| Objective:To study the protection of noninvasive delayed limb ischemic preconditioning(NDLIP)against myocardial ischemia reperfusion(I/R)injury in rats,and to investigate the possible contribution of mitochondrial ATP-sensitive potassium channel(mitoKATP)in the delayed protection of NDLIP.Method:Healthy male Wistar rats were divided randomly into 5 groups.(1) I/R group(n=22):rats were subjected to 30 min occlusion of the left coronary artery anterior descending(LAD)followed by 120 min of reperfusion.(2)early myocardial ischemic preconditioning(EMIP)group(n=22):rats were subjected to 3 cycles of 5 min of ischemia and reperfusion on the LAD before 30 min ischemia and 120 min reperfusion.(3)NDLIP group(n=22):rats were subjected to 3 cycles of 5 min of ischemia and reperfusion on the left hind limb for 3 days to induce NDLIP.On the forth day,30 min ischemia and 120 min reperfusion of LAD was performed.(4)I/R+5-HD group(n=22): 5-Hydroxydecanoate Na(5-HD),a special blocker of mitoKATP,was given by intravenous bolus injection(9 mg/kg)10 min before 30 min ischemia and by intravenous infusion from 25 min of ischemia to the end of reperfusion(1 mg/kg).(5)NDLIP+5-HD group(n=22):5-HD was given to rats subjected to NDLIP for 3 days by intravenous bolus injection(9 mg/kg)10 min before 30 min ischemia and by intravenous infusion from 25 min of ischemia to the end of reperfusion(1 mg/kg).Delayed protections of NDLIP were estimated according to the changes of ECG,myocardial cell death,myocardial antioxidative ability,blood vessel endothelial function and coagulation.Heart rate(HR),mean arterial blood pressure(MAP)and ECG during 30 min ischemia and 120 min reperfusion were monitored.Myocardial infarct size was determined based on 2,3,5-triphenyltetrazolium chloride staining.Myocardial apoptosis was detected using the TUNEL method.Expressions of apoptosis-associated protein Fas and FasL were measured using the immunohistochemical method.Changes of myocardial morphology were observed after HE staining.Activities of myocardial tissue myeloperoxidase(MPO),total-superoxide dismutase(T-SOD), manganese-superoxide dismutase(Mn-SOD),glutathione peroxidase(GSH-PX) and xanthine oxidase(XOD),content of myocardial malonaldehyde(MDA)and concentration of nitric oxide(NO)in serum were detected by spectrophotometer. Concentration of endothelin-1(ET-1)in serum was detected by ELISA method. Mn-SOD mRNA was measured by RT-PCR method.Prothrombin time(PT), activated partial thromboplastin time(APTT)and plasma fibrinogen(FIB)were determined by coagulation method.Results:1.Effects of NDLIP on changes of ECG induced by myocardial I/R injuryCompared with I/R group,the extent of ST-segment increase was degraded(P＜0.01),onsets of ventricular premature contraction(VPC)and ventricular tachycardia(VT)were delayed(VPC:5.33±2.14 min vs 11.85±1.79 min and 8.25±2.15 min,respectively,P＜0.01;VT:6.40±2.77 min vs 13.25±1.72 min and 10.16±3.29 min,respectively P＜0.01),durations of them were shortened(VPC:12.09±2.63 min vs 5.13±2.18 min and 6.65±2.20 min, respectively,P＜0.01;VT:10.45±4.24 min vs 3.26±1.69 min and 4.06±1.92 min, respectively,P＜0.01),incidences of VT and ventricular fibrillation(VF)were decreased(VT:81.82%vs 22.73%and 50%,respectively,P＜0.05;VF:54.54%vs 13.64%and 18.18%,respectively,P＜0.05)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 death induced by myocardial I/R injuryCompared with I/R group,myocardial infarct size was diminished (IS/AAR%:32.4±10.1%vs 19.0±5.9%and 21.4±9.4%,respectively,P＜0.05), activity of MPO was attenuated(1.05±0.16 U/g vs 0.89±0.05 U/g and 0.77±0.11 U/g,respectively,P＜0.05),apoptosis was decreased(TUNEL:31.66±3.74%vs 20.67±1.30%and 22.09±1.76%,respectively,P＜0.01),positive indexes of Fas and FasL were lessened(Fas:28.51±1.31%vs 21.91±2.15%and 22.21±1.84%, respectively,P＜0.01,FasL:26.81±1.43%vs 19.75±1.80%and 19.95±1.74%, respectively,P＜0.01),and the injury of myocardial morphous was improved in EMIP group and NDLIP group.The anti-death protection of NDLIP was as effective as that of EMIP.3.Effects of NDLIP on myocardial antioxidative activity after myocardial I/R injuryCompared with I/R group,activities of T-SOD(144.78±16.47U/mgprot vs 162.26±6.41 U/mgprot and 162.67±14.21 U/mgprot,respectively,P＜0.05), Mn-SOD(46.37±12.18 U/mgprot vs 76.49±17.57 U/mgprot and 75.67±3.36 U/mgprot,respectively,P＜0.01)and GSH-PX(79.38±7.95 U/gprot vs 90.62±9.80 U/gprot and 92.74±7.76 U/gprot,respectively,P＜0.05)were augmented, expression of Mn-SOD mRNA was increased(Mn-SOD/β-actin%:41.36±14.91% vs 61.46±10.73%and 79.18±21.92%,respectively,P＜0.05),activity of XOD (91.20±10.69 U/gprot vs 81.34±7.23 U/gprot and 76.29±8.77 U/gprot,respectively, P＜0.05)and content of MDA(2.01±0.17 nmol/mgprot vs 1.79±0.18 nmol/mgprot and 1.68±0.11 nmol/mgprot,respectively,P＜0.05)were decreased in EMIP group and NDLIP group.The antioxidative protection of NDLIP was as effective as that of EMIP.4.Effects of NDLIP on blood vessel endothelial function before and after myocardial I/R injuryBefore 30 min ischemia,compared with I/R group,NO concentration in serum was increased(11.88±2.46μmol/L vs 14.85±2.00μmol/L and 15.09±2.82μmol/L,respectively,P＜0.05),ratio of ET-1 and NO was decreased (0.2526±0.0673 vs 0.2005±0.0528 and 0.1834±0.0490,respectively,P＜0.05)in EMIP group and NDLIP group.After 120 rain reperfusion,the increase in release of ET-1 was decreased(4.10±0.30 ng/ml vs 3.50±0.34 ng/ml and 3.21±0.49 ng/ml,respectively,P＜0.01),the concentration of NO was preserved (7.21±1.29μmol/L vs 10.23±0.82μmol/L and 11.18±2.52μmol/L,respectively, P＜0.01),the increase in ET-1/NO was diminished(0.5790±0.1571 vs 0.3448±0.0492 and 0.3105±0.0695,respectively,P＜0.05)in EMIP group and NDLIP group.The endothelial protection of NDLIP was as effective as that of EMIP.5.Effects of NDLIP on coagulation before and after myocardial I/R injury Before 30 min ischemia,there were no significant differences on coagulation indicators among three groups.After 120 min reperfusion, compared with I/R group,the increase of plasma FIB concentration was decreased(238.28±22.19 mg/dl vs 214.73±22.33 mg/dl and 216.54±14.52 mg/dl, respectively,P＜0.05)in EMIP group and NDLIP group.There were no siginificant differences on PT and APTT among three groups.6.Role of mitOKATPin protection induced by NDLIPCompared with I/R group,there were no significant differences on all indicators in I/R+5-HD group,which suggested that 5-HD has no effect on I/R injury.Protections induced by NDLIP as shown above were abolished or attenuated in NDLIP+5-HD group.Compared with NDLIP group,the extent of ST-segment increase was decreased(P＜0.05),onsets of VPC and VT were shifted to an earlier time(VPC:8.25±2.15 min vs 6.28±2.29 min,P＜0.01;VT: 10.16±3.29 min vs 6.81±2.85 min,P＜0.01),durations of them were lengthened(VPC: 6.65±2.20 min vs 10.31±5.14 min,P＜0.01;VT:4.06±1.92 min vs 9.35±4.17 min, P＜0.01),incidences of VT and VF were increased(VT:50%vs 81.82%,P＜0.05;VF: 18.18%vs 50%,P＜0.01).Myocardial infarct size was enlarged(21.4±9.4%vs 34.9±14.7%,P＜0.05),activity of MPO(0.77±0.11 U/g vs 0.98±0.24 U/g,P＜0.05), myocardial cell apoptosis(22.09±1.76%vs 27.28±1.38%,P＜0.01),positive indexes of Fas and FasL(Fas:22.21±1.84%vs 26.53±1.56%,P＜0.01;FasL: 19.95±1.74%vs 24.65±1.85%,P＜0.01)were increased,myocardial morphology injury is aggravated.Activities ofT-SOD(162.67±14.21 U/mgprot vs 145.07±14.02 U/mgprot,P＜0.05),Mn-SOD(75.67±3.36 U/mgprot vs 44.75±15.33 U/mgprot, P＜0.01)and GSH-PX(92.74±7.76 U/gprot vs 79.00±7.51 U/gprot,P＜0.01)were decreased,expression of Mn-SOD mRNA(79.18±21.92%vs 54.62±15.31%, P＜0.05)was decreased,activity of XOD(76.29±8.77 U/gprot vs 94.73±10.66 U/gprot,P＜0.01)and content of MDA(1.68±0.11 nmol/mgprot vs 2.10±0.23 nmol/mgprot,P＜0.01)were increased.NO concentration was decreased (11.18±2.52μmol/L vs 7.71±1.47μmol/L,,P＜0.01),ET-1 concentration in serum (3.21±0.49 ng/ml vs 3.91±0.22 ng/ml,P＜0.01)and ratio of ET-1 and NO (0.3105±0.0695 vs 0.5372±0.0976,P＜0.01)were increased.Plasma FIB concentration was increased(216.54±14.52 mg/dl vs 234.23±22.40 mg/dl, P＜0.05).Conclusion:1.NDLIP decreased the extent of ST-segment increase induced by I/R injury,delayed onsets of VPC and VT,shortened durations of them,decrease incidence of malignant ventricular arrhythmia during ischemia.It suggested NDLIP had the delayed antiarrhythmic effect,which was as effective as that of EMIP.Changes of MAP and HR caused by ischemia reperfusion injury were not influenced by NDLIP.2.NDLIP decreased myocardial infarct size and cell apoptosis,attenuated myocardial morphology injury induced by I/R injury.The anti-cell death effect of NDLIP was as effective as that of EMIP.The mechanism was involved in the decrease of MPO activity and the down regulation of expressions of apoptosis-associated protein Fas and FasL.3.NDLIP increased myocardial antioxidative ability after I/R injury, decreased peroxidative damage.The antioxidative protection of NDLIP was as effective as that of EMIP.4.In normal condition,NDLIP increased the concentration of NO in serum,slanted the balance between ET-1 and NO toward NO.After I/R injury, NDLIP decreased the endothelial function disorder,increased NO concentration in serum,decreased elevated ET-1 concentration and maintained the balance between ET-1 and NO to approach normal level.The endothelial protection of NDLIP was as effective as that of EMIP.5.NDLIP had no effect on coagulation in normal condition,but it decreased the extent of plasma FIB increase after I/R injury,which may be one of the mechanisms of NDLIP delayed myocardial protection.6.The opening of mitoKATPmediated the delayed protections induced by NDLIP. |
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