TLR4对氧化低密度脂蛋白作用巨噬细胞后凋亡和炎症因子的影响  

Effects of TLR4 on release of apoptosis and inflammatory cytokines in oxidized LDL treated-macrophages

作　　者：石中全[1] 李国利[1] 胡艳玲[1] 马丽华[1] SHI Zhong-Quan;LI Guo-Li;HU Yan-Ling;MA Li-Hua(Department of Basic Medicine,Chongqing Three Gorges Medical College,Chongqing 404120,China)

机构地区：[1]重庆三峡医药高等专科学校基础医学部,重庆404120

出　　处：《中国免疫学杂志》2018年第7期984-989,995,共7页Chinese Journal of Immunology

基　　金：重庆市教委科学技术研究项目(No.KJ1725390)资助

摘　　要：目的:研究Toll样受体4(TLR4)对氧化低密度脂蛋白(ox-LDL)作用后巨噬细胞凋亡和炎症因子的影响。方法:巨噬细胞Ana-1分为Con(不做处理)、ox-LDL(50 pg/ml ox-LDL处理)、TAK-242(50 pg/ml ox-LDL和20μmol/L的TLR4阻断剂TAK-242处理)。流式细胞术检测细胞凋亡,Western blot检测Bcl-2相关X蛋白(Bax)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)、核因子-κBp65(NF-κBp65)、TLR4蛋白水平,二氯二氢荧光素-乙酰乙酸酯(DCFH-DA)法检测细胞中ROS水平,ELISA检测培养液上清中TNF-α、IL-6、IL-1β水平。结果:ox-LDL处理后的Ana-1细胞中TLR4表达水平升高,而TLR4阻断剂TAK-242可以部分降低细胞中TLR4水平。ox-LDL细胞凋亡率从(6.12±0.95)%升高到(45.35±2.71)%,而TAK-242作用后的凋亡率降低至(29.59±4.23)%,同时ox-LDL细胞中Bax、Cleaved Caspase-3、NF-κBp65水平和ROS水平均明显高于Con(P<0.05),而TAK-242细胞中Bax、Cleaved Caspase-3、NF-κBp65水平ROS水平均明显低于ox-LDL(P<0.05)。ox-LDL细胞培养液上清中TNF-α、IL-6、IL-1β水平均明显高于Con(P<0.05),而TAK-242细胞培养液上清中TNF-α、IL-6、IL-1β水平均明显低于ox-LDL(P<0.05)。结论:巨噬细胞经过ox-LDL作用后细胞中TLR4表达升高,细胞凋亡增多,细胞分泌的炎症因子水平升高,细胞中ROS水平和NF-κBp65水平均升高,而敲低TLR4能够抑制ox-LDL的上述作用。Objective：To investigate the effects of TLR4 on apoptosis and inflammatory cytokines in macrophages after oxidized LDL treated. Methods：Macrophage Ana-1 was divided into Con（ without treatment）,ox-LDL（ 50 pg/ml ox-LDL treatment）,TAK-242（50 pg/ml ox-LDL and 20 mol/L TLR4 blocking agent TAK-242）. Apoptosis was detected by flow cytometry,the levels of Bax,Cleaved Caspase-3,NF-κBp65 and TLR4 protein were detected by Western blot,and the level of ROS in cells was detected by DCFH-DA,detection of TNF-α,IL-6 and IL-1β levels in culture supernatant by ELISA. Results：The expression level of TLR4 increased in ox-LDL treated Ana-1 cells,the TLR4 blocker TAK-242 partially decreased the level of TLR4 in the cells. The apoptosis rate of ox-LDL cells increased from（ 6. 12±0. 95） % to（ 45. 35± 2. 71） %,the apoptotic rate decreased to（ 29. 59± 4. 23） % after TAK-242 treatment,at the same time,the levels of Bax,Cleaved Caspase-3,NF-κBp65 and ROS in ox-LDL cells were significantly higher than those in Con（ P〈0. 05）. However,the levels of Bax,Cleaved Caspase-3 and NF-κBp65,ROS in TAK-242 cells were significantly lower than those in oxLDL（ P〈0. 05）. The levels of TNF-α,IL-6 and IL-1β in the supernatant of ox-LDL cells were significantly higher than those in Con（ P〈0. 05）. However,the levels of TNF-α,IL-6 and IL-1β in supernatant of TAK-242 cells were significantly lower than those in ox-LDL（P〈0. 05）. Conclusion：The expression of TLR4 in macrophages increase after ox-LDL treatment,increase apoptosis,the level of inflammatory cytokines secreted by cells increase,the levels of ROS and NF-κBp65 in the cells are increase,knockdown of TLR4 can inhibit the above effects of ox-LDL.

关 键 词：巨噬细胞 凋亡 炎症因子 TLR4 ROS 

分 类 号：R285.5[医药卫生—中药学]