| 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 ratsPart 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 ratsAim This study was designed to determine the optimal therapeutic dose and time of ethyl pyruvate in rats myocardial ischemia reperfusion modelMethods 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 pyruvateAim: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 ratsAim 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. |
