMGB1、TNF-α、IL-6表达显著增高,磷酸化P38水平显著增高(P<0.05);与Control组相比,给予EP治疗后心肌梗死面积显著下降(35.0±3.1% vs. 45.7±3.5%, P<0.05),心功能明显改善,HMGB1、TNF-α、IL-6及磷酸化P38水平显著降低,而当HMGB1(100μg/kg)与EP同时给药时,EP的心肌保护效益完全消失。
结论心肌缺血再灌注前给予适时适量EP能抑制炎症因子HMGB1、TNF-α、IL-6的表达,减少炎性心肌缺血-再灌注损伤;同时给予外源性HMGB1则可完全消除EP的心肌保护作用;EP通过影响HMGB1与受体结合后的信号转导通路关键蛋白p38的磷酸化、阻断或降低HMGB1介导的促炎反应及炎性心肌缺血-再灌注损伤。
四、丙酮酸乙酯对心肌缺血再灌注后氧自由基及线粒体的影响
目的探讨丙酮酸乙酯(EP)干预缺血再灌注损伤是否可影响氧自由基的代谢;观察EP干预缺血再灌注损伤是否影响线粒体结构与功能。
方法清洁级健康雄性SD大鼠分为4个组,每组6只,给予缺血30分钟,再灌注3小时:Sham组(冠脉只穿线不结扎);Control组(再灌注前2分钟给予RLS静脉注射);EP治疗组(EP组,再灌注前2分钟给予EP40mg/kg静脉注射)。EP+苍术苷组(EP+Atr组,EP40mg/kg加苍术苷5mg/kg静脉注射)。检测大鼠左心功能变化,心肌梗死面积,心肌MDA、MPO、SOD水平,电镜检测心肌超微结构变化。
结果与Sham组相比,Control组心肌MDA(6.3±0.7 nmol/mg vs. 1.6±0.3 nmol/mg,P<0.05)、MPO(0.35±0.04 U/g vs. 0.95±0.12 U/g,P<0.05)显著升高,SOD显著下降(65±10 U/mg vs. 315±46 U/mg,P<0.05),线粒体受损明显;与Control组相比,EP治疗组显著减小心肌梗死面积(44.5±4.5% vs.33.7±4.1%, P<0.05),抑制MDA(3.2±0.5 nmol/mg vs. control,P<0.05)、MPO(0.67±0.06 U/g vs. control,P<0.05)增高,提升SOD水平(189±25 U/mg vs. control,P<0.05),减轻心肌线粒体损伤;当苍术苷与EP同时给药时,EP的心肌保护效益完全消失。
结论心肌缺血再灌注前予EP干预可抑制自由基生成、增加对其的清除能力;心肌缺血再灌注前予EP干预可抑制mPTP过度开放、减轻线粒体损伤;心肌缺血再灌注前予EP干预可减少再灌损伤及心梗面积、改善心功能。
Early reperfusion of ischemic myocardium is the most effective treatment to limit the infarct size and improve the clinical outcome. However, the process of restoring blood flow to the ischemic myocardium has the potential to induce additional injuries and reduce myocardial salvage. Oxidative stress and Inflammation play an important role in the pathogenesis of myocardial reperfusion injury, however, the the trigger and the mechanism of inflammation propagation have not been identified.The current study was to investigate:(1) The current study was to investigate the role of HMGB1 in rat myocardial I/R model,(2)the optimal dose and time choice of ethyl pyruvate(EP) in rat myocardium after ischemia reperfusion, (3)the potential mechenism that HMGB1 induced myocardial ischemia reperfusion injury and the effect of ethyl pyruvate(EP), (4) Effect of ethyl pyruvate on reactive oxygen species and mitochondria in myocardial ischemia reperfusion in rats
Part I: Role of high-mobility group box-1 in myocardial ischemia reperfusion injury Aim This study was designed to investigate potential role of high-mobility group box-1(HMGB1)after ischemia reperfusion injury and effect of exogenous recombined HMGB1 in rat myocardium.
Methods Part one: Male Sprague Dawley rats were assigned to five groups (n=6) . Rats were executed before ischemia, 30 minutes after ischemia, 30 minutes after reperfusion, 24 hours after after reperfusion, 48 hours after reperfusion, respectively. Immunohistochemistry and western blot were conducted to detecte the expression of HMGB1. Part two: Male Sprague Dawley rats were subjected to 30 minutes myocardial ischemia and 48 hours reperfusion. Rats were assigned to four group (n=6 per group): Control group, HMGB1-1μg group, HMGB1-10μg and HMGB1-100μg group; PBS (vehicle) or three doses of recombined HMGB1 (1μg/kg, 10μg/kg, 100μg/kg) was injected intravenously before reperfusion respectively). Cardiac function was measured and myocardial necrosis was evaluated by triphenyltetrazolium chloride (TTC) staining. Western blot and quantitative Real-time PCR were conducted to evaluate HMGB1, RAGE、TLR-2、TLR-4 expression levels.
Results Part one: HMGB1 was mildly stained and the expression was predominantly in the nucleus of cardiac myocyte in non-ischemic myocardium. After 30 minutes ischemia and 48 hours reperfusion, strong expression of HMGB1 was observed both in cytoplasm of cardiac myocyte and the infiltrated inflammatory cells. Expression of HMGB1 was significantly increased after 30 minutes ischemia and was still elevated 48 hours after reperfusion. Part two: No significant difference of infarct size,±dp/dt max, RAGE、TLR-2、TLR-4 among the control, HMGB1-1μg and HMGB1-10μg group was determined. Compared with the control group, infarct size (IS) was markedly increased in the HMGB1-100 group (56.4±5.1% vs. 44.6±3.7%, P<0.05), accompanied by impaired LV +dp/dt max (2199±185 mmHg/s vs. 2890±217mmHg/s, P<0.05), -dp/dt max (1536±122mmHg/s vs.1979±190 mmHg/s, P<0.05) and elevated level of TNF-αand IL-6. Compared with the control group, TLR-2, TLR-4 and RAGE were significantly upregulated in the HMGB1-100μg group. The increase of RAGE was most obvious.
Conclusions In this rat in vivo ischemia/reperfusion (I/R) experiment, myocardial ischemia reperfusion resulted in increased expression of cardiac HMGB1.Exogenous HMGB1 amplified myocardial inflammation and increased infarcted size. HMGB1 significantly exacerbate myocardial damage after ischemia reperfusion mainly signal via RAGE.
Part II: Dose range and therapeutic window of ethyl pyruvate in myocardial ischemia reperfusion in rats
Aim This study was designed to determine the optimal therapeutic dose and time of ethyl pyruvate in rats myocardial ischemia reperfusion model
Methods Male Sprague Dawley rats were subjected to 30 minutes myocardial ischemia and 48 hours reperfusion. In the dose-effect part, rats were assigned to six groups (n=6 per group): Control group, 10mg/kg, 20mg/kg, 40mg/kg group, 80mg/kg group and 160mg/kg group; PBS (vehicle) or the five different doses of ethyl pyruvate was injected intravenously before reperfusion respectively). In the time-effect part, rats were assigned to four groups (n=6 per group): Control group (Rats received a intravenous bolus Ringer’s solution), Pre-ischemia group(IS group, rats received a intravenous bolus of ethyl pyruvate (40mg/kg) one minute before ischemia), Reperfusion group (R group, ethyl pyruvate was intravenously injected one minute before reperfusion), and the Post-reperfusion group (R30 group, ethyl pyruvate was intravenously injected one minute 30 minutes after reperfusion). Cardiac function was measured and myocardial necrosis was evaluated by triphenyltetrazolium chloride (TTC) staining.
Results In the dose-effect part, compared to the control group, infarct size (IS) was significantly decreased in the 20 mg/kg group(36.1±1.6% vs. 45.9±4.2%, P<0.05)with a significant improved cardiac function. IS further decrease to 28.7±1.5% in the 40mg/kg group with an improved cardiac function(+dp/dt max 3972±382 mmHg/s vs. control, -dp/dt max 3270±352 mmHg/s vs. control). Compared to the 40mg/kg group, no significant change of IS was found in the 80mg/kg or the 160mg/kg. In the time-effect part, compared to the control group, infarct size (IS) was significantly decreased in IS group(29.0±2.5% vs. 46.4±3.8%, P<0.05), R group (35.3±2.8% vs. 46.4±3.8%, P<0.05)but not in the R30 group(42.7±3.4% vs. 46.4±3.8%, P>0.05).
Conclusions Ethyl pyruvate protect rats heart from ischemia reperfusion injury in a dose-dependent manner. The optimal dose of ethyl pyruvate used in rat myocardial ischemia reperfusion model was 40mg/kg. The cardiac-protective effect of ethyl pyruvate was better when it was injected before ischemia than it’s injected after reperfusion. Two minutes prior to ischemia or before reperfusion is the best therapeutic window for ethyl pyruvate to protect myocardail ischemia reperfusion injury.
Part III Mechenism of HMGB1 induced myocardial ischemia reperfusion injury and the effect of ethyl pyruvate
Aim:This study was designed to investigate the effect of ethyl pyruvate on HMGB1、TNF-α、IL-6 in rats myocardial ischemia reperfusion model and the potential mechamism of ethyl pyruvate that mediated cytokines expression after ischemia reperfusion.
Methods Male Sprague Dawley rats were subjected to 30 minutes myocardial ischemia and 48 hours reperfusion. Rats were assigned to four group (n=12 per group): Sham group, control group (Rats received a intravenous bolus Ringer’s solution), EP group (rats received a intravenous bolus of ethyl pyruvate 40mg/kg two minutes before ischemia), EP+HMGB1 group (rats received a intravenous bolus of ethyl pyruvate (40mg/kg) and rhHMGB1 (100μg/kg) two minutes before ischemia). Cardiac function was measured and myocardial necrosis was evaluated by triphenyltetrazolium chloride (TTC) staining. Phosphorylation of p38 was evaluated by western blot. Serum level of HMGB1、TNF-α、IL-6 was evaluated by ELISA.
Results Compared with the sham group, serum level of HMGB1, TNF-α, IL-6 were all significantly upregulated with impaired LV +dp/dt max (2740±212 vs. 4630±369 mmHg/s, P<0.05), -dp/dt max (2092±193 vs. 3546±302 mmHg/s, P<0.05) in the control group. Compared to the control group, infarct size (IS) was significantly decreased in the EP group (45.7±3.5% vs.35.0±3.1%, P<0.05). EP significantly inhibited the elevated HMGB1 level; suppressed the activated TNF-α, IL-6, and preserved cardiac function. This cardioprotection was abolished by rhHMGB1.
Conclusions:Ethyl pyruvate can depressed the expression of HMGB1、TNF-α、IL-6 and decreased myocardial ischemia reperfusion injury when it is properly used, this befefit can totally blocked by exogerous HMGB1. Ethyl pyruvate can afford strong protection against heart ischemia reperfusion injury, and these benefits are related to a reduction of p 38 phospholation and HMGB1 induced pro-inflammatory reaction.
Part IV: Effect of ethyl pyruvate on reactive oxygen species and mitochondria in myocardial ischemia reperfusion in rats
Aim This study was designed to investigate the effect of ethyl pyruvate on reactive oxygen species in rats myocardial ischemia reperfusion model and the effect on the structure and function of cardiomyocyte mitochondria.
Methods Male Sprague Dawley rats were subjected to 30 minutes myocardial ischemia and 3 hours reperfusion. Rats were assigned to four group (n=6 per group): Sham group, Control group (Rats received a intravenous bolus Ringer’s solution), EP group (rats received a intravenous bolus of ethyl pyruvate 40mg/kg two minutes before ischemia), EP+Atr group (rats received a intravenous bolus of ethyl pyruvate (40mg/kg) and atractyloside (5mg/kg) two minutes before ischemia). Cardiac function was measured and myocardial necrosis was evaluated by triphenyltetrazolium chloride (TTC) staining and cardiomyocyte ultrastructure were evaluated. Myocardium homogenate was used to determine the activity of myeloperoxidase (MPO) superoxide dismutase (SOD) and level of malondialdehyde (MDA).
Results Compared with the sham group, MDA and MPO were all significantly upregulated (MDA: 6.3±0.7 nmol/mg vs. 1.6±0.3 nmol/mg P<0.05,MPO: 0.35±0.04 U/g vs. 0.95±0.12 U/g,P<0.05),SOD was significantly inhibited(65±10 U/mg vs. 315±46 U/mg,P<0.05),acompanied by an impaired cardiac function and higher mitochondrial score in the control group. EP significantly suppressed the activated MDA (3.2±0.5 nmol/mg vs. control,P<0.05), MPO (0.67±0.06 U/g vs. control,P<0.05), increased the level of SOD (189±25 U/mg vs. control,P<0.05), decreased mitochondrial damage, and preserved cardiac function. This cardioprotection was abolished by atractyloside.
Conclusions Ethyl pyruvate can decrease the level of reactive oxygen species when it’s properly used. Ethyl pyruvate can inhibit the opening of mitochondrial permeability transition pore opening. Ethyl pyruvate attenuated myocardial ischemia reperfusion injury when it was used before reperfusion.
一、心肌缺血再灌注后高迁移率族蛋白1的表达及其作用
目的探讨高迁移率族蛋白1(high mobility group box 1,HMGB1)在心肌缺血-再灌注后的时空表达特点;观察外源性HMGB1对心肌缺血再灌注损伤的影响及其可能机制。
方法第I部分:清洁级健康雄性SD大鼠分为5组,每组6只,分别于心肌缺血前、缺血30分钟、再灌注30分钟、再灌注24小时、再灌注48小时处死大鼠,留取标本检测HMGB1的表达。第II部分:清洁级健康雄性SD大鼠分为4组,每组6只,均给予缺血30分钟,再灌注48小时:对照组(Control),HMGB1-1μg /kg组(HMGB1-1μg),HMGB1-10μg /kg组(HMGB1-10μg),HMGB1-100μg /kg组(HMGB1-100μg),后3组在缺血再灌注后注射相应剂量rhHMGB1。采用有创血流动力学检测大鼠左心功能变化,TTC染色计算心肌梗死面积,免疫组化、Real-time PCR、Western Blot检测HMGB1及其受体RAGE、TLR-2、TLR-4的表达。
结果第I部分:在正常心肌组织中,HMGB1蛋白定位于心肌细胞核内;心肌缺血再灌注后,HMGB1不仅在心肌细胞核内表达,而且大量表达于梗死区域内的炎性细胞;HMGB1在大鼠心肌缺血30分钟后显著增高,并持续至再灌注48小时。第II部分:与Control组相比,HMGB1-1μg组、HMGB1-10μg组心肌梗死面积、±dp/dt max、RAGE、TLR-2、TLR-4无显著差异(P >0.05);但与Control组相比,HMGB1-100μg组心肌梗死面积显著增大(56.4±5.1% vs. 44.6±3.7%,P<0.05),±dp/dt max显著恶化(2199±185 mmHg/s vs. 2890±217mmHg/s; 1536±122mmHg/s vs.1979±190 mmHg/s;p<0.05);HMGB1、TNF-α及IL-6水平显著增高,与Control组相比,HMGB1-100μg组RAGE、TLR-2、TLR-4水平明显增高,其中以RAGE升高幅度最为明显。
结论心肌缺血再灌注后心肌及炎症细胞胞核浆HMGB1表达显著增高,外源性HMGB1进一步放大心肌炎症反应、增大梗死面积;HMGB1主要通过RAGE受体介导加重炎症反应及心肌损伤。
二、丙酮酸乙酯干预心肌缺血再灌注损伤的剂量及时机
目的观察不同剂量丙酮酸乙酯(Ethyl pyruvate, EP)对缺血-再灌注心肌的保护作用即量效关系研究;在合适的剂量下,探索其有效的治疗窗口即时效关系研究。
方法量效关系部分:清洁级健康雄性SD大鼠分为6组,每组6只,给予缺血30分钟,再灌注48小时,具体分组如下:Control组、10mg/kg组、20mg/kg组、40mg/kg组、80mg/kg组、160mg/kg组。Control组给予乳酸林格斯液静脉注射,其余各组均于缺血前2分钟给予相应剂量的EP静脉注射。时效关系部分:清洁级健康雄性SD大鼠分为4个组,每组6只,给予缺血30分钟,再灌注48小时,具体分组如下:Control组(缺血前2分钟给予乳酸林格斯液注射),缺血前2分钟给药组(Is),再灌注前2分钟给药组(R),再灌注后30分钟给药组(R30)。除Control组外,其余各组均给予EP(40mg/kg)于相应的时间点静脉注射。采用有创血流动力学检测大鼠左心功能变化,TTC染色计算心肌梗死面积。
结果在量效关系部分,与Control组相比,心梗面积在20mg/kg组就显著减小(36.1±1.6% vs. 45.9±4.2%, P<0.05),心功能显著改善,当剂量增加到40mg/kg时,心梗面积进一步减小(28.7±1.5% vs. 45.9±4.2%, P<0.05),心功能进一步改善,当给药剂量进一步增加到80mg/kg、160mg/kg时,心梗面积未见进一步减小。在时效关系部分,与Control组相比,Is组、R组均可显著减少心肌梗死面积(29.0±2.5%, 35.3±2.8 vs. 46.4±3.8%, P<0.05),改善心功能;R30组心梗面积有减小趋势,但无统计学差异(42.7±3.4% vs. 46.4±3.8%, P>0.05)。
结论40mg/kg的EP是治疗大鼠心肌缺血-再灌注损伤的最佳剂量;缺血前预先应用EP的疗效优于再灌注即刻给药,当给药时间延迟到再灌注后30分钟则EP的心肌保护作用消失;缺血前2分钟至再灌注前2分钟是EP保护心肌缺血-再灌注损伤的最佳时间窗。
三、HMGB1介导炎性缺血-再灌注损伤的机制及EP的作用
目的观察丙酮酸乙酯(EP)干预对炎症因子表达及炎症性心肌缺血-再灌注损伤的影响;研究EP干预对心肌缺血-再灌注后炎症因子表达变化的调控机制。
方法清洁级健康雄性SD大鼠分为4组,每组12只,均给予缺血30分钟,再灌注48小时:1.假手术组(Sham),只穿线不结扎;2.对照组(Control),于再灌注前给予PBS 0.5ml+RLS 0.5ml静脉注射;3. EP治疗组(EP),再灌注前2分钟给予静脉注射EP(40mg/kg);4. EP+HMGB1组(EP+HMGB1):缺血再灌注前2分钟静脉注射EP(40mg/kg)+重组HMGB1(100μg /kg)。采用有创血流动力学检测大鼠左心功能变化,TTC染色计算心肌梗死面积,ELISA法检测血清HMGB1、TNF-α、IL-6,Western Blot检测心肌磷酸化P38的表达。
结果与Sham组相比,Control组心肌梗死面积显著增加,心功能显著恶化,血清HMGB1、TNF-α、IL-6表达显著增高,磷酸化P38水平显著增高(P<0.05);与Control组相比,给予EP治疗后心肌梗死面积显著下降(35.0±3.1% vs. 45.7±3.5%, P<0.05),心功能明显改善,HMGB1、TNF-α、IL-6及磷酸化P38水平显著降低,而当HMGB1(100μg/kg)与EP同时给药时,EP的心肌保护效益完全消失。
结论心肌缺血再灌注前给予适时适量EP能抑制炎症因子HMGB1、TNF-α、IL-6的表达,减少炎性心肌缺血-再灌注损伤;同时给予外源性HMGB1则可完全消除EP的心肌保护作用;EP通过影响HMGB1与受体结合后的信号转导通路关键蛋白p38的磷酸化、阻断或降低HMGB1介导的促炎反应及炎性心肌缺血-再灌注损伤。
四、丙酮酸乙酯对心肌缺血再灌注后氧自由基及线粒体的影响
目的探讨丙酮酸乙酯(EP)干预缺血再灌注损伤是否可影响氧自由基的代谢;观察EP干预缺血再灌注损伤是否影响线粒体结构与功能。
方法清洁级健康雄性SD大鼠分为4个组,每组6只,给予缺血30分钟,再灌注3小时:Sham组(冠脉只穿线不结扎);Control组(再灌注前2分钟给予RLS静脉注射);EP治疗组(EP组,再灌注前2分钟给予EP40mg/kg静脉注射)。EP+苍术苷组(EP+Atr组,EP40mg/kg加苍术苷5mg/kg静脉注射)。检测大鼠左心功能变化,心肌梗死面积,心肌MDA、MPO、SOD水平,电镜检测心肌超微结构变化。
结果与Sham组相比,Control组心肌MDA(6.3±0.7 nmol/mg vs. 1.6±0.3 nmol/mg,P<0.05)、MPO(0.35±0.04 U/g vs. 0.95±0.12 U/g,P<0.05)显著升高,SOD显著下降(65±10 U/mg vs. 315±46 U/mg,P<0.05),线粒体受损明显;与Control组相比,EP治疗组显著减小心肌梗死面积(44.5±4.5% vs.33.7±4.1%, P<0.05),抑制MDA(3.2±0.5 nmol/mg vs. control,P<0.05)、MPO(0.67±0.06 U/g vs. control,P<0.05)增高,提升SOD水平(189±25 U/mg vs. control,P<0.05),减轻心肌线粒体损伤;当苍术苷与EP同时给药时,EP的心肌保护效益完全消失。
结论心肌缺血再灌注前予EP干预可抑制自由基生成、增加对其的清除能力;心肌缺血再灌注前予EP干预可抑制mPTP过度开放、减轻线粒体损伤;心肌缺血再灌注前予EP干预可减少再灌损伤及心梗面积、改善心功能。
Early reperfusion of ischemic myocardium is the most effective treatment to limit the infarct size and improve the clinical outcome. However, the process of restoring blood flow to the ischemic myocardium has the potential to induce additional injuries and reduce myocardial salvage. Oxidative stress and Inflammation play an important role in the pathogenesis of myocardial reperfusion injury, however, the the trigger and the mechanism of inflammation propagation have not been identified.The current study was to investigate:(1) The current study was to investigate the role of HMGB1 in rat myocardial I/R model,(2)the optimal dose and time choice of ethyl pyruvate(EP) in rat myocardium after ischemia reperfusion, (3)the potential mechenism that HMGB1 induced myocardial ischemia reperfusion injury and the effect of ethyl pyruvate(EP), (4) Effect of ethyl pyruvate on reactive oxygen species and mitochondria in myocardial ischemia reperfusion in rats
Part I: Role of high-mobility group box-1 in myocardial ischemia reperfusion injury Aim This study was designed to investigate potential role of high-mobility group box-1(HMGB1)after ischemia reperfusion injury and effect of exogenous recombined HMGB1 in rat myocardium.
Methods Part one: Male Sprague Dawley rats were assigned to five groups (n=6) . Rats were executed before ischemia, 30 minutes after ischemia, 30 minutes after reperfusion, 24 hours after after reperfusion, 48 hours after reperfusion, respectively. Immunohistochemistry and western blot were conducted to detecte the expression of HMGB1. Part two: Male Sprague Dawley rats were subjected to 30 minutes myocardial ischemia and 48 hours reperfusion. Rats were assigned to four group (n=6 per group): Control group, HMGB1-1μg group, HMGB1-10μg and HMGB1-100μg group; PBS (vehicle) or three doses of recombined HMGB1 (1μg/kg, 10μg/kg, 100μg/kg) was injected intravenously before reperfusion respectively). Cardiac function was measured and myocardial necrosis was evaluated by triphenyltetrazolium chloride (TTC) staining. Western blot and quantitative Real-time PCR were conducted to evaluate HMGB1, RAGE、TLR-2、TLR-4 expression levels.
Results Part one: HMGB1 was mildly stained and the expression was predominantly in the nucleus of cardiac myocyte in non-ischemic myocardium. After 30 minutes ischemia and 48 hours reperfusion, strong expression of HMGB1 was observed both in cytoplasm of cardiac myocyte and the infiltrated inflammatory cells. Expression of HMGB1 was significantly increased after 30 minutes ischemia and was still elevated 48 hours after reperfusion. Part two: No significant difference of infarct size,±dp/dt max, RAGE、TLR-2、TLR-4 among the control, HMGB1-1μg and HMGB1-10μg group was determined. Compared with the control group, infarct size (IS) was markedly increased in the HMGB1-100 group (56.4±5.1% vs. 44.6±3.7%, P<0.05), accompanied by impaired LV +dp/dt max (2199±185 mmHg/s vs. 2890±217mmHg/s, P<0.05), -dp/dt max (1536±122mmHg/s vs.1979±190 mmHg/s, P<0.05) and elevated level of TNF-αand IL-6. Compared with the control group, TLR-2, TLR-4 and RAGE were significantly upregulated in the HMGB1-100μg group. The increase of RAGE was most obvious.
Conclusions In this rat in vivo ischemia/reperfusion (I/R) experiment, myocardial ischemia reperfusion resulted in increased expression of cardiac HMGB1.Exogenous HMGB1 amplified myocardial inflammation and increased infarcted size. HMGB1 significantly exacerbate myocardial damage after ischemia reperfusion mainly signal via RAGE.
Part II: Dose range and therapeutic window of ethyl pyruvate in myocardial ischemia reperfusion in rats
Aim This study was designed to determine the optimal therapeutic dose and time of ethyl pyruvate in rats myocardial ischemia reperfusion model
Methods Male Sprague Dawley rats were subjected to 30 minutes myocardial ischemia and 48 hours reperfusion. In the dose-effect part, rats were assigned to six groups (n=6 per group): Control group, 10mg/kg, 20mg/kg, 40mg/kg group, 80mg/kg group and 160mg/kg group; PBS (vehicle) or the five different doses of ethyl pyruvate was injected intravenously before reperfusion respectively). In the time-effect part, rats were assigned to four groups (n=6 per group): Control group (Rats received a intravenous bolus Ringer’s solution), Pre-ischemia group(IS group, rats received a intravenous bolus of ethyl pyruvate (40mg/kg) one minute before ischemia), Reperfusion group (R group, ethyl pyruvate was intravenously injected one minute before reperfusion), and the Post-reperfusion group (R30 group, ethyl pyruvate was intravenously injected one minute 30 minutes after reperfusion). Cardiac function was measured and myocardial necrosis was evaluated by triphenyltetrazolium chloride (TTC) staining.
Results In the dose-effect part, compared to the control group, infarct size (IS) was significantly decreased in the 20 mg/kg group(36.1±1.6% vs. 45.9±4.2%, P<0.05)with a significant improved cardiac function. IS further decrease to 28.7±1.5% in the 40mg/kg group with an improved cardiac function(+dp/dt max 3972±382 mmHg/s vs. control, -dp/dt max 3270±352 mmHg/s vs. control). Compared to the 40mg/kg group, no significant change of IS was found in the 80mg/kg or the 160mg/kg. In the time-effect part, compared to the control group, infarct size (IS) was significantly decreased in IS group(29.0±2.5% vs. 46.4±3.8%, P<0.05), R group (35.3±2.8% vs. 46.4±3.8%, P<0.05)but not in the R30 group(42.7±3.4% vs. 46.4±3.8%, P>0.05).
Conclusions Ethyl pyruvate protect rats heart from ischemia reperfusion injury in a dose-dependent manner. The optimal dose of ethyl pyruvate used in rat myocardial ischemia reperfusion model was 40mg/kg. The cardiac-protective effect of ethyl pyruvate was better when it was injected before ischemia than it’s injected after reperfusion. Two minutes prior to ischemia or before reperfusion is the best therapeutic window for ethyl pyruvate to protect myocardail ischemia reperfusion injury.
Part III Mechenism of HMGB1 induced myocardial ischemia reperfusion injury and the effect of ethyl pyruvate
Aim:This study was designed to investigate the effect of ethyl pyruvate on HMGB1、TNF-α、IL-6 in rats myocardial ischemia reperfusion model and the potential mechamism of ethyl pyruvate that mediated cytokines expression after ischemia reperfusion.
Methods Male Sprague Dawley rats were subjected to 30 minutes myocardial ischemia and 48 hours reperfusion. Rats were assigned to four group (n=12 per group): Sham group, control group (Rats received a intravenous bolus Ringer’s solution), EP group (rats received a intravenous bolus of ethyl pyruvate 40mg/kg two minutes before ischemia), EP+HMGB1 group (rats received a intravenous bolus of ethyl pyruvate (40mg/kg) and rhHMGB1 (100μg/kg) two minutes before ischemia). Cardiac function was measured and myocardial necrosis was evaluated by triphenyltetrazolium chloride (TTC) staining. Phosphorylation of p38 was evaluated by western blot. Serum level of HMGB1、TNF-α、IL-6 was evaluated by ELISA.
Results Compared with the sham group, serum level of HMGB1, TNF-α, IL-6 were all significantly upregulated with impaired LV +dp/dt max (2740±212 vs. 4630±369 mmHg/s, P<0.05), -dp/dt max (2092±193 vs. 3546±302 mmHg/s, P<0.05) in the control group. Compared to the control group, infarct size (IS) was significantly decreased in the EP group (45.7±3.5% vs.35.0±3.1%, P<0.05). EP significantly inhibited the elevated HMGB1 level; suppressed the activated TNF-α, IL-6, and preserved cardiac function. This cardioprotection was abolished by rhHMGB1.
Conclusions:Ethyl pyruvate can depressed the expression of HMGB1、TNF-α、IL-6 and decreased myocardial ischemia reperfusion injury when it is properly used, this befefit can totally blocked by exogerous HMGB1. Ethyl pyruvate can afford strong protection against heart ischemia reperfusion injury, and these benefits are related to a reduction of p 38 phospholation and HMGB1 induced pro-inflammatory reaction.
Part IV: Effect of ethyl pyruvate on reactive oxygen species and mitochondria in myocardial ischemia reperfusion in rats
Aim This study was designed to investigate the effect of ethyl pyruvate on reactive oxygen species in rats myocardial ischemia reperfusion model and the effect on the structure and function of cardiomyocyte mitochondria.
Methods Male Sprague Dawley rats were subjected to 30 minutes myocardial ischemia and 3 hours reperfusion. Rats were assigned to four group (n=6 per group): Sham group, Control group (Rats received a intravenous bolus Ringer’s solution), EP group (rats received a intravenous bolus of ethyl pyruvate 40mg/kg two minutes before ischemia), EP+Atr group (rats received a intravenous bolus of ethyl pyruvate (40mg/kg) and atractyloside (5mg/kg) two minutes before ischemia). Cardiac function was measured and myocardial necrosis was evaluated by triphenyltetrazolium chloride (TTC) staining and cardiomyocyte ultrastructure were evaluated. Myocardium homogenate was used to determine the activity of myeloperoxidase (MPO) superoxide dismutase (SOD) and level of malondialdehyde (MDA).
Results Compared with the sham group, MDA and MPO were all significantly upregulated (MDA: 6.3±0.7 nmol/mg vs. 1.6±0.3 nmol/mg P<0.05,MPO: 0.35±0.04 U/g vs. 0.95±0.12 U/g,P<0.05),SOD was significantly inhibited(65±10 U/mg vs. 315±46 U/mg,P<0.05),acompanied by an impaired cardiac function and higher mitochondrial score in the control group. EP significantly suppressed the activated MDA (3.2±0.5 nmol/mg vs. control,P<0.05), MPO (0.67±0.06 U/g vs. control,P<0.05), increased the level of SOD (189±25 U/mg vs. control,P<0.05), decreased mitochondrial damage, and preserved cardiac function. This cardioprotection was abolished by atractyloside.
Conclusions Ethyl pyruvate can decrease the level of reactive oxygen species when it’s properly used. Ethyl pyruvate can inhibit the opening of mitochondrial permeability transition pore opening. Ethyl pyruvate attenuated myocardial ischemia reperfusion injury when it was used before reperfusion.
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