胆固醇通过NADPH氧化酶诱导ROS升高,NF-κB活化进而导致内皮细胞损伤  

Cholesterol induces endothelial cells injury by increasing production of reactive oxygen species and activating NF-κB

作　　者：权媛[1,2] 钱民章[1] 

机构地区：[1]遵义医学院生物化学教研室,贵州遵义563003 [2]上海中医药大学07级博士研究生,上海200032

出　　处：《中国病理生理杂志》2010年第8期1521-1526,共6页Chinese Journal of Pathophysiology

基　　金：贵州省科技厅基金资助项目(No.黔科合J字[2009]2178号)

摘　　要：目的:研究胆固醇对人脐静脉内皮细胞(HUVECs)的损伤是否与细胞内活性氧(ROS)升高有关,明确此种情况下ROS的主要来源。方法:体外培养HUVECs-12,设置正常对照组、溶媒(无水乙醇)组、胆固醇组、N-乙酰半胱氨酸(NAC)作用1 h后再加入胆固醇作用48 h组。以DCFH-DA为荧光探针,流式细胞术检测细胞内ROS水平;免疫细胞化学方法检测细胞内核因子-κB亚单位p65核移位阳性细胞数;分别用比色法、硝酸酶还原法及ELISA检测细胞培养液中乳酸脱氢酶(LDH)活性、一氧化氮(NO)及单核细胞趋化蛋白-1(MCP-1)浓度。并观察加入产生ROS的4种酶抑制剂二联苯碘(DPI)、鱼藤酮(rotenone)、奥苷嘌醇(oxypurinol)、N-硝基-L-精氨酸甲酯(L-NAME)后细胞内ROS水平。结果:(1)与正常对照组比较,50 mg/L胆固醇能升高细胞内ROS(P<0.01),并能激活细胞内NF-κB p65的核移位(P<0.01),升高细胞培养液中LDH活性、MCP-1浓度,降低NO浓度(P<0.01);(2)与胆固醇作用组相比,NADPH氧化酶抑制剂DPI预作用组细胞内ROS明显降低(P<0.01),retenone也可以部分抑制ROS的产生(P<0.05),oxypurinol和L-NAME预作用则几乎不影响胆固醇所致的ROS生成增加(P>0.05)。结论:一定浓度的游离胆固醇能引起内皮细胞内ROS升高,激活细胞内NF-κB,进而导致内皮细胞损伤;此情况下细胞内产生的ROS主要来源于NADPH氧化酶。AIM： To study the increased level of reactive oxygen species in human umbilical vein endothelial cells（HUVECs） and their correlation with the injury caused by cholesterol on HUVECs,and to clarify the original source of intracellular ROS.METHODS： The cells of HUVECs-12 were cultured in F12 medium with 10% FBS and divided into normal control group（without any treatment）,solvent group（treated with 0.25% dehydrated alcohol）,cholesterol group（treated with 50 mg/L cholesterol） and N-acetyl-L-cysteine（NAC） group（pretreated with 10 mmol/L NAC for 1 h and then treated with 50 mg/L cholesterol for 48 h）.The intracellular ROS levels were determined by flow cytometry（FCM） with DCFH-DA as fluorescent probe.Nuclear translocation of NF-κB subunit p65 was detected by immunocytochemistry staining.LDH activity and concentration of nitric oxide in the supernatant of the cell culture were also determined.The concentration of MCP-1 protein in cultured supernatant was measured by ELISA.The intracellular levels of ROS and the changes after adding 4 kinds of enzyme inhibitors（NADPH oxidase inhibitor diphenyl iodide,mitochondrial respiratory chain enzyme complex inhibitor rotenone,NOS inhibitor L-NAME and xanthine oxidase inhibitor oxypurinol） were observed.RESULTS：（1）Compared to the normal control cells,50 mg/L cholesterol increased intracellular ROS（P0.01） and activated the nuclear translocation of NF-κB p65.A significant increases in LDH activity and the MCP-l protein were also observed.The NO level decreased in the cells.（2）Compared to the cholesterol control cells,diphenyl iodide decreased intracellular ROS significantly（P0.01）.Retenone also inhibited the generation of ROS partially（P0.05）.The other inhibitors almost did not affect the level of ROS caused by cholesterol（P0.05）.CONCLUSION： Free cholesterol increases ROS generation in endothelial cells,activates intracellular NF-κB,thus leading to endothelial cell injury.NADPH oxidase was the main source of ROS generat

关 键 词：胆固醇 人脐静脉内皮细胞 活性氧 NADPH氧化酶 

分 类 号：R363[医药卫生—病理学]