异丙酚对缺氧损伤人血管内皮细胞蛋白激酶C表达及钙离子浓度的影响

Effects of propofol on expression of protein kinase C and calcium ion concentration in human vascular endothelial cells induced by hypoxia injury

目的:探讨异丙酚对缺氧损伤所致人血管内皮细胞凋亡、细胞内钙稳态和蛋白激酶C(PKC)表达的影响。方法:依文献方法进行人脐静脉内皮细胞原代和继代培养,建立人脐静脉内皮细胞缺氧复氧损伤模型。将培养至融合状态的细胞随机分组:C组(正常对照组)、HR组(缺氧30min后复氧6h组)和PR组(异丙酚25、50、100μmol/L组)。采用流式细胞仪检测细胞凋亡,Western-blot测定PKC蛋白的表达,MetaFlour单细胞内钙测定系统测定细胞内钙浓度变化。结果:PR组细胞凋亡降低,与HR组(14.7±1.0)%比较差异有统计学意义(P<0.01),PR50μmol/L组(9.4±0.6)%、PR100μmol/L组(9.5±0.6)%与PR25μmol/L组(12.3±0.7)%比较差异有统计学意义(P<0.01);PR组细胞内钙离子浓度明显降低,与HR组(117.3±6.0)%比较差异有统计学意义(P<0.01),PR50μmol/L组(48.2±4.4)%、PR100μmol/L组(47.0±6.3)%与PR25μmol/L组(80.3±7.6)%比较差异有统计学意义(P<0.01);PR组PKC蛋白表达升高,与HR组(6.3±0.8)%比较差异有统计学意义(P<0.01),PR50μmol/L组(27.0±2.4)%、PR100μmol/L组(27.5±2.6)%与PR25μmol/L组(19.0±1.7)%比较差异有统计学意义(P<0.01)。结论:缺氧复氧导致血管内皮细胞凋亡增加,与抑制PKC表达、升高细胞内钙离子浓度有关。异丙酚抑制缺氧复氧所致的人血管内皮细胞凋亡,可能与其活化细胞内PKC,降低钙超载,维持细胞内钙稳态有关。

AIM： To investigate the effects of propofol on the expression of protien kinase C and intracellular calcium homeostasis in human vascular endothelial cells induced by hypoxia-reoxygenation injury. METHODS： The endothelial cells were separated from human umbilical veins. The cells were cultured in normal medium or the medium containing different concentrations of propofol. The cells were further incubated in hypoxic condition for 30 min and then in normal condition for 6 h to induce hypoxia-reoxygenation injury. The cells were divided into groups： the control group （C group）, the hypoxia-reoxy-genation group （ HR group） and propofol groups （PR 25, 50, 100 μmol/L groups）. The expression of PKC and the concentration of intracellular calcium were measured by Western-blot analysis and method, respectively. RESULTS： The number of apoptosis increased after reoxygenation （ 14.7 ± 1.0% ）. Propofol inhibited the apoptosis significantly （ P 〈 0.01 ）, and the inhibited effect of propofol on 50μmol/L（9.4 ± 0.6）% and 100 μmol/L（9.5 ± 0.6）%was more significant compared with the propofol on 25 μmol/L（ 12.3 ± 0.7） % （ P 〈0.01 ）. The expression of PKC was significantly inhibited after reoxygenation （ P 〈 0.01）. The concentration of intracellular calcium induced by hypoxia-reoxygenation injury was up-regulated （ 117.3 ± 6.0） %. Propofol attenuated the concentration of intracellular calcium significantly （ P 〈 0.01） and was more effective at 50 and 100 μmol/L （P 〈 0.01）. Propofol at a concentration of 25 μmol/L could significantly （ 19.0 ± 1.7） % upregulate the expression of PKC （ P 〈 0.01）. Propofol at concentrations of 50 μmol/L （27.0 ±2.4 ）% and 100 μmol/L （ 27.5 ± 2.6） % showed a similar result but is significant compared with PR25 group （ P 〈 0.01 ）. CONCLUSION： Hypoxia-reoxygenation injury induces apoptosis, which may be related to inhibiting the expression of PKC and increasing the concentration of intracellular calcium. Propofol inhibits the apoptosis of endothelial cells induced by hypoxia-reoxygenation injury, and the mechanism is possibly related to the activation of PKC and depression of calcium overload.