可溶性尿酸通过下调UCP2蛋白激活心肌细胞中NLRP3炎性小体的实验研究  

Soluble Uric Acid Activates NLRP3 Inflammasome in Myocardial Cells Through Down-regulating UCP2

作　　者：王冠丽 袁红敏 王珍凤 宗贝贝 叶阳 张军[1] 张海龙[1] WANG Guan-li;YUAN Hong-min;WANG Zhen-feng;ZONG Bei-bei;YE Yang;ZHANG Jun;ZHANG Hai-long(Henan Key Laboratory of Cellular and Molecular Immunology,Joint National Laboratory for Antibody Drug Engineering,Henan University,Kaifeng 475004,China;Department of Thyroid Breast Surgery,Huaihe Hospital,Henan University,Kaifeng 475004,China)

机构地区：[1]河南大学抗体药物开发技术国家地方联合工程实验室河南省细胞与分子免疫重点实验室,开封475004 [2]河南大学淮河医院乳腺甲状腺外科,开封475004

出　　处：《四川大学学报（医学版）》2018年第4期512-517,共6页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金面上项目(No.81670271)资助

摘　　要：目的观察尿酸处理下大鼠心肌细胞系H9C2的损伤和NLRP3炎性小体的活化情况,探讨可溶性尿酸活化NLRP3炎性小体的机制。方法采用不同质量浓度的尿酸处理H9C2 12h、24h和48h,通过MTS和乳酸脱氢酶(LDH)检测细胞活力,以反映细胞损伤情况,同时采用流式细胞术(FCM)分析H9C2凋亡情况。通过Western blot检测NLRP3炎性小体相关分子[NLRP3、凋亡相关斑点样蛋白(ASC)和Caspase-1]的蛋白水平。分离线粒体和胞质,Western blot检测细胞色素C的释放情况,评价线粒体损伤情况。MTS和LDH检测活性氧(ROS)抑制剂N-乙酰基-L-半胱氨酸(NAC)对细胞活力的影响,real time-PCR及Western blot检测NAC对NLRP3炎性小体活化的改善情况。最后采用Western blot和免疫荧光检测解偶联蛋白2(UCP2)蛋白的表达。结果随着尿酸浓度的增加,细胞损伤和凋亡加剧,且具有时间依赖性;尿酸可上调NLRP3炎性小体相关分子的表达,并导致线粒体受损;NAC可改善细胞损伤并抑制NLRP3炎性小体相关mRNA和蛋白的活化;尿酸还可下调UCP2的表达。结论尿酸下调UCP2蛋白的表达,诱导线粒体损伤,激活NLRP3炎性小体,导致心肌细胞损伤。Objective To determine the H9 C2 cell damage and NLRP3 inflammasome activation trigged by soluble uric acid（UA）.Methods H9 C2 cells were treated with UA.The cellular damage was examined after12 h,24 hand 48 hof treatment using MTS and lactic dehydrogenase（LDH）.The apoptosis of H9 C2 cells was analyzed by flow cytometry（FCM）.NLRP3 inflammasome activation was reflected by the protein levels of NLRP3,apoptosis-associated speck-like protein containing a CARD（ASC）and Caspase-1 detected by Western blot.The mitochondria and cytoplasm were separated and the release of cytochrome C was detected by Western blot to analyze the damage of mitochondria.The impacts of NAC,a ROS inhibitor,on the cell viability and NLRP3 inflammasome activation were analyzed.The expression of UCP2 was detected by Western blot and immunofluorescence（IF）.Results Dose response and time dependent effects of UA on cellular damage and cell apoptosis was observed.UA up-regulated the expression of NLRP3 inflammasome-related molecules.UA damaged the mitochondria.NAC improved the cell viability and inhibited NLRP3 inflammasome activation.UA down-regulated the expression of UCP2.Conclusion Soluble UA can down-regulate the expression of UCP2,damage the mitochondria and activate NLRP3 inflammasome,resulting in cellular damage of H9 C2 cells.

关 键 词：可溶性尿酸 解偶联蛋白2 NLRP3炎性小体 心肌细胞损伤 

分 类 号：R392[医药卫生—免疫学]