舒芬太尼预处理对大鼠腹主动脉血管内皮细胞损伤的早期保护研究
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摘要
研究目的(1)研究缺血预处理及不同剂量的舒芬太尼预处理对大鼠腹主动脉缺血再灌注后血管内皮细胞损伤是否具有早期保护作用;(2)研究阿片受体、一氧化氮及一氧化氮合酶是否介导了舒芬太尼预处理的早期血管内皮细胞保护作用;(3)用免疫组化及反转录聚合酶链式反应方法检测缺血预处理及舒芬太尼预处理早期内皮型一氧化氮合酶及诱生性一氧化氮合酶表达的变化,探索两者在早期保护中的作用。
研究方法实验分为三部分:(1)SD大鼠随机分为6组:NA组、I/R组、IPC组、SUE1组、SUL2组和SUL3组。假手术组(NA组)制备好腹主动脉套线模型后不做任何处理,缺血再灌注损伤组(I/R组)给予缺血30 min、再灌注120 min,缺血预处理组(IPC组)缺血前给予三次5 min缺血、5 min再灌注的预处理,舒芬太尼预处理组(SUL1组、SUL2组和SUL3组)缺血前分别给予舒芬太尼5μg/kg、10μg/kg和20μg/kg预处理,分别测定血浆内血浆可溶性血栓调节蛋白浓度及假性血管血友病因子含量,行循环内皮细胞计数,并用光学显微镜和电子显微镜观察血管内皮细胞形态结构的变化,确定缺血预处理及不同剂量的舒芬太尼预处理对大鼠腹主动脉缺血再灌注后血管内皮细胞损伤是否具有早期保护作用;(2)单独应用阿片受体阻滞剂纳洛酮(NLX组)、一氧化氮前体左旋精氨酸(L-Arg组)、一氧化氮合酶阻滞剂左旋硝基精氨酸甲酯(L-NAME组)或联合舒芬太尼10μg/kg预处理(N-SUL组、LA-SUL组、LN-SUL组)后行腹主动脉缺血再灌注,并与假手术组(NA组)、缺血再灌注损伤组(UR组)、缺血预处理组(IPC组)和舒芬太尼10μg/kg预处理组(SUL组)比较,以血浆内血浆可溶性血栓调节蛋白浓度、血浆内假性血管血友病因子含量和循环内皮细胞计数为判断血管内皮细胞损伤的指标,并检测血浆一氧化氮浓度、组织匀浆一氧化氮浓度及组织匀浆一氧化氮合酶活性,确定阿片受体、一氧化氮及一氧化氮合酶是否介导了舒芬太尼预处理的早期血管内皮细胞保护作用;(3)用免疫组化及反转录聚合酶链式反应方法检测假手术组(NA组)、缺血再灌注损伤组(I/R组)、缺血预处理组(IPC组)及舒芬太尼10μg/kg预处理组(SUL组)大鼠腹主动脉内皮型一氧化氮合酶及诱生型一氧化氮合酶表达的变化,并探索两者在血管内皮细胞早期保护中的作用。
研究结果(1)实验模型缺血再灌注成功,与NA组相比,其余五组腹主动脉阻断期间平均动脉压升高,心率减慢,开放再灌注时平均动脉压降低,心率增加,再灌注1 h内平均动脉压和心率逐渐恢复至基础值,I/R组平均动脉压及心率波动较IPC组、SUL1组、SUL2组和SUL3组更为明显,该四组血浆内血浆可溶性血栓调节蛋白浓度、血浆内假性血管血友病因子含量和循环内皮细胞计数明显低于I/R组,光镜和电镜结果亦显示该四组血管内皮细胞损伤减轻,SUL2组血浆内血浆可溶性血栓调节蛋白浓度、血浆内假性血管血友病因子含量和循环内皮细胞计数低于SUL1组和SUL3组,光镜和电镜下损伤亦好于该两组;(2)NLX组血浆内血浆可溶性血栓调节蛋白浓度、血浆内假性血管血友病因子含量、循环内皮细胞计数、血浆一氧化氮浓度、组织匀浆一氧化氮浓度及组织匀浆一氧化氮合酶活性与I/R组无明显区别,N-SUL组各指标好于I/R组,但较IPC组及SUL组差,L-Arg组各指标与IPC组及SUL组无明显区别,LA-SUL组好于IPC组及SUL组,L-NAME组及LN-SUL组各指标较I/R组、IPC组及SUL组差;(3)免疫组化显示阳性结果为胞浆内棕色反应产物,IPC组与SUL组的内皮型一氧化氮合酶阳性细胞数多于NA组及I/R组,I/R组诱生型一氧化氮合酶多于其余各组,反转录聚合酶链式反应结果显示IPC组和SUL组内皮型一氧化氮合酶mRNA的表达较NA组与I/R组增强,各组诱生型一氧化氮合酶的表达均较低,无显著差异。
研究结论(1)缺血预处理及舒芬太尼预处理对大鼠腹主动脉缺血再灌注后血管内皮细胞损伤均具有早期保护作用,10μg/kg是相对最佳的预处理保护剂量;(2)纳洛酮能部分阻断保护作用,表明阿片受体可能部分介导了舒芬太尼预处理的早期保护作用,左旋硝基精氨酸甲酯完全阻断了保护作用,表明一氧化氮及结构型一氧化氮合酶在舒芬太尼预处理的早期保护作用中可能起主要作用;(3)缺血预处理及舒芬太尼预处理使早期血管内皮细胞内皮型一氧化氮合酶表达增加,而诱生型一氧化氮合酶在早期保护过程中未发生明显变化。
Objective (1) To investigate whether ischemic preconditioning and sufentanil preconditioning with various doses could induce the early protective effect on vascular endothelial cells after abdominal aorta ischemic-reperfusion in rats; (2) To investigate the roles of opioid receptor, nitric oxide and nitric oxide synthase in the early protective effect of vascular endothelial cells which is induced by sufentanil preconditioning; (3) To detect the expression of endothelial nitric oxide synthase and inducible nitric oxide synthase in the early protective phase by the methods of immunohistochemistry and reverse transcription Polymerase Chain Reaction, then identify their contribution in this effect.
Methods The experiment consisted of three parts: (1) Sprague-Dawley rats were divided into six groups, including group NA, group I/R, group IPC, group SUL1, group SUL2 and group SUL3, group NA was given nothing just segregated the abdominal aorta and the inferior vena cava, group I/R was given ischemia of thirty minutes and reperfusion of one hundred and twenty minutes, group IPC was added ischemia of five minutes and reperfusion of five minutes three times, group SUL1, group SUL2 and group SUL3 were given sufentanil fiveμg/kg, tenμg/kg and twentyμg/kg separatedly before ischemic-reperfusion, plasmic soluble Thrombomodulin and von Willebrand Factor were determined, Circular Endothelial Cells were calculated, and the injury of vessel was examined with optical and electron microscope, then investigated whether ischemic preconditioning and sufentanil preconditioning with various doses could induce the early protective effect on vascular endothelial cells; (2) opioid receptor antagnist naloxone, nitric oxide precursor L-Arginine and nitric oxide synthase antagnist N_(60)-Nitro-Arginine Methyl Ester were used both respectively (group NLX, group L-Arg and group L-NAME) and combinedly with sufentanil tenμg/kg (group N-SUL, group LA-SUL and group LN-SUL) for preconditioning before ischemic-reperfusion, and group NA, group I/R, group IPC and group SUL (tenμg/kg) were also done simultaneously, we determined plasmic soluble Thrombomodulin, plasmic von Willebrand Factor and Circular Endothelial Cells calculation to evaluate the injury of vascular endothelial cells, plasmic nitric oxide, nitric oxide of aorta homogenate and nitric oxide synthase activity of aorta homogenate were also determined, then investigated the roles of opioid receptor, nitric oxide and nitric oxide synthase in the early protective effect of vascular endothelial cells which is induced by sufentanil preconditioning; (3) We detected the expression of endothelial nitric oxide synthase and inducible nitric oxide synthase in the early protective phase by the methods of immunohistochemistry and reverse transcription Polymerase Chain Reaction of four groups (group NA, group I/R, group IPC and group SUL), then identify their contribution in this effect.
Results (1) The abdominal aorta ischemic-reperfusion model was succeeded, compared with group NA, mean arterial pressure of the other five groups was rising during block phase associated with lower heart rate, then became opposite during reperfusion phase, mean arterial pressure and heart rate became normal in one hour after reperfusion, the fluctuation of group I/R was more severe than group IPC, group SUL1, group SUL2 and group SUL3, the concentration of plasmic soluble Thrombomodulin, plasmic von Willebrand Factor and Circular Endothelial Cells calculation of group I/R was also higher than group IPC, group SUL1, group SUL2 and group SUL3, and the images of vessel examined by optical and electron microscope also showed less injury in group IPC, group SUL1, group SUL2 and group SUL3, the concentration of plasmic soluble Thrombomodulin, plasmic von Willebrand Factor, Circular Endothelial Cells calculation and images of optical and electron microscope of group SUL2 were better than group SUL1 and group SUL3; (2) the concentration of plasmic soluble Thrombomodulin, plasmic von Willebrand Factor, Circular Endothelial Cells calculation, plasmic nitric oxide, nitric oxide of aorta homogenate and nitric oxide synthase activity of aorta homogenate in group NLX were not different to those indices in group I/R, indices of group N-SUL were better than those of group I/R, but worse than those of group IPC and group SUL, indices of group L-Arg were not different to those of group IPC and group SUL, indices of group LA-SUL were better than those of group IPC and group SUL, indices of group L-NAME and group LN-SUL were the worst. (3) The positive result of immunohistochemistry was brown color in cytoplasm, the positive cells of endothelial nitric oxide synthase in group IPC and group SUL were more than those cells in group NA and group I/R, the positive cells of inducible nitric oxide synthase in group I/R were more than other groups, the results of reverse transcription Polymerase Chain Reaction showed that expression of endothelial nitric oxide synthase in group IPC and group SUL were better than group NA and group I/R, expression of inducible nitric oxide synthase in each group were lower and had no differences.
Conclusion (1) ischemic preconditioning and sufentanil preconditioning with various doses could induce the early protective effect on vascular endothelial cells after abdominal aorta ischemic-reperfusion in rats, tenμg/kg of sufentanil is the most effective dose relatively; (2) Naloxone could partly block the protective effect while N_(60)-Nitro-Arginine Methyl Ester could totally block the effect, it is indicated that opioid receptor maybe partly mediates the early protective effect of vascular endothelial cells which is induced by sufentanil preconditioning, while nitric oxide and nitric oxide synthase could be the major effective factors in the early protective phase; (3) the expression of endothelial nitric oxide synthase is increased during the early protective effect of ischemic preconditioning and sufentanil preconditioning, inducible nitric oxide synthase doesn't play a part in the early effect.
研究方法实验分为三部分:(1)SD大鼠随机分为6组:NA组、I/R组、IPC组、SUE1组、SUL2组和SUL3组。假手术组(NA组)制备好腹主动脉套线模型后不做任何处理,缺血再灌注损伤组(I/R组)给予缺血30 min、再灌注120 min,缺血预处理组(IPC组)缺血前给予三次5 min缺血、5 min再灌注的预处理,舒芬太尼预处理组(SUL1组、SUL2组和SUL3组)缺血前分别给予舒芬太尼5μg/kg、10μg/kg和20μg/kg预处理,分别测定血浆内血浆可溶性血栓调节蛋白浓度及假性血管血友病因子含量,行循环内皮细胞计数,并用光学显微镜和电子显微镜观察血管内皮细胞形态结构的变化,确定缺血预处理及不同剂量的舒芬太尼预处理对大鼠腹主动脉缺血再灌注后血管内皮细胞损伤是否具有早期保护作用;(2)单独应用阿片受体阻滞剂纳洛酮(NLX组)、一氧化氮前体左旋精氨酸(L-Arg组)、一氧化氮合酶阻滞剂左旋硝基精氨酸甲酯(L-NAME组)或联合舒芬太尼10μg/kg预处理(N-SUL组、LA-SUL组、LN-SUL组)后行腹主动脉缺血再灌注,并与假手术组(NA组)、缺血再灌注损伤组(UR组)、缺血预处理组(IPC组)和舒芬太尼10μg/kg预处理组(SUL组)比较,以血浆内血浆可溶性血栓调节蛋白浓度、血浆内假性血管血友病因子含量和循环内皮细胞计数为判断血管内皮细胞损伤的指标,并检测血浆一氧化氮浓度、组织匀浆一氧化氮浓度及组织匀浆一氧化氮合酶活性,确定阿片受体、一氧化氮及一氧化氮合酶是否介导了舒芬太尼预处理的早期血管内皮细胞保护作用;(3)用免疫组化及反转录聚合酶链式反应方法检测假手术组(NA组)、缺血再灌注损伤组(I/R组)、缺血预处理组(IPC组)及舒芬太尼10μg/kg预处理组(SUL组)大鼠腹主动脉内皮型一氧化氮合酶及诱生型一氧化氮合酶表达的变化,并探索两者在血管内皮细胞早期保护中的作用。
研究结果(1)实验模型缺血再灌注成功,与NA组相比,其余五组腹主动脉阻断期间平均动脉压升高,心率减慢,开放再灌注时平均动脉压降低,心率增加,再灌注1 h内平均动脉压和心率逐渐恢复至基础值,I/R组平均动脉压及心率波动较IPC组、SUL1组、SUL2组和SUL3组更为明显,该四组血浆内血浆可溶性血栓调节蛋白浓度、血浆内假性血管血友病因子含量和循环内皮细胞计数明显低于I/R组,光镜和电镜结果亦显示该四组血管内皮细胞损伤减轻,SUL2组血浆内血浆可溶性血栓调节蛋白浓度、血浆内假性血管血友病因子含量和循环内皮细胞计数低于SUL1组和SUL3组,光镜和电镜下损伤亦好于该两组;(2)NLX组血浆内血浆可溶性血栓调节蛋白浓度、血浆内假性血管血友病因子含量、循环内皮细胞计数、血浆一氧化氮浓度、组织匀浆一氧化氮浓度及组织匀浆一氧化氮合酶活性与I/R组无明显区别,N-SUL组各指标好于I/R组,但较IPC组及SUL组差,L-Arg组各指标与IPC组及SUL组无明显区别,LA-SUL组好于IPC组及SUL组,L-NAME组及LN-SUL组各指标较I/R组、IPC组及SUL组差;(3)免疫组化显示阳性结果为胞浆内棕色反应产物,IPC组与SUL组的内皮型一氧化氮合酶阳性细胞数多于NA组及I/R组,I/R组诱生型一氧化氮合酶多于其余各组,反转录聚合酶链式反应结果显示IPC组和SUL组内皮型一氧化氮合酶mRNA的表达较NA组与I/R组增强,各组诱生型一氧化氮合酶的表达均较低,无显著差异。
研究结论(1)缺血预处理及舒芬太尼预处理对大鼠腹主动脉缺血再灌注后血管内皮细胞损伤均具有早期保护作用,10μg/kg是相对最佳的预处理保护剂量;(2)纳洛酮能部分阻断保护作用,表明阿片受体可能部分介导了舒芬太尼预处理的早期保护作用,左旋硝基精氨酸甲酯完全阻断了保护作用,表明一氧化氮及结构型一氧化氮合酶在舒芬太尼预处理的早期保护作用中可能起主要作用;(3)缺血预处理及舒芬太尼预处理使早期血管内皮细胞内皮型一氧化氮合酶表达增加,而诱生型一氧化氮合酶在早期保护过程中未发生明显变化。
Objective (1) To investigate whether ischemic preconditioning and sufentanil preconditioning with various doses could induce the early protective effect on vascular endothelial cells after abdominal aorta ischemic-reperfusion in rats; (2) To investigate the roles of opioid receptor, nitric oxide and nitric oxide synthase in the early protective effect of vascular endothelial cells which is induced by sufentanil preconditioning; (3) To detect the expression of endothelial nitric oxide synthase and inducible nitric oxide synthase in the early protective phase by the methods of immunohistochemistry and reverse transcription Polymerase Chain Reaction, then identify their contribution in this effect.
Methods The experiment consisted of three parts: (1) Sprague-Dawley rats were divided into six groups, including group NA, group I/R, group IPC, group SUL1, group SUL2 and group SUL3, group NA was given nothing just segregated the abdominal aorta and the inferior vena cava, group I/R was given ischemia of thirty minutes and reperfusion of one hundred and twenty minutes, group IPC was added ischemia of five minutes and reperfusion of five minutes three times, group SUL1, group SUL2 and group SUL3 were given sufentanil fiveμg/kg, tenμg/kg and twentyμg/kg separatedly before ischemic-reperfusion, plasmic soluble Thrombomodulin and von Willebrand Factor were determined, Circular Endothelial Cells were calculated, and the injury of vessel was examined with optical and electron microscope, then investigated whether ischemic preconditioning and sufentanil preconditioning with various doses could induce the early protective effect on vascular endothelial cells; (2) opioid receptor antagnist naloxone, nitric oxide precursor L-Arginine and nitric oxide synthase antagnist N_(60)-Nitro-Arginine Methyl Ester were used both respectively (group NLX, group L-Arg and group L-NAME) and combinedly with sufentanil tenμg/kg (group N-SUL, group LA-SUL and group LN-SUL) for preconditioning before ischemic-reperfusion, and group NA, group I/R, group IPC and group SUL (tenμg/kg) were also done simultaneously, we determined plasmic soluble Thrombomodulin, plasmic von Willebrand Factor and Circular Endothelial Cells calculation to evaluate the injury of vascular endothelial cells, plasmic nitric oxide, nitric oxide of aorta homogenate and nitric oxide synthase activity of aorta homogenate were also determined, then investigated the roles of opioid receptor, nitric oxide and nitric oxide synthase in the early protective effect of vascular endothelial cells which is induced by sufentanil preconditioning; (3) We detected the expression of endothelial nitric oxide synthase and inducible nitric oxide synthase in the early protective phase by the methods of immunohistochemistry and reverse transcription Polymerase Chain Reaction of four groups (group NA, group I/R, group IPC and group SUL), then identify their contribution in this effect.
Results (1) The abdominal aorta ischemic-reperfusion model was succeeded, compared with group NA, mean arterial pressure of the other five groups was rising during block phase associated with lower heart rate, then became opposite during reperfusion phase, mean arterial pressure and heart rate became normal in one hour after reperfusion, the fluctuation of group I/R was more severe than group IPC, group SUL1, group SUL2 and group SUL3, the concentration of plasmic soluble Thrombomodulin, plasmic von Willebrand Factor and Circular Endothelial Cells calculation of group I/R was also higher than group IPC, group SUL1, group SUL2 and group SUL3, and the images of vessel examined by optical and electron microscope also showed less injury in group IPC, group SUL1, group SUL2 and group SUL3, the concentration of plasmic soluble Thrombomodulin, plasmic von Willebrand Factor, Circular Endothelial Cells calculation and images of optical and electron microscope of group SUL2 were better than group SUL1 and group SUL3; (2) the concentration of plasmic soluble Thrombomodulin, plasmic von Willebrand Factor, Circular Endothelial Cells calculation, plasmic nitric oxide, nitric oxide of aorta homogenate and nitric oxide synthase activity of aorta homogenate in group NLX were not different to those indices in group I/R, indices of group N-SUL were better than those of group I/R, but worse than those of group IPC and group SUL, indices of group L-Arg were not different to those of group IPC and group SUL, indices of group LA-SUL were better than those of group IPC and group SUL, indices of group L-NAME and group LN-SUL were the worst. (3) The positive result of immunohistochemistry was brown color in cytoplasm, the positive cells of endothelial nitric oxide synthase in group IPC and group SUL were more than those cells in group NA and group I/R, the positive cells of inducible nitric oxide synthase in group I/R were more than other groups, the results of reverse transcription Polymerase Chain Reaction showed that expression of endothelial nitric oxide synthase in group IPC and group SUL were better than group NA and group I/R, expression of inducible nitric oxide synthase in each group were lower and had no differences.
Conclusion (1) ischemic preconditioning and sufentanil preconditioning with various doses could induce the early protective effect on vascular endothelial cells after abdominal aorta ischemic-reperfusion in rats, tenμg/kg of sufentanil is the most effective dose relatively; (2) Naloxone could partly block the protective effect while N_(60)-Nitro-Arginine Methyl Ester could totally block the effect, it is indicated that opioid receptor maybe partly mediates the early protective effect of vascular endothelial cells which is induced by sufentanil preconditioning, while nitric oxide and nitric oxide synthase could be the major effective factors in the early protective phase; (3) the expression of endothelial nitric oxide synthase is increased during the early protective effect of ischemic preconditioning and sufentanil preconditioning, inducible nitric oxide synthase doesn't play a part in the early effect.
引文
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