氧合血红蛋白诱导脑微血管内皮细胞凋亡中半胱天冬酶3和细胞色素C的变化

Caspase-3 and Cyt-C activity in apoptosis of brain microvascular endothelial cells induced by oxyhemoglobin

目的:探讨氧合血红蛋白(oxyhemoglobin,OxyHb)诱导体外培养的大鼠脑微血管内皮细胞(brain microvas-cular endothelial cells,BMvECs)凋亡的可能机制。方法:将原代培养的大鼠BMvECs分别与10μmol/L和100μmol/L的OxyHb共孵育,在孵育后6、12和24h分别以流式细胞术、荧光分光光度计法和Western印迹检测线粒体跨膜电位(mitochondrial transmembrane potentials,ΔΨm)、半胱天冬酶3(caspase-3)和细胞色素C(cytochrome C,Cyt-C)的表达水平。结果:经10μmol/L和100μmol/LOxyHb刺激6～24h后,与对照组相比,OxyHb能显著降低血管内皮细胞ΔΨm,而增强Cyt-C和caspase-3的表达,并呈刺激时间和刺激浓度依赖性。与10μmol/LOxyHb分别孵育6、12和24h,caspase-3的活性分别为0.103±0.010,0.126±0.010和0.234±0.031,而在OxyHb100μmol/L组,其6、12和24h caspase-3的活性分别为0.154±0.012,0.205±0.020和0.302±0.015。结论:ΔΨm的下降、caspase-3的激活以及Cyt-C的释放可能是OxyHb诱导BMvECs凋亡的重要机制。

Objective：To investigate the mechanism of apoptosis induced by oxyhemoglobin （OxyHb） in cultured rat brain microvascular endothelial cells（BMvECs）. Methods： Primary cultured rat BMvECs were incubated with 10 μmol/L and 100 μmol/L OxyHb for various periods of time（6 h, 12 h, and 24 h）. The caspase-3 activity was detected by spectrophotometric method, mitochondrial transmembrane potentials were determined by flow cytometry and cytochrome C （Cyt-C） was measured by Western blot. Results： OxyHb could significantly inhibit mitochondrial transmembrane potentials and increase the expression of Cyt-C and caspase-3 compared with control group, depending on the incubation concentrations and incubation time. The activity of caspase-3 was 0.103±0.010,0. 126±0.010 and 0.234±0. 031 after treatment with the OxyHb at concentration of 10 μmol/L for 6 h, 12 h and 24 h,respectively, while at the OxyHb concentration of 100 μmol/ L, it was 0. 154±0. 012,0. 205±0. 020 and 0. 302±0. 015, respectively. Conclusion： OxyHb can induce BMvECs apoptosis by suppressing mitochondrial transmembrane potentials and increasing the caspase-3 activity and releasing of Cyt-C.