高尿酸通过下调UCP2表达水平引起线粒体损伤的研究

Study on mitochondrial damage induced by hyperuric acid through down regulation of UCP2 expression level

目的在细胞水平探究高尿酸对线粒体解偶联蛋白2(UCP2)表达水平的影响,以探索高尿酸对HL7702细胞线粒体损伤作用的可能机制。方法通过实时荧光定量聚合酶链式反应(Q-PCR)和Western blot法分析3个不同浓度尿酸(400、800、1200μmol/L)对线粒体UCP2表达的影响,免疫荧光、荧光探针法等技术分析UCP2相关的线粒体损伤指标活性氧(ROS)、线粒体膜电位、三磷酸腺苷(ATP)合成的变化情况。结果与对照组相比,随着尿酸浓度的升高,3个浓度组(400、800、1200μmol/L)的线粒体UCP2 mRNA转录水平分别为对照组的46.30%、16.40%和5.00%(P<0.01),随着尿酸浓度的升高,线粒体UCP2蛋白表达也逐渐减少。ROS生成量随着尿酸浓度的升高呈现明显增加的趋势,3个浓度组(400、800、1200μmol/L)的ROS荧光强度分别为对照组的133、184和253倍(P<0.01),线粒体膜电位和ATP合成率与对照组比较则呈现明显降低趋势,3个浓度组(400、800、1200μmol/L)线粒体膜电位分别为对照组的14.53%、8.30%和4.70%(P<0.01),ATP合成率为对照组的83.45%、69.92%和55.64%(P<0.01)。结论随着尿酸浓度的增加,高尿酸对线粒体UCP2的mRNA和蛋白表达存在着抑制作用,并造成线粒体损伤。

Objective To explore the effect of high uric acid on the expression of mitochondrial uncoupling protein 2(UCP2)at the cellular level,so as to explore the possible mechanism of high uric acid on cell damage.Methods Three different concentrations of uric acid(400,800,1200μmol/L)were added to the cultured cells,and the expression of UCP2 in mitochondria was analyzed by Q-PCR and Western blot,and the changes of UCP2 related mitochondrial damage indexes reactive oxygen species(ROS),mitochondrial membrane potential and adenosine triphosphate(ATP)synthesis were analyzed by immunofluorescence,fluorescence probe and other related techniques.Results Compared with the control group,with the increase of uric acid concentration,the transcriptional levels of mitochondrial UCP2 mRNA in the three concentration groups(400,800,1200μmol/L)was 46.30%,16.40%and 5.00%of the control group,respectively(P<0.01).With the increase of uric acid concentration,the expression of UCP2 protein in mitochondria decreased gradually.ROS production increased significantly with the increase of uric acid concentration,and The fluorescence intensity of ROS in the three concentration groups(400,800,1200μmol/L)was 133times,184times and 253times of the control group,respectively(P<0.01).Compared with the control group,the mitochondrial membrane potential and ATP synthesis rate showed a significant downward trend,the mitochondrial membrane potential in the three concentration groups(400,800,1200μmol/L)was 14.53%,8.30%and 4.70%of the control group,respectively(P<0.01),and the ATP synthesis rate was 83.45%,69.92%and 55.64%of the control group respectively(P<0.01).Conclusion With the increase of uric acid concentration,high uric acid inhibited the mRNA and protein expression of mitochondrial UCP2,and caused mitochondrial damage.