摘要
目的探讨热损伤过程中异丙酚对内皮细胞的抗氧化作用以及对线粒体的保护效应。方法采用43℃培养2h后37℃、5%CO2常规培养6h的方法建立人脐静脉内皮细胞(HUVECs)热打击模型;实验分为37℃组、37℃+脂肪乳组(阴性对照组)、37℃+异丙酚组、43℃组、43℃+异丙酚组、H2O2+异丙酚组(阳性对照组)6组,其中异丙酚、脂肪乳均于热打击或H2O2作用前先行与细胞孵育。应用CCK-8法检测细胞活力;流式细胞术检测细胞内活性氧(ROS)、线粒体膜电位(ΔΨm)以及线粒体通透性转换孔(MPTP)的变化;荧光素-荧光素酶法检测ATP含量;Caspase活性试剂盒检测caspase-9和caspase-3活性的变化。结果热打击后,HUVECs活力明显降低,线粒体功能受损(P<0.05);经异丙酚预处理的HUVECs在热打击后保持了细胞活力,胞内ROS水平较单纯43℃热打击组明显降低(P<0.05),线粒体膜电位下降的细胞占比(JC-1单体所占比例)明显减少(P<0.05),线粒体通透性转换孔开放,细胞内ATP含量明显升高(P<0.05),同时线粒体损伤相关的caspase-9/3所介导的线粒体凋亡通路的激活也被抑制。结论异丙酚在热损伤过程中具有抗氧化损伤、抗凋亡以及线粒体保护作用。
Objective To explore the protective effect of propofol on endothelial cells during heat stress and its protective effect to mitochondra. Methods Heat stress model of human umbilical vein endothelial cell was established when cells were incubated at 43℃ for 2h, then further incubted at 37℃, 5%CO2 for 6h. The experimental group was subdivided into six groups, including 37℃ group, 37℃ plus intralipid group (negative control group), 37℃ plus propofol group, 43℃ plus propofol group, 43℃ plus intralipid group, H2O2 plus propofol group (positive control group); Pretreated with 50μmol/L propofol, 0.2ml intralipid or 25μmol/L H2O2 before heat stress at 43℃, while the cells in the control group were incubated at 37℃. Cell viability was tested by CCK-8. ROS, mitochondrial membrane potential and the changes in mitochondrial permeability transition pore were determined by flow cytometry. The level of ATP was detected by fluorescein-luciferase. The changes of caspase-9 and caspase-3 were analyzed by Caspase Activity Assay Kit. Results HUVESs cell viability and damage of mitochondra were significantly decreased after heat stress. Compared with 43℃ heat stress group, pretreatment with propofol induced the recovery of cell viability and the ROS levels were significantly decreased in HUVEC cells (P〈0.05). Meanwhile, the number of cells representing the decrease of mitochondrial membrane potential (the proportion of JC-1 monomer) was significantly decreased (P〈0.05) by propofol. The average fluorescence intensity of calcein which representing the MPTP changes and intracellular ATP content was significantly increased (P〈0.05). In addition, the activation of mitochondrial apoptotic pathway mediated by caspase-9/3 was also inhibited. Conclusions Propofol have anti-oxidative, anti-apoptosis and mitochondria protective effect against endothelial cell injury during heat stress.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2017年第6期500-505,共6页
Medical Journal of Chinese People's Liberation Army
基金
广东省自然科学基金面上项目(2014A030313601)
广东省自然科学基金研究团队项目(S2013030013217)
广东省科技计划项目(2014A020212490,2013B031800010)
南方医科大学校科研启动计划项目(PY2015NO29,QD2016N017)~~
关键词
中暑
内皮细胞
氧化性应激
heat stroke
endothelial cells
oxidative stress