法舒地尔对oxLDL损伤内皮细胞功能的影响
摘要
目的:探讨法舒地尔对氧化型低密度脂蛋白(oxLDL)致内皮细胞损伤的保护作用。
方法:培养人脐静脉内皮细胞(HUVECs),加入不同浓度的法舒地尔(Fasudil)(0μmol/L、1μmol/L、10μmol/L、100μmol/L)共孵育24h后,用western-blotting方法检测各组细胞eNOS表达量;加入含不同刺激物的培养液,分成对照组、法舒地尔组、oxLDL组、法舒地尔加oxLDL组,孵育24h后用western-blotting方法检测各组细胞eNOS生成量,用硝酸酶还原法检测各组细胞培养液中NO的代谢产物NO2-/NO3-的量来反映的NO释放量。
结果:HUVECs eNOS蛋白表达量随培养基中Fasudil浓度的升高而增加,Fasudil终浓度为1.0μmol/L、10μmol/L、100μmol/L时eNOS蛋白表达量与对照组(0μmol /L)相比分别升高至121±8﹪、147±13﹪、181±16﹪,呈浓度依赖性递增,各组间差异显著,p<0.05。与不同刺激物共孵育的HUVECs eNOS蛋白表达量:与对照组相比,法舒地尔组显著增加,为基础值的171±15﹪;oxLDL组下降,为基础值的67±6﹪;法舒地尔加oxLDL组增加,为基础值的133±11﹪;各组间差异显著,p<0.01。NO生成量与对照组(48.12±5.34)相比,法舒地尔组(72.52±6.21)升高(p<0.01),法舒地尔加oxLDL组(41.31±5.11)下降(p<0.05),oxLDL组显著降低(26.26±4.54)(p<0.01)。
结论:oxLDL使培养的人脐静脉内皮细胞eNOS蛋白表达量及NO生成量降低,法舒地尔可逆转或部分纠正其作用,对内皮细胞功能具有保护作用。
Objective To investigate whether fasudil can protect the function of cultivated human umbilical vein endothelial cells(HUVECs) induced by oxidized low density lipoprotein.
Methods HUVECs were cultivated in four groups with different concentrations of fasudil (0μmol/L,1μmol/L,10μmol/L, 100μmol/L) respectively for 24hs and then the expression amount of eNOS in HUVECs of each group was detected by western-blotting. The HUVECs cultivated in vitro were divided into four groups which were treated by fasudil, oxLDL, fasudil + oxLDL, and normal culture medium respectively. After 24hs, the amount of eNOS in each group was detected by western-blotting. And the amount of the NO2-/NO3- , which is the metabolic product of NO and indicates the release amount of NO, was detected by the nitrate reductase assay (NRA).
Result In a concentration depend manner, treatment with fasudil (1μmol/L, 10μmol/L, 100μmol/L) increased eNOS protein expression to 121±8﹪,147±13﹪,181±16﹪respectively than the contrast group (fasudil 0μmol/L).The differences between each group were significant, p<0.05. Compared with the basal level in group control, the eNOS protein level was significantly increased in group fasudil(171±15﹪) (p<0.01) and in group fasudil + oxLDL (133±11﹪) (p<0.01), decreased in group oxLDL(67±6﹪) (p<0.01). Compared with group contro(l48.12±5.34),the production of NO was significantly increased in group fasudil(72.52±6.21) (p<0.01), decreased in group oxLDL(26.26±4.54)(p<0.01)and in group fasudil + oxLDL(41.31±5.11) (p<0.05).
Conclusion OxLDL decreased the production of eNOS and NO in cultivated human umbilical vein endothelial cells, while fasudil could reverse or partly rectify these effects. Fasudil could protect the function of cultivated HUVECs.
方法:培养人脐静脉内皮细胞(HUVECs),加入不同浓度的法舒地尔(Fasudil)(0μmol/L、1μmol/L、10μmol/L、100μmol/L)共孵育24h后,用western-blotting方法检测各组细胞eNOS表达量;加入含不同刺激物的培养液,分成对照组、法舒地尔组、oxLDL组、法舒地尔加oxLDL组,孵育24h后用western-blotting方法检测各组细胞eNOS生成量,用硝酸酶还原法检测各组细胞培养液中NO的代谢产物NO2-/NO3-的量来反映的NO释放量。
结果:HUVECs eNOS蛋白表达量随培养基中Fasudil浓度的升高而增加,Fasudil终浓度为1.0μmol/L、10μmol/L、100μmol/L时eNOS蛋白表达量与对照组(0μmol /L)相比分别升高至121±8﹪、147±13﹪、181±16﹪,呈浓度依赖性递增,各组间差异显著,p<0.05。与不同刺激物共孵育的HUVECs eNOS蛋白表达量:与对照组相比,法舒地尔组显著增加,为基础值的171±15﹪;oxLDL组下降,为基础值的67±6﹪;法舒地尔加oxLDL组增加,为基础值的133±11﹪;各组间差异显著,p<0.01。NO生成量与对照组(48.12±5.34)相比,法舒地尔组(72.52±6.21)升高(p<0.01),法舒地尔加oxLDL组(41.31±5.11)下降(p<0.05),oxLDL组显著降低(26.26±4.54)(p<0.01)。
结论:oxLDL使培养的人脐静脉内皮细胞eNOS蛋白表达量及NO生成量降低,法舒地尔可逆转或部分纠正其作用,对内皮细胞功能具有保护作用。
Objective To investigate whether fasudil can protect the function of cultivated human umbilical vein endothelial cells(HUVECs) induced by oxidized low density lipoprotein.
Methods HUVECs were cultivated in four groups with different concentrations of fasudil (0μmol/L,1μmol/L,10μmol/L, 100μmol/L) respectively for 24hs and then the expression amount of eNOS in HUVECs of each group was detected by western-blotting. The HUVECs cultivated in vitro were divided into four groups which were treated by fasudil, oxLDL, fasudil + oxLDL, and normal culture medium respectively. After 24hs, the amount of eNOS in each group was detected by western-blotting. And the amount of the NO2-/NO3- , which is the metabolic product of NO and indicates the release amount of NO, was detected by the nitrate reductase assay (NRA).
Result In a concentration depend manner, treatment with fasudil (1μmol/L, 10μmol/L, 100μmol/L) increased eNOS protein expression to 121±8﹪,147±13﹪,181±16﹪respectively than the contrast group (fasudil 0μmol/L).The differences between each group were significant, p<0.05. Compared with the basal level in group control, the eNOS protein level was significantly increased in group fasudil(171±15﹪) (p<0.01) and in group fasudil + oxLDL (133±11﹪) (p<0.01), decreased in group oxLDL(67±6﹪) (p<0.01). Compared with group contro(l48.12±5.34),the production of NO was significantly increased in group fasudil(72.52±6.21) (p<0.01), decreased in group oxLDL(26.26±4.54)(p<0.01)and in group fasudil + oxLDL(41.31±5.11) (p<0.05).
Conclusion OxLDL decreased the production of eNOS and NO in cultivated human umbilical vein endothelial cells, while fasudil could reverse or partly rectify these effects. Fasudil could protect the function of cultivated HUVECs.
引文
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