无创性延迟肢体缺血预适应对糖尿病大鼠心肌缺血再灌注损伤保护作用的研究
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摘要
目的：研究无创性延迟肢体缺血预适应(NDLIP)对糖尿病(DM)大鼠心肌缺血再灌注(I／R)损伤的保护作用。
方法：健康雄性Wistar大鼠经一次尾静脉注射2％链脲佐菌素(STZ)溶液45 mg／kg，造成急性糖尿病模型，随机分为4组。(1)假手术组(Sham组，n=13)：经冠状动脉左前降支(LAD)下穿线后，旷置；(2)I／R组(n=24)：LAD实施30 min缺血／120 min再灌注；(3)早期心肌缺血预适应组(EMIP组，n=24)：心脏LAD先行3次5 min缺血／5 min再灌注，随后实施30 min缺血／120 min再灌注；(4)NDLIP组(n=24)：左后肢实施3次5 min缺血／5min再灌注，每天1次，连续3天，第4天对心脏LAD实施30 min缺血／120 min再灌注。从ECG、心肌细胞损伤与死亡、心肌抗氧化能力、血管内皮功能、及纤溶系统功能及心肌能量变化等方面评价NDLIP延迟心脏保护作用。监测LAD 30 min缺血／120 min再灌注期间心率(HR)、平均动脉压(MAP)、ECG变化；TTC染色法测定心肌梗死面积，HE染色观察心肌形态学改变，TUNEL法检测心肌细胞凋亡，实时定量PCR和免疫印迹法分别检测凋亡相关因子Fas、FasL的转录和蛋白水平的表达；比色法测定心肌组织髓过氧化物酶(MPO)、总超氧化物歧化酶(T-SOD)、锰-超氧化物歧化酶(Mn-SOD)、谷胱甘肽过氧化物酶(GSH-PX)、黄嘌呤氧化酶(XOD)活力，丙二醛(MDA)含量，以及血清一氧化氮(NO)浓度；RT-PCR法检测心肌Mn-SOD mRNA含量；免疫抑制法检测血清肌酸激酶MB型同工酶(CK-MB)活性；ELISA法检测心肌肌钙蛋白I(cTnI)、内皮素-1(ET-1)、组织型纤溶酶原激活物(t-PA)和纤溶酶原激活物抑制剂-1(PAI-1)含量；HPLC-紫外法检测心肌组织ATP、ADP和AMP含量并计算腺苷酸池和能荷。
结果：
1．NDLIP对DM大鼠心肌I／R损伤所致心脏生理功能变化的影响
与I／R组比较，NDLIP组I／R期间ST-段抬高幅度降低(P＜0．05)，EMIP组和NDLIP组室早(VPC)、室速(VT)出现时间推迟(P＜0．05或P＜0．01)，持续时间缩短(P＜0．01；P＜0．05)，VPC、VT和室颤(VF)发生率降低(P＜0．05；P＜0．01；P＜0．01)，对I／R期间HR和MAP变化的影响与I／R组比较无显著性差异。
2．NDLIP对DM大鼠心肌I／R所致细胞损伤和坏死的影响
与I／R组比较，EMIP组和NDLIP组再灌注末血清CK-MB活性升高幅度降低(P＜0．01)，cTnI含量增幅下降(P＜0．05和P＜0．01)，心肌梗死面积缩小(P＜0．01)，MPO活性降低(P＜0．05)，心肌形态学损伤减轻。EMIP和NDLIP保护作用相当。
3．NDLIP对DM大鼠心肌I／R损伤所致细胞凋亡的影响
与I／R组比较，EMIP组和NDLIP组心肌细胞凋亡减少(P＜0．01)，凋亡相关蛋白Fas、FasL表达减少(P＜0．01或P＜0．05)。EMIP和NDLIP保护作用相当。
4．NDLIP对DM大鼠心肌I／R后抗氧化能力的影响
与I／R组比较，EMIP组和NDLIP组心肌T-SOD(P＜0．01)、Mn-SOD(P＜0．01)、GSH-PX(P＜0．01)活力增强，Mn-SOD mRNA表达增加(P＜0．01)，XOD活性(P＜0．01)和MDA含量(P＜0．01)降低。EMIP和NDLIP保护作用相当。
5．NDLIP对大鼠DM心肌I／R前后血管内皮功能的影响
缺血前，与I／R组比较，EMIP组和NDLIP组血清NO浓度升高(P＜0．05)，ET-1／NO比值降低(P＜0．05)。再灌注后，EMIP组和NDLIP组血清ET-1升高程度降低(P＜0．01)，NO浓度得到保留(P＜0．05和P＜0．01)，ET-1／NO比值升高程度降低(P＜0．01)。EMIP和NDLIP保护作用相当。
6．NDLIP对DM大鼠心肌I／R前后纤溶因子的影响
缺血前，各组血清t-PA和PAI-1的含量均无显著性差异。再灌注后，各组血清t-PA含量与缺血前比较亦无显著差异。与I／R组比较，EMIP组和NDLIP组再灌注后血清PAI-1的升高程度显著降低(P＜0．05)。
7．NDLIP对DM大鼠心肌I／R后能量状态的影响
与I／R组比较，EMIP组和NDLIP组I／R后心肌ATP、ADP、AMP和TAN含量明显增加(P＜0．01；P＜0．01和P＜0．05；P＜0．05；P＜0．01)，ECP显著提高(P＜0．05)，各组间AMP含量无明显差异。
结论：
1．NDLIP明显降低DM大鼠心肌I／R所致ST-段抬高幅度，推迟缺血期VPC、VT出现时间，缩短其持续时间，降低恶性室性心律失常发生率，具有延迟抗心律失常作用，其保护作用强度与EMIP相当。NDLIP对DM大鼠心肌I／R所致MAP和HR变化无影响。
2．NDLIP能减少DM大鼠心肌I／R所致CK-MB和cTnI释出，缩小心肌梗死面积，改善心肌形态学损伤，其抗细胞损伤与坏死的延迟保护作用强度与EMIP相当。机制可能与降低MPO活力有关。
3．NDLIP能减少DM大鼠I／R后心肌细胞凋亡，下调凋亡相关因子Fas、FasL mRNA和蛋白的表达，其抗细胞凋亡的延迟保护作用强度与EMIP相当。这可能是其限制心肌梗死面积的延迟相心脏保护作用机制之一。
4．NDLIP能增强DM大鼠I／R心肌的抗氧化能力，减轻过氧化损伤，此作用强度与EMIP相当。
5．NDLIP能增加DM大鼠基础状态下的血清NO浓度，使ET-1与NO之间的平衡向NO移动。I／R后，NDLIP减轻血管内皮功能障碍，保留血清NO浓度，降低ET-1升高幅度，使ET-1与NO之间的平衡接近正常，其保护作用强度与EMIP相当。
6．NDLIP对DM大鼠基础状态下的血清PAI-1含量和I／R前后t-PA的含量均无影响，但能降低I／R后血清PAI-1的增加程度，可能是其延迟心脏保护作用机制之一。其作用强度与EMIP相当。
7．NDLIP能增加DM大鼠I／R后心肌磷酸腺苷含量，提高组织能荷，其作用强度与EMIP相当。
Objective:To study the protection of noninvasive delayed limb ischemicpreconditioning (NDLIP)against myocardial ischemia reperfusion (I/R)injuryin streptozotocin(STZ)-induced diabetic rats.
Method:Healthy male Wistar rats were administered once intravenousinjection of STZ solution at a dose of 45 mg/kg,to induce acute diabetes mellitus.Then the diabetic rats were assigned randomly into four groups.(1)Shamgroup (n=13):rats were threaded a silk suture under the left coronary arteryanterior descending (LAD)and laid up throughout the experiment.(2)I/Rgroup (n=24):rats were subjected to 30 min LAD occlusion of the left coronaryartery anterior descending (LAD)followed by 120 min of reperfusion.(3)earlymyocardial ischemic preconditioning (EMIP)group (n=24):rats weresubjected to three episodes of 5 rain LAD occlusion followed by 5 minreperfusion before 30 min ischemia and 120 min reperfusion.(4)NDLIP group(n=24):rats were subjected to three episodes of 5 min ischemia of left hind limbfollowed by 5 min reperfusion daily for three consecutive days to induce NDLIP.On the forth day,30 min ischemia followed by 120 min reperfusion of LADwas performed.Delayed protective effects of NDLIP were evaluated accordingto the changes of ECG,injury and death of myocardial cell,myocardial antioxidativeability,blood vessel endothelial function,function of fibrinolysis and myocardialenergy.Heart rate (HR),mean arterial blood pressure (MAP)and ECG during 30min ischemia and 120 min reperfusion were monitored.Myocardial infarct size wasdetermined based on 2,3,5-triphenyltetrazolium chloride staining.Myocardialapoptosis was detected using the TUNEL method.Expressions ofapoptosis-associated protein Fas and FasL were measured by the real-timequantitative PCR and by Western blotting Blotting technique.Changes of myocardialmorphology were observed after by HE staining.Activities of myocardial tissuemyeloperoxidase (MPO),total-superoxide dismutase (T-SOD),manganese-superoxide dismutase (Mn-SOD),glutathione peroxidase (GSH-PX)andxanthine oxidase (XOD),content of myocardial malonaldehyde (MDA)and concentration of nitric oxide (NO)in serum were detected by spectrophotometer.Activity of creatine kinase-MB type isoenzyme (CK-MB)in serum was determinedby immunological inhibition method.Serum concentrations of cardiac troponin I(cTnI),endothelin-1 (ET-1),tissue plasminogen activator (t-PA)and plasminogenactivator inhibitor-1 (PAI-1)were detected by ELISA method.Mn-SOD mRNA wasmeasured by reverse transcription-PCR method.The contents of ATP,ADP,AMP inmyocardium were detected by HPLC assay,and then the total adenine nucleotide andenergy charge potential were calculated.
Results:
1.Effects of NDLIP on changes of cardiac physiological functioninduced by myocardial I/R injury
Compared with I/R group,the extent of ST-segment was degraded(P＜0.01),onsets of ventricular premature contraction (VPC)and ventriculartachycardia (VT)were delayed (P＜0.05 or P＜0.01),durations of them wereshortened (P＜0.01;P＜0.05),incidences of VT and ventricular fibrillation (VF)were decreased (P＜0.05;P＜0.01;P＜0.01)in EMIP group and NDLIP group.Changes of MAP and HR caused by I/R injury were not influenced by NDLIP
2.Effects of NDLIP on myocardial cell injury and necrosis induced bymyocardial I/R injury
Compared with I/R group,the amplitude of increase in the activity ofCK-MB and serum content of cTnI were reduced (P＜0.01;P＜0.05 and P＜0.01)at the end of reperfusion,myocardial infarct size was diminished (P＜0.01),activity of MPO was attenuated (P＜0.05),and the injury of myocardialmorphous was improved in EMIP group and NDLIP group.The protection of NDLIPwas as effective as that of EMIP
3.Effects of NDLIP on myocardial cell apoptosis induced by myocardial I/Rinjury
Compared with I/R group,apoptosis of cadiocytes was decreased (P＜0.01),expression of Fas and FasL were lessened (P＜0.01or P＜0.05)in EMIP groupand NDLIP group.The anti-apoptosis protection of NDLIP was as effective as that ofEMIP
4.Effects of NDLIP on myocardial antioxidative activity after myocardial I/Rinjury
Compared with I/R group,activities of T-SOD (P＜0.01),Mn-SOD(P＜0.01)and GSH-PX (P＜0.01)were augmented,expression of Mn-SODmRNA was increased (P＜0.01),activity of XOD (P＜0.01)and content of MDA(P＜0.01)were decreased in EMIP group and NDLIP group.The antioxidativeprotection of NDLIP was as effective as that of EMIP.
5.Effects of NDLIP on blood vessel endothelial function before and aftermyocardial I/R injury
Before 30 min ischemia,serum concentration of NO was increased inEMIP group and NDLIP group compared with I/R group (P＜0.05),ratio ofET-1 and NO was decreased (P＜0.05).After 120 min reperfusion,the increasein content of ET-1 was reduced (P＜0.01),the concentration of NO waspreserved (P＜0.05 and P＜0.01),the increase in ET-1/NO was diminished(P＜0.01)in EMIP group and NDLIP group.The protection of NDLIP onendothelial function was as effective as that of EMIP.
6.Effects of NDLIP on fibrinolytic factors before and after myocardial I/Rinjury
Before 30 min ischemia,there were no significant differences on serumconcentrations of t-PA and PAI-1 among the three groups.At the end ofreperfusion,the increase in concentration of serum PAI-1 was decreased inEMIP group and NDLIP group compared with I/R group (P＜0.05),while nostatistical change in the content of t-PA was observed among the groups.
7.Effects of NDLIP on the level of myocardial energy after I/R injury
Compared with I/R group,the levels of ATP(P＜0.01),ADP (P＜0.01 andP＜0.05,respectively),AMP(P＜0.05)and TAN(P＜0.01)in myocardium wereelevated in EMIP group and NDLIP group.As a result,the ECP wassignificantly increased in the two groups (P＜0.05).No significant deferencein the content of AMP was observed among the groups.
Conclusion:
1.NDLIP decreased the extent of ST-segment increase induced by I/R injury,delayed onsets and shortened durations of VPC and VT,and decreasesthe incidence of severe ventricular arrhythmia during ischemia.The effect ofNDLIP on anti-ventricular arrhythmias was similar to that of EMIP in diabeticmyocardium.Changes of MAP and HR caused by I/R injury were not influenced byNDLIP.
2.NDLIP decreased the release of myocardial CK-MB and cTnI anddiminished myocardial infarct size and improved myocardial morphology afterI/R injury.The protective effect of NDLIP against cell injury and necrosis wasas effective as that of EMIP in diabetic myocardium.The decline in activity ofMPO may contribute to the protection.
3.NDLIP decreased the apoptotic index and down regulated expression ofapoptotic proteins Fas and FasL to the extent of that by EMIP in diabeticmyocardium suffered from I/R injury,which may be one of the mechanisms ofdelayed myocardial protection of NDLIP contributed to the decrease ofmyocardial infarct size.
4.NDLIP enhanced myocardial antioxidative function after I/R injury todecrease peroxidative injury to the extent of EMIP in diabetic myocardium.
5.NDLIP increased the basic serum concentration of NO,which made thebalance between ET-1 and NO shift toward NO.Preservation of NO,declinein serum elevation of ET-1 and maintainance of balance between ET-1 and NOinduced by NDLIP after I/R injury in diabetic myocardium indicated animprovement in endothelial function,which reached to the extent of that byEMIP
6.NDLIP exhibited no effect on the serum contents of both t-PA andPAI-1 in basic condition and that of t-PA after I/R injury,but it decreased theelevation in serum PAI-1 to the extent of EMIP,which may be one of themechanisms of delayed myocardial protection of NDLIP,and did no change inserum t-PA induced by I/R injury in diabetic myocardium.
7.NDLIP increased the contents of ATP,ADP and TAN and raised theECP in diabetic myocardium after I/R injury,which reached to the extent ofthat by EMIP
方法：健康雄性Wistar大鼠经一次尾静脉注射2％链脲佐菌素(STZ)溶液45 mg／kg，造成急性糖尿病模型，随机分为4组。(1)假手术组(Sham组，n=13)：经冠状动脉左前降支(LAD)下穿线后，旷置；(2)I／R组(n=24)：LAD实施30 min缺血／120 min再灌注；(3)早期心肌缺血预适应组(EMIP组，n=24)：心脏LAD先行3次5 min缺血／5 min再灌注，随后实施30 min缺血／120 min再灌注；(4)NDLIP组(n=24)：左后肢实施3次5 min缺血／5min再灌注，每天1次，连续3天，第4天对心脏LAD实施30 min缺血／120 min再灌注。从ECG、心肌细胞损伤与死亡、心肌抗氧化能力、血管内皮功能、及纤溶系统功能及心肌能量变化等方面评价NDLIP延迟心脏保护作用。监测LAD 30 min缺血／120 min再灌注期间心率(HR)、平均动脉压(MAP)、ECG变化；TTC染色法测定心肌梗死面积，HE染色观察心肌形态学改变，TUNEL法检测心肌细胞凋亡，实时定量PCR和免疫印迹法分别检测凋亡相关因子Fas、FasL的转录和蛋白水平的表达；比色法测定心肌组织髓过氧化物酶(MPO)、总超氧化物歧化酶(T-SOD)、锰-超氧化物歧化酶(Mn-SOD)、谷胱甘肽过氧化物酶(GSH-PX)、黄嘌呤氧化酶(XOD)活力，丙二醛(MDA)含量，以及血清一氧化氮(NO)浓度；RT-PCR法检测心肌Mn-SOD mRNA含量；免疫抑制法检测血清肌酸激酶MB型同工酶(CK-MB)活性；ELISA法检测心肌肌钙蛋白I(cTnI)、内皮素-1(ET-1)、组织型纤溶酶原激活物(t-PA)和纤溶酶原激活物抑制剂-1(PAI-1)含量；HPLC-紫外法检测心肌组织ATP、ADP和AMP含量并计算腺苷酸池和能荷。
结果：
1．NDLIP对DM大鼠心肌I／R损伤所致心脏生理功能变化的影响
与I／R组比较，NDLIP组I／R期间ST-段抬高幅度降低(P＜0．05)，EMIP组和NDLIP组室早(VPC)、室速(VT)出现时间推迟(P＜0．05或P＜0．01)，持续时间缩短(P＜0．01；P＜0．05)，VPC、VT和室颤(VF)发生率降低(P＜0．05；P＜0．01；P＜0．01)，对I／R期间HR和MAP变化的影响与I／R组比较无显著性差异。
2．NDLIP对DM大鼠心肌I／R所致细胞损伤和坏死的影响
与I／R组比较，EMIP组和NDLIP组再灌注末血清CK-MB活性升高幅度降低(P＜0．01)，cTnI含量增幅下降(P＜0．05和P＜0．01)，心肌梗死面积缩小(P＜0．01)，MPO活性降低(P＜0．05)，心肌形态学损伤减轻。EMIP和NDLIP保护作用相当。
3．NDLIP对DM大鼠心肌I／R损伤所致细胞凋亡的影响
与I／R组比较，EMIP组和NDLIP组心肌细胞凋亡减少(P＜0．01)，凋亡相关蛋白Fas、FasL表达减少(P＜0．01或P＜0．05)。EMIP和NDLIP保护作用相当。
4．NDLIP对DM大鼠心肌I／R后抗氧化能力的影响
与I／R组比较，EMIP组和NDLIP组心肌T-SOD(P＜0．01)、Mn-SOD(P＜0．01)、GSH-PX(P＜0．01)活力增强，Mn-SOD mRNA表达增加(P＜0．01)，XOD活性(P＜0．01)和MDA含量(P＜0．01)降低。EMIP和NDLIP保护作用相当。
5．NDLIP对大鼠DM心肌I／R前后血管内皮功能的影响
缺血前，与I／R组比较，EMIP组和NDLIP组血清NO浓度升高(P＜0．05)，ET-1／NO比值降低(P＜0．05)。再灌注后，EMIP组和NDLIP组血清ET-1升高程度降低(P＜0．01)，NO浓度得到保留(P＜0．05和P＜0．01)，ET-1／NO比值升高程度降低(P＜0．01)。EMIP和NDLIP保护作用相当。
6．NDLIP对DM大鼠心肌I／R前后纤溶因子的影响
缺血前，各组血清t-PA和PAI-1的含量均无显著性差异。再灌注后，各组血清t-PA含量与缺血前比较亦无显著差异。与I／R组比较，EMIP组和NDLIP组再灌注后血清PAI-1的升高程度显著降低(P＜0．05)。
7．NDLIP对DM大鼠心肌I／R后能量状态的影响
与I／R组比较，EMIP组和NDLIP组I／R后心肌ATP、ADP、AMP和TAN含量明显增加(P＜0．01；P＜0．01和P＜0．05；P＜0．05；P＜0．01)，ECP显著提高(P＜0．05)，各组间AMP含量无明显差异。
结论：
1．NDLIP明显降低DM大鼠心肌I／R所致ST-段抬高幅度，推迟缺血期VPC、VT出现时间，缩短其持续时间，降低恶性室性心律失常发生率，具有延迟抗心律失常作用，其保护作用强度与EMIP相当。NDLIP对DM大鼠心肌I／R所致MAP和HR变化无影响。
2．NDLIP能减少DM大鼠心肌I／R所致CK-MB和cTnI释出，缩小心肌梗死面积，改善心肌形态学损伤，其抗细胞损伤与坏死的延迟保护作用强度与EMIP相当。机制可能与降低MPO活力有关。
3．NDLIP能减少DM大鼠I／R后心肌细胞凋亡，下调凋亡相关因子Fas、FasL mRNA和蛋白的表达，其抗细胞凋亡的延迟保护作用强度与EMIP相当。这可能是其限制心肌梗死面积的延迟相心脏保护作用机制之一。
4．NDLIP能增强DM大鼠I／R心肌的抗氧化能力，减轻过氧化损伤，此作用强度与EMIP相当。
5．NDLIP能增加DM大鼠基础状态下的血清NO浓度，使ET-1与NO之间的平衡向NO移动。I／R后，NDLIP减轻血管内皮功能障碍，保留血清NO浓度，降低ET-1升高幅度，使ET-1与NO之间的平衡接近正常，其保护作用强度与EMIP相当。
6．NDLIP对DM大鼠基础状态下的血清PAI-1含量和I／R前后t-PA的含量均无影响，但能降低I／R后血清PAI-1的增加程度，可能是其延迟心脏保护作用机制之一。其作用强度与EMIP相当。
7．NDLIP能增加DM大鼠I／R后心肌磷酸腺苷含量，提高组织能荷，其作用强度与EMIP相当。
Objective:To study the protection of noninvasive delayed limb ischemicpreconditioning (NDLIP)against myocardial ischemia reperfusion (I/R)injuryin streptozotocin(STZ)-induced diabetic rats.
Method:Healthy male Wistar rats were administered once intravenousinjection of STZ solution at a dose of 45 mg/kg,to induce acute diabetes mellitus.Then the diabetic rats were assigned randomly into four groups.(1)Shamgroup (n=13):rats were threaded a silk suture under the left coronary arteryanterior descending (LAD)and laid up throughout the experiment.(2)I/Rgroup (n=24):rats were subjected to 30 min LAD occlusion of the left coronaryartery anterior descending (LAD)followed by 120 min of reperfusion.(3)earlymyocardial ischemic preconditioning (EMIP)group (n=24):rats weresubjected to three episodes of 5 rain LAD occlusion followed by 5 minreperfusion before 30 min ischemia and 120 min reperfusion.(4)NDLIP group(n=24):rats were subjected to three episodes of 5 min ischemia of left hind limbfollowed by 5 min reperfusion daily for three consecutive days to induce NDLIP.On the forth day,30 min ischemia followed by 120 min reperfusion of LADwas performed.Delayed protective effects of NDLIP were evaluated accordingto the changes of ECG,injury and death of myocardial cell,myocardial antioxidativeability,blood vessel endothelial function,function of fibrinolysis and myocardialenergy.Heart rate (HR),mean arterial blood pressure (MAP)and ECG during 30min ischemia and 120 min reperfusion were monitored.Myocardial infarct size wasdetermined based on 2,3,5-triphenyltetrazolium chloride staining.Myocardialapoptosis was detected using the TUNEL method.Expressions ofapoptosis-associated protein Fas and FasL were measured by the real-timequantitative PCR and by Western blotting Blotting technique.Changes of myocardialmorphology were observed after by HE staining.Activities of myocardial tissuemyeloperoxidase (MPO),total-superoxide dismutase (T-SOD),manganese-superoxide dismutase (Mn-SOD),glutathione peroxidase (GSH-PX)andxanthine oxidase (XOD),content of myocardial malonaldehyde (MDA)and concentration of nitric oxide (NO)in serum were detected by spectrophotometer.Activity of creatine kinase-MB type isoenzyme (CK-MB)in serum was determinedby immunological inhibition method.Serum concentrations of cardiac troponin I(cTnI),endothelin-1 (ET-1),tissue plasminogen activator (t-PA)and plasminogenactivator inhibitor-1 (PAI-1)were detected by ELISA method.Mn-SOD mRNA wasmeasured by reverse transcription-PCR method.The contents of ATP,ADP,AMP inmyocardium were detected by HPLC assay,and then the total adenine nucleotide andenergy charge potential were calculated.
Results:
1.Effects of NDLIP on changes of cardiac physiological functioninduced by myocardial I/R injury
Compared with I/R group,the extent of ST-segment was degraded(P＜0.01),onsets of ventricular premature contraction (VPC)and ventriculartachycardia (VT)were delayed (P＜0.05 or P＜0.01),durations of them wereshortened (P＜0.01;P＜0.05),incidences of VT and ventricular fibrillation (VF)were decreased (P＜0.05;P＜0.01;P＜0.01)in EMIP group and NDLIP group.Changes of MAP and HR caused by I/R injury were not influenced by NDLIP
2.Effects of NDLIP on myocardial cell injury and necrosis induced bymyocardial I/R injury
Compared with I/R group,the amplitude of increase in the activity ofCK-MB and serum content of cTnI were reduced (P＜0.01;P＜0.05 and P＜0.01)at the end of reperfusion,myocardial infarct size was diminished (P＜0.01),activity of MPO was attenuated (P＜0.05),and the injury of myocardialmorphous was improved in EMIP group and NDLIP group.The protection of NDLIPwas as effective as that of EMIP
3.Effects of NDLIP on myocardial cell apoptosis induced by myocardial I/Rinjury
Compared with I/R group,apoptosis of cadiocytes was decreased (P＜0.01),expression of Fas and FasL were lessened (P＜0.01or P＜0.05)in EMIP groupand NDLIP group.The anti-apoptosis protection of NDLIP was as effective as that ofEMIP
4.Effects of NDLIP on myocardial antioxidative activity after myocardial I/Rinjury
Compared with I/R group,activities of T-SOD (P＜0.01),Mn-SOD(P＜0.01)and GSH-PX (P＜0.01)were augmented,expression of Mn-SODmRNA was increased (P＜0.01),activity of XOD (P＜0.01)and content of MDA(P＜0.01)were decreased in EMIP group and NDLIP group.The antioxidativeprotection of NDLIP was as effective as that of EMIP.
5.Effects of NDLIP on blood vessel endothelial function before and aftermyocardial I/R injury
Before 30 min ischemia,serum concentration of NO was increased inEMIP group and NDLIP group compared with I/R group (P＜0.05),ratio ofET-1 and NO was decreased (P＜0.05).After 120 min reperfusion,the increasein content of ET-1 was reduced (P＜0.01),the concentration of NO waspreserved (P＜0.05 and P＜0.01),the increase in ET-1/NO was diminished(P＜0.01)in EMIP group and NDLIP group.The protection of NDLIP onendothelial function was as effective as that of EMIP.
6.Effects of NDLIP on fibrinolytic factors before and after myocardial I/Rinjury
Before 30 min ischemia,there were no significant differences on serumconcentrations of t-PA and PAI-1 among the three groups.At the end ofreperfusion,the increase in concentration of serum PAI-1 was decreased inEMIP group and NDLIP group compared with I/R group (P＜0.05),while nostatistical change in the content of t-PA was observed among the groups.
7.Effects of NDLIP on the level of myocardial energy after I/R injury
Compared with I/R group,the levels of ATP(P＜0.01),ADP (P＜0.01 andP＜0.05,respectively),AMP(P＜0.05)and TAN(P＜0.01)in myocardium wereelevated in EMIP group and NDLIP group.As a result,the ECP wassignificantly increased in the two groups (P＜0.05).No significant deferencein the content of AMP was observed among the groups.
Conclusion:
1.NDLIP decreased the extent of ST-segment increase induced by I/R injury,delayed onsets and shortened durations of VPC and VT,and decreasesthe incidence of severe ventricular arrhythmia during ischemia.The effect ofNDLIP on anti-ventricular arrhythmias was similar to that of EMIP in diabeticmyocardium.Changes of MAP and HR caused by I/R injury were not influenced byNDLIP.
2.NDLIP decreased the release of myocardial CK-MB and cTnI anddiminished myocardial infarct size and improved myocardial morphology afterI/R injury.The protective effect of NDLIP against cell injury and necrosis wasas effective as that of EMIP in diabetic myocardium.The decline in activity ofMPO may contribute to the protection.
3.NDLIP decreased the apoptotic index and down regulated expression ofapoptotic proteins Fas and FasL to the extent of that by EMIP in diabeticmyocardium suffered from I/R injury,which may be one of the mechanisms ofdelayed myocardial protection of NDLIP contributed to the decrease ofmyocardial infarct size.
4.NDLIP enhanced myocardial antioxidative function after I/R injury todecrease peroxidative injury to the extent of EMIP in diabetic myocardium.
5.NDLIP increased the basic serum concentration of NO,which made thebalance between ET-1 and NO shift toward NO.Preservation of NO,declinein serum elevation of ET-1 and maintainance of balance between ET-1 and NOinduced by NDLIP after I/R injury in diabetic myocardium indicated animprovement in endothelial function,which reached to the extent of that byEMIP
6.NDLIP exhibited no effect on the serum contents of both t-PA andPAI-1 in basic condition and that of t-PA after I/R injury,but it decreased theelevation in serum PAI-1 to the extent of EMIP,which may be one of themechanisms of delayed myocardial protection of NDLIP,and did no change inserum t-PA induced by I/R injury in diabetic myocardium.
7.NDLIP increased the contents of ATP,ADP and TAN and raised theECP in diabetic myocardium after I/R injury,which reached to the extent ofthat by EMIP
引文
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