TLR4对ox-LDL作用后的血管内皮细胞ROS及线粒体膜电位的影响

The effect of TLR4 on the ROS and mitochondrial membrane potential of vascular endothelial cells after ox-LDL action

目的探讨Toll样受体4(TLR4)对氧化低密度脂蛋白(ox-LDL)作用后的血管内皮细胞活性氧(ROS)及线粒体膜电位的影响。方法用qRT-PCR和Western blot分别测定ox-LDL作用后的血管内皮细胞中TLR4 mRNA和蛋白水平。在血管内皮细胞中转染TLR4小干扰RNA和小干扰RNA阴性对照,qRT-PCR和Western blot分别测定血管内皮细胞中TLR4 mRNA和蛋白水平。用ox-LDL作用转染以后的血管内皮细胞,流式细胞术测定细胞凋亡情况,荧光素酶基因报告系统测定三磷酸腺苷(ATP)含量,二氯二氢荧光素-乙酰乙酸酯(DCFH-DA)法测定ROS含量,JC-1法测定线粒体膜电位,Western blot测定细胞浆及线粒体中细胞色素C(Cytochrome C)蛋白水平。结果 ox-LDL作用后的血管内皮细胞中TLR4 mRNA和蛋白水平均明显高于未经处理的细胞(P<0.05)。TLR4小干扰RNA转染以后的细胞中TLR4 mRNA和蛋白水平均明显低于未经转染的细胞(P<0.05),而转染小干扰RNA阴性对照后细胞中mRNA和蛋白水平与未经转染的细胞相比没有明显变化(P>0.05)。ox-LDL处理以后的细胞凋亡增多,细胞线粒体膜电位和ATP水平降低,细胞中ROS水平升高,线粒体中Cytochrome C蛋白减少,胞浆中Cytochrome C蛋白增多,与未经处理的细胞相比差异有统计学意义(P<0.05)。下调TLR4后的血管内皮细胞经ox-LDL处理以后,细胞凋亡率降低,线粒体膜电位和ATP水平升高,细胞中ROS水平降低,线粒体中Cytochrome C蛋白增多,胞浆中Cytochrome C蛋白减少,与单纯ox-LDL处理的细胞相比,差异有统计学意义(P<0.05)。结论 TLR4表达下调后可以提高ox-LDL处理后血管内皮细胞线粒体膜电位,促进细胞ATP合成,减少细胞中ROS的积累,减少线粒体中Cytochrome C蛋白的释放,减少线粒体损伤,减少细胞凋亡发生。

Objective To investigate the effect of TLR4 on the ROS and mitochondrial membrane potential of vascular endothelial cells after ox-LDL action. Methods The levels of TLR4 mRNA and protein in vascular endothelial cells after ox-LDL were measured by qRT-PCR and Western blot respectively. Transfection of TLR4 small interference RNA and small interference RNA negative control in vascular endothelial cells was performed,the levels of TLR4 mRNA and protein in vascular endothelial cells were measured by qRT-PCR and Western blot respectively. Vascular endothelial cells after transfection with ox-LDL were transfected,cell apoptosis was measured by flow cytometry,the content of ATP was determined by the luciferase gene report system,the content of ROS was determined by DCFH-DA method,the JC-1 method was used to determine the mitochondrial membrane potential,determination of Cytochrome C protein in cytoplasm and mitochondria by Western blot. Results The levels of TLR4 mRNA and protein in vascular endothelial cells after ox-LDL were significantly higher than those of untreated cells（ P〈0. 05）. The levels of TLR4 mRNA and protein in cells transfected by TLR4 small interference RNA were significantly lower than those of untransfected cells（ P〈0. 05）. The levels of mRNA and protein in the cells transfected with RNA negative control were not significantly different from those in the untransfected cells（ P〈0. 05）. The number of apoptotic cells increased after ox-LDL treatment,the cell mitochondrial membrane potential and ATP level were reduced,the level of ROS in the cells was elevated,the decrease of Cytochrome C protein in mitochondria,the increase of Cytochrome C protein in the cytoplasm,the difference was statistically significant compared with the untreated cells（ P〈0. 05）. After down regulation of TLR4,the vascular endothelial cells were treated by ox-LDL,the rate of apoptosis was reduced,mitochondrial membrane potential and ATP level were increased,the level of ROS in the cells was reduced,the number of Cytochrome C proteins in mitochondria was increased,the decrease of Cytochrome C protein in the cytoplasm,compared with the cells treated with ox-LDL alone,the difference was statistically significant（ P〈0. 05）. Conclusion The down regulation of TLR4 can inhibit the abnormal decrease of mitochondrial membrane potential induced by ox-LDL,and promote the synthesis of ATP in cells,reduce the accumulation of ROS in cells,reduce the release of Cytochrome C protein in mitochondria,reduce mitochondrial damage,decrease the occurrence of apoptosis.