缺血预适应、瑞舒伐他汀和通心络减少猪急性心肌缺血再灌注后无再流及机制研究

【作者】 程宇彤；

【导师】 杨跃进； 孟宪敏；

【作者基本信息】 中国协和医科大学 ， 内科学， 2009， 博士

【摘要】 (一)缺血预适应减少猪心肌缺血后无再流与内源性一氧化氮有关【目的】:观察缺血预适应对猪缺血再灌注后无再流、心肌坏死及心肌凋亡的影响,探讨作为内皮功能标志的内源性一氧化氮(NO)在其中的作用。【方法】:35只中华小型猪分为假手术组、安慰剂组、一氧化氮合酶抑制剂N-硝基-左旋精氨酸(L-NNA)组、缺血预适应组、缺血预适应联合L-NNA组。开胸冠状动脉结扎1.5小时,松解3小时制备急性心肌梗死再灌注模型。术中监测动脉血压、心室压、心率及冠脉血流量,比色法测定心肌组织一氧化氮合酶(NOS)活性;用面积求积法测定心肌的缺血、坏死及无再流范围;髓过氧化物酶(MPO)法测定心肌MPO活力;病理组织染色计数中性粒细胞浸润程度;免疫印迹法测定心肌内皮型一氧化氮合酶(eNOS)、血管内皮钙粘连素(VE-cadherin)、半胱天冬酶—3(caspase-3)含;TUNEL法检测心肌细胞凋亡发生率;在电镜下观察微血管内皮损伤情况。【结果】:L-NNA组、缺血预适应联合L-NNA处理组各有一只动物在再灌注期间因低血压死亡;在缺血前,L-NNA组收缩期动脉血压和心功能指标左室心室内压力变化率峰值(+dp/dt)明显高于安慰剂组(P＜0.05);各组血压心率乘积(RPP)及左室舒张末压无明显差异(所有P＞0.05);在再灌注期间,安慰剂组心率较假手术组增快(所有P＜0.05);再灌注180分钟时RPP和心功能指标在各组间无明显差异(P＞0.05)。假手术组(0.98±0.20 u/mg)心肌组织cNOS活性明显高于安慰剂组无再流(0.24±0.04 u/mg)和再流区(0.30±0.05 u/mg)心肌,假手术组(0.31±0.06u/mg)iNOS活性明显低于安慰剂组再流区(0.73±0.08 u/mg)和无再流区(0.90±0.14 u/mg)(所有P＜0.05);与安慰剂相比,L-NNA处理(0.13±0.04 u/mg)明显降低再流区心肌组织eNOS活性(P＜0.05);缺血预适应(0.62±0.07 u/mg,0.55±0.04 u/mg)明显增加再流区和无再流区心肌组织eNOS活性(所有P＜0.05);而缺血预适应联合L-NNA处理组eNOS活性(0.42±0.07 u/mg,0.34±0.03 u/mg)与安慰剂组无明显差异;四个缺血模型组间iNOS活性无差异(P＞0.05)。四个缺血模型组间心肌缺血面积无明显差异(P＞0.05);L-NNA组(97±3%,34.5±1.8%)心肌坏死面积、心肌凋亡指数与安慰剂组(90±3%.28.2±2.4%)无明显差异(P＞0.05),缺血预适应(78±2%,16.5±0.9%)及联合使用L-NNA(79±2%,18.6±1.7%)明显减少心肌坏死面积和心肌凋亡指数(所有P＜0.05),减少了再流区活性caspase-3蛋白含量。L-NNA(70±2%)、缺血预适应联合L-NNA(67±1%)组无再流面积与安慰剂组(70±4%)比较无明显差异;缺血预适应组无再流面积56±2%,与以上3组均有明显差异(P＜0.05);与安慰剂相比,缺血预适应减少了再流区中性粒细胞浸润程度(MPO活性0.64±0.15 vs.1.77±0.09IU/g,中性粒细胞浸润评分1.14±0.14 vs.2.00±0.31,所有P＜0.05),保护了血管内皮结构的完整性(微血管内皮超微结构损伤减轻和无再流区VE-cadherin表达增加)。而缺血预适应联合NOS抑制剂L-NNA组心肌再流区MPO活性(1.63±0.16 IU/g)、中性粒细胞浸润评分(2.00±0.26)与缺血预适应组相比明显降低(P＜0.05)。【结论】:缺血预适应减少无再流的作用与内源性NO有关,而内源性NO不是缺血预适应减少心肌坏死和凋亡的主要机制。(二)瑞舒伐他汀减少猪心肌缺血后无再流和再灌注损伤与内源性一氧化氮有关【目的】:观察瑞舒伐他汀预处理对猪缺血再灌注后无再流、心肌坏死及心肌凋亡的影响,探讨作为内皮功能标志的内源性一氧化氮(NO)在其中的作用。【方法】:35只中华小型猪随机分为假手术组、安慰剂组、一氧化氮合酶抑制剂N-硝基-左旋精氨酸(L-NNA)处理组、瑞舒伐他汀组、瑞舒伐他汀联合L-NNA组。开胸结扎冠状动脉1.5小时,松解3小时制备急性心肌梗死再灌注模型。术中监测动脉血压、心室压、心率及冠脉血流量,比色法测定心肌组织NOS活性;用面积求积法测定心肌的缺血、坏死及无再流面积;髓过氧化物酶(MPO)法测定心肌MPO活力;病理组织染色计数中性粒细胞浸润程度;免疫印迹法测定心肌内皮型一氧化氮合酶(eNOS)、血管内皮钙粘连素(VE-cadherin)、半胱天冬酶—3(caspase-3)水平;TUNEL法检测心肌细胞凋亡发生率;在电镜下观察微血管内皮损伤情况;监测瑞舒伐他汀血药浓度和血脂水平。【结果】:L-NNA组和瑞舒伐他汀联合L-NNA组各有一只动物在再灌注期间死于低血压;缺血前,L-NNA和瑞舒伐他汀联合L-NNA组收缩期动脉血压和心功能指标左心室内压力变化率峰值(+dp/dt)明显高于安慰剂组(P＜0.05);各组血压心率乘积(RPP)及左室舒张末压无明显差异(所有P＞0.05);在再灌注期间,安慰剂组心率较假手术组增快(所有P＜0.05);再灌注180分钟时,RPP和心功能指标在各组间无明显差异(所有P＞0.05)。假手术组(0.98±0.20 u/mg)心肌组织cNOS活性明显高于安慰剂组无再流(0.24±0.04 u/mg)和再流区(0.30±0.05 u/mg)心肌,假手术组(0.31±0.06u/mg)iNOS活性明显低于安慰剂组再流区(0.73±0.08 u/mg)和无再流区心肌(0.90±0.14 u/mg)(所有P＜0.05);与安慰剂相比,L-NNA处理(0.13±0.04 u/mg)明显降低再流区心肌组织eNOS活性(P＜0.05),瑞舒伐他汀(0.52±0.04 u/mg)明显增加再流区心肌组织eNOS活性(P＜0.05),而瑞舒伐他汀联合L-NNA(0.42±0.06u/mg)虽有增加eNOS活性趋势,但未达统计学差异;四个缺血模型组间iNOS活性、eNOS蛋白含量无差异。四个缺血模型组间心肌缺血面积无明显差异(P＞0.05);L-NNA组(97±3%,34.5±1.8%)心肌坏死面积、心肌凋亡指数与安慰剂(90±3%,28.2±2.4%)无明显差异(P＞0.05);与安慰剂相比,瑞舒伐他汀明显减少心肌坏死面积(79±3%)、再流区心肌凋亡(17.4±1.6%)(所有P＜0.05)和活性caspase-3蛋白含量,而联合使用L-NNA组心肌坏死面积(88±1%)和心肌凋亡(26.9±2.3%)与瑞舒伐他汀组相比明显增加(所有P＜0.05)。L-NNA(70±2%)组无再流面积与安慰剂组(70±4%)比较无明显差异(P＞0.05);与安慰剂相比,瑞舒伐他汀减少了无再流面积(54±4%),增加了再灌注180分钟时冠脉血流量,减少再流区中性粒细胞浸润程度(再流区MPO活力是0.69±0.12 vs1.77±0.09IU/g,中性粒细胞浸润评分1.00±0.14 vs 2.00±0.31,所有P＜0.05),保护了血管内皮结构的完整性(微血管超微结构损伤减轻和无再流区VE-cadherin表达增加);瑞舒伐他汀联合L-NNA组无再流面积(70±2%)、心肌再流区MPO活力(1.56±0.13 IU/g)、中性粒细胞浸润评分(1.83±0.17)、冠脉血流量等指标与瑞舒伐他汀组相比均有明显差异(所有P＜0.05)。给药后3 h和4.5 h,瑞舒伐他汀组血药浓度分别为7.53±0.55 ug/L和9.92±1.42 ug/L;瑞舒伐他汀组血浆总胆固醇和低密度脂蛋白浓度分别为1.91±0.06mmol/L和1.11±0.07)mmol/L明显低于安慰剂组2.54±0.17 mmol/L和1.63±0.17mmol/L(P＜0.05)。【结论】:瑞舒伐他汀减少心肌缺血后无再流和心肌再灌注损伤作用与内源性NO有关。常规剂量的瑞舒伐他汀服用4天就可以明显降低血脂水平。(三)通心络减少猪心肌缺血后无再流和再灌注损伤与内源性一氧化氮有关【目的】:观察中药方剂通心络对猪缺血再灌注后无再流、心肌坏死及心肌凋亡的影响,探讨作为内皮功能标志的内源性一氧化氮(NO)在其中的作用。【方法:35只中华小型猪随机分为假手术组、安慰剂组、一氧化氮合酶抑制剂N-硝基-左旋精氨酸(L-NNA)组、通心络超微粉(0.4g/kg)组和通心络联合L-NNA组。开胸结扎冠状动脉1.5小时,松解3小时制备急性心肌梗死再灌注模型。术中监测动脉血压、心室压、心率及冠脉血流量,比色法测定心肌组织NOS活性;用面秋求积法测定心肌的缺血、坏死及无再流面积;髓过氧化物酶(MPO)法测定心肌MPO活力;病理组织染色计数中性粒细胞浸润程度;免疫印迹法测定心肌内皮型一氧化氮合酶(eNOS)、血管内皮钙粘连素(VE-cadherin)、半胱灭冬酶—3(caspase-3)水平;TUNEL法检测心肌细胞凋亡发生率;在电镜下观察微血管内皮受损伤情况。【结果】:L-NNA组一只动物在再灌注期间死于低血压,通心络联合L-NNA组一只动物在缺血期间死于心室颤动;缺血前,L-NNA和通心络联合L-NNA组收缩期动脉血压和左心室内压力变化率峰值(+dp/dt)明显高于安慰剂组(P＜0.05),各组血压心率乘积(RPP)及左室舒张末压无明显差异;在缺血后再灌注期间,安慰剂较假手术组心率增快;再灌注180分钟时RPP和心功能指标在各组间无明显差异(所有P＞0.05)。假手术组(0.98±0.20 u/mg)心肌组织cNOS活性明显高于安慰剂组无再流(0.24±0.04 u/mg)和再流区(0.30±0.05 u/mg)心肌组织,假手术组(0.31±0.06u/mg)iNOS活性明显低于安慰剂组再流区(0.73±0.08 u/mg)和无再流区(0.90±0.14)u/mg)(所有P＜0.05);与安慰剂相比,L-NNA处理(0.13±0.04 u/mg)明显降低再流区心肌组织eNOS活性(P＜0.05),通心络(0.51±0.07 u/mg)明显增加再流区心肌组织eNOS活性(P＜0.05);通心络联合L-NNA处理eNOS活性(0.38±0.05 u/mg)与安慰剂相比无差异;四个缺血模型组间iNOS活性、eNOS蛋白含量无差异。四个缺血模型组间心肌缺血面积无明显差异(P＞0.05);L-NNA组(97±3%,34.5±1.8%)心肌坏死面积、心肌凋亡指数与安慰剂(90±3%,28.2±2.4%)无明显差异(P＞0.05);与安慰剂相比,通心络(78±3%)明显减少心肌坏死面积、心肌凋亡指数(15.5±2.2%)(所有P＜0.05)和活性caspase-3蛋白含量;而联合使用L-NNA组心肌坏死面积(95±3%),心肌凋亡凋亡指数(30±0.8%)与通心络组相比有明显差异(所有P＜0.05)。L-NNA(70±2%)组无再流面积与安慰剂组(70±4%)比较无明显差异;与安慰剂相比,通心络减少了无再流面积(47±5%),增加了再灌注120、180分钟时冠脉血流量,减少中性粒细胞浸润程度(心肌再流区、无再流区MPO活力分别是1.12±0.15 vs 1.77±0.09IU/g和1.30±0.11 vs 2.07±0.17IU/g,再流区中性粒细胞浸润评分0.86±0.26 vs 2.00±0.31,所有P＜0.05),保护了血管内皮结构的完整性(微血管内皮超微结构损伤减轻和无再流区VE-cadherin表达增加)。而通心络联合L-NNA组无再流面积(63±3%)、心肌再流区、无再流区MPO活力(分别是1.57±0.05和1.64±0.33 IU/g)、再流区中性粒细胞浸润评分(1.64±0.33),无再流区VE-cadherin、再灌注120和180分钟时冠脉血流量等指标,与通心络组相比有明显差异(所有P＜0.05)。【结论】:通心络减少心肌缺血再灌注损伤作用与内源性NO有关。更多还原

【Abstract】 【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.更多还原