Ischemic Preconditioning, Ruishu Cutting The Statins Tongxinluo Reduce No-reflow And Mechanisms Of Acute Myocardial Ischemia And Reperfusion,

【Objective】To explore whether ischemic preconditioning(IP) can protect myocardium against ischemia/reperfusion injury and to investigate the involvement of endogenous nitric oxide and therefore neutrophil accumulation.【Methods】35 open-chest pigs were divided to four groups of sham,placebo,nitric oxide synthase inhibitor NG-nitro-L-Arginine(L-NNA),IP and IP co-administration with L-NNA(10 mg/kg iv).IP was mimiced by three episodes of 5 minutes of ischemia followed by 5 minutes of reperfusion before left anterior descending(LAD) coronary artery ligation for 90 minutes followed by reperfusion for 3 hours.Area of no reflow and necrosis and risk region were determined pathologically by planimetry.The endothelial nitric oxide synthase activity and myeloperoxidase(MPO) activity of the myocardium were measured by colorimetric method.Histologic analysis was performed to determine the degree of neutrophil accumulation.Myocardial endothelial nitric oxide synthase (eNOS) and VE-cadherin were measured by western blotting.The myocardial apoptosis in the reflow region was detected with TUNEL assay and Western blot analysis on caspase-3.【Result】2 animals died during procedure and were excluded out from L-NNA and IP co-administration with L-NNA groups respectively.There were significant differences in blood pressure and peak of left ventricular pressure elevation(+dp/dt) in L-NNA and IP co-administration with L-NNA group compared with placebo group.There were no differences in rate pressure product at baseline among all groups.There were no differences in +dp/dt at the end of reperfusion and size of area at risk between four ischemic groups(P＞0.05).Compared with placebo,L-NNA decreased eNOS activity in reflow area(P＜0.05) but did not alter inductible nitric oxide synthase(iNOS) activity and eNOS protein abundance;Compared with placebo,IP increased eNOS activity both in reflow area and no reflow area(all P＜0.05).There were no differences in eNOS activity both in reflow and no reflow region between IP co-administrated with L-NNA and placebo group. There were no differences in iNOS activity between four ischemic groups. Both IP and IP co-administrated with L-NNA limited the infarct size from 90±3%of the area at risk in the group of placebo to 78±2%and 79±2%respectively(all P＜0.05).IP (16.5±0.9%)and IP co-administrated with L-NNA(18.6±1.7%) decreased myocardial apoptosis index in the reflow region compared with placebo(28.2±2.4%)(all P＜0.05). Actived caspase-3 content was also reduced in IP and IP co-administrated with L-NNA groups.Whereas L-NNA treatment had no differences in infarct size,myocardial apoptosis index and actived caspase-3 content compared with placebo(all P＞0.05). IP limited the no reflow size from 70±4%of the area at risk in the group of placebo to 56±2%(all P＜0.05).IP also attenuated MPO activity and neutrophil accumulation score in the reflow region compared with placebo group(P＜0.05 respectively).IP preserved the level of VE-cadherin in risk region when compared with placebo.Whereas L-NNA and IP co-administrated with L-NNA did not alter no-reflow size,MPO activity and neutrophil accumulation score in the reflow region compared with placebo.【Conclusions】The no-reflow limitation effect of IP was suggested to be dependent on endogenous NO production and may therefore attained by reducing the neutrophil accumulation and preserving microvascular integrity.Whereas endogenous NO production was not the major mechanism via which IP reduced myocardial necrosis and apoptosis. 【Objective】To explore whether pre-treatment with rosuvastatin can protects myocardium against ischemia/reperfusion injury and to investigate the involvement of endogenous nitric oxide and therefore neutrophil accumulation.【Methods】35 open-chest pigs were divided to four groups of sham,placebo,nitric oxide synthase inhibitor NG-nitro-L-Arginine(L-NNA),rosuvastatin and rosuvastatin co-administration L-NNA(10 mg/kg iv).Rosuvastatin was administrated 4 days before left anterior descending(LAD) coronary artery ligation for 90 minutes followed by reperfusion for 3 hours.The serum total cholesterol and low density lipoprotein were measured before ligation.Area of no reflow and necrosis and risk region were determined pathologically by planimetry.The endothelial nitric oxide synthase activity and myeloperoxidase(MPO) activity of the myocardium was measured by colorimetric method.Histologic analysis was performed to determine the degree of neutrophil accumulation.Myocardial endothelial nitric oxide synthase(eNOS) and VE-cadherin was measured by western blotting.The myocardial apoptosis in the reflow region was detected with TUNEL assay.Caspase-3 in the reflow region was measured by Western blot analysis.【Result】2 animals died during procedure and were excluded out from L-NNA and rosuvastatin co-administration with L-NNA groups.There were significant differences in blood pressure and rate of rise of left ventricular pressure(+dp/dt) in L-NNA and rosuvastatin co-administration with L-NNA groups compared with placebo group.There were no differences in rate pressure product at baseline among all groups(P＞0.05). There were no differences in +dp/dt at the end of reperfusion and size of area at risk between four ischemic groups(P＞0.05).Compared with placebo,L-NNA decreased eNOS activity in reflow area(P＜0.05) but did not alter iNOS activity and eNOS protein abundance;rosuvastatin increased eNOS activity in reflow area(P＜0.05);Rosuvastatin co-administrated with L-NNA had a trend toward increasing eNOS activity in reflow area.There were no differences in inductible NOS activity(P＞0.05) and eNOS protein abundance between rosuvastatin,rusuvastatin co-administration with L-NNA and placebo groups.Rosuvastatin increased the local LAD blood flow at 180 min of reperfusion and limited the infarct and no reflow size from 90±3%and 70±4%of the area at risk in the group of placebo to 79±3%and 54±4%(all P＜0.05).Whereas L-NNA and rosuvastatin co-administrated with L-NNA treatments had no differences in infarct size and no reflow size compared with placebo(all P＞0.05).Rosuvastatin decreased actived Caspase-3 abundance in the reflow region compared with placebo.Rosuvastatin also decreased myocardial apoptosis index from 28.2±2.4%of placebo to 17.4±1.6%(P＜0.05).L-NNA and rosuvastatin co-administrated with L-NNA treatments had no differences in myocardial apoptosis index(P＞0.05) and actived caspase-3 content compared with placebo.Rosuvastatin attenuated MPO activity and neutrophil accumulation score in the reflow region(0.69±0.12 IU/g,1.00±0.14) when compared with placebo group(1.77±0.09 IU/g, 2.00±0.31)(all P＜0.05).Rosuvastatin also preserved the level of VE-cadherin in risk region when compared with placebo.Whereas L-NNA and rosuvastatin co-administrated with L-NNA did not alter MPO activity and neutrophil accumulation score in the reflow region compared with placebo.The concentration of rosuvastatin in plasma at 3 and 4.5 hours after the last administration was 7.53±0.55 ug/L and 9.92±1.42 ug/L respectively.The serum total cholesterol and low density lipoprotein were decreased by rosuvastatin compared with placebo(all P＜0.05).【Conclusions】Pre-treatment with rosuvastatin lowered lipid level and had cardioprotective effect following ischemia and reperfusion.The cardioprotective effect is suggested to be dependent on endogenous NO production and therefore attained by reducing the neutrophil accumulation and preserving microvascular integrity. 【Objective】To explore whether pre-treatment with traditional Chinese medicine Tongxinluo powder can protect myocardium against ischemia/reperfusion injury and to investigate the involvement of endogenous nitric oxide and therefore neutrophil accumulation.【Methods】35 open-chest pigs were divided to four groups of sham,placebo,nitric oxide synthase inhibitor NG-nitro-L-Arginine(L-NNA),Tongxinluo(0.4g/kg) and Tongxinluo co-administration L-NNA(10 mg/kg iv).Tongxinluo was intragastrically administrated 3 hours before left anterior descending(LAD) coronary artery ligation for 90 minutes followed by reperfusion for 3 hours.Area of no reflow and necrosis and risk region were determined pathologically by planimetry.The myeloperoxidase(MPO) activity of the myocardium was measured by colorimetric method and histologic analysis was performed to determine the degree of neutrophil accumulation.Myocardial endothelial nitric oxide synthase(eNOS) and VE-cadherin were measured by western blotting.The myocardial apoptosis in the refiow region was detected with TUNEL assay. Caspase-3 in the reflow region was measured by Western blot analysis.【Result】2 animals died during procedure and were excluded out from L-NNA and Tongxinluo co-administration with L-NNA groups.There were significant differences in blood pressure and rate of rise of left ventricular pressure(+dp/dt) in L-NNA and Tongxinluo co-administration with L-NNA groups compared with placebo group.There were no differences in rate pressure product at baseline among all groups(P＞0.05). There were no differences in +dp/dt at the end of reperfusion and size of area at risk between four ischemic groups(P＞0.05).Compared with placebo,L-NNA decreased eNOS activity in reflow area(P＜0.05) but did not alter iNOS activity and eNOS protein abundance;Tongxiluo increased eNOS activity in reflow area(P＜0.05) but did not alter inductible nitric oxide synthase(iNOS) activity and eNOS protein abundance.There were no differences in eNOS,iNOS activity and eNOS protein abundance between Tongxinluo co-administrated with L-NNA and placebo groups.Tongxinluo increased the local LAD blood flow at 120 rain and 180 min of reperfusion and limited the infarct and no reflow size from 90±3%and 70±4%of the area at risk in the group of placebo to 78±3%and 47±5%(all P＜0.05).In addition,L-NNA and Tongxinluo co-administrated with L-NNA treatments had significant differences in infarct size and no reflow size compared with Tongxinluo(all P＜0.05).Tongxinluo decreased actived caspase-3 abundance in the reflow region compared with placebo.Tongxinluo also decreased myocardial apoptosis index from 28.2±2.4%of placebo to 15.5±2.2%(P＜0.05).Whereas L-NNA treatment and Tongxinluo co-administrated with L-NNA treatments had no differences in myocardial apoptosis index and actived caspase-3 content compared with placebo(all P＞0.05).Tongxinluo attenuated MPO activity and neutrophil accumulation score in histologic sections both in the reflow(1.12±0.15 IU/g,0.86±0.26) and MPO activity in no reflow region(1.30±0.11 IU/g) when compared with placebo group(1.77±0.09 IU/g, 2.00±0.31 and 2.07±0.17 IU/g)(all P＜0.05).Tongxinluo also preserved the level of VE-cadherin in risk region compared with placebo.Whereas L-NNA and Tongxinluo co-administrated with L-NNA did not alter MPO activity and neutrophil accumulation score in the reflow region compared with placebo.【Conclusions】Pre-treatment with Tongxinluo had a cardioprotective effect following ischemia/reperfusion.The cardioprotective effect was suggested to be dependent on endogenous NO production.Reduction of no reflow size may therefore attained by reducing the neutrophil accumulation and preserving microvascular integrity.