P2X7R介导可溶性尿酸诱导的肾小管上皮细胞焦亡  

Effect of P2X7R on regulation of pyroptosis in renal tubular epithelial cells by soluble uric acid

作　　者：何文姬 谢恺庆 HE Wenji;XIE Kaiqing(Department of Nephrology,the Second Affiliated Hospital,Guangxi Medical University,Guangxi Zhuang Autonomous Region,Nanning530005,China;Department of Nephrology,Affiliated Cancer Hospital,Guangxi Medical University,Guangxi Zhuang Autonomous Region,Nanning530007,China)

机构地区：[1]广西医科大学第二附属医院肾内科,广西南宁530005 [2]广西医科大学附属肿瘤医院肾内科,广西南宁530007

出　　处：《中国医药导报》2025年第7期19-25,共7页China Medical Herald

基　　金：广西自然科学基金资助项目(2018GXNSFAA050057)。

摘　　要：目的 探讨嘌呤能受体P2X7(P2X7R)与可溶性尿酸诱导的肾小管上皮细胞(HK-2)焦亡的关系。方法 不同浓度可溶性尿酸(0、1、5、10、20 mg/dl)和不同作用时间(0、12、24、48、72 h)干预HK-2细胞,采用CCK-8法测定细胞活力。使用可溶性尿酸和/或A438079(P2X7R抑制剂)刺激HK-2细胞,分为对照组、可溶性尿酸组、A438079组和可溶性尿酸+A438079组。采用CCK-8法测定细胞活力;RT-q PCR和免疫荧光检测P2X7R m RNA表达量;Western blot法检测Cleaved IL-18、Cleaved IL-1β、Cleaved Caspase-1、GSDMD-N蛋白表达水平;RT-q PCR检测炎症因子[白细胞介素(IL)-1β、IL-18]、NLRP3炎症小体(包含模式识别受体NLRP3、Caspase-1、及凋亡相关斑点样蛋白(ASC)、GSDMD的m RNA表达水平;光镜下观察细胞焦亡形态)。结果 与0 mg/dl可溶性尿酸比较,10、20 mg/dl可溶性尿酸细胞活力降低(P<0.05)。10 mg/dl可溶性尿酸作用于细胞,与0 h比较,24、48、72 h细胞活力降低(P<0.05)。与对照组比较,可溶性尿酸组细胞膜上P2X7R表达强度增加(P<0.01);与可溶性尿酸组比较,可溶性尿酸+A438079组的P2X7R表达强度下降(P<0.01)。RT-q PCR结果显示,与对照组比较,可溶性尿酸组P2X7R m RNA表达量升高(P<0.01);与可溶性尿酸组比较,可溶性尿酸+A438079组的P2X7R m RNA表达量下降(P<0.01)。与对照组比较,可溶性尿酸组细胞活力下降(P<0.01);与可溶性尿酸组比较,可溶性尿酸+A438079组细胞活力升高(P<0.01)。与对照组比较,可溶性尿酸组IL-18、IL-1β、GSDMD、Caspase-1、NLRP3和ASC的m RNA表达量升高(P<0.05);与对照组比较,可溶性尿酸组Cleaved IL-18、Cleaved IL-1β、GSDMD、Cleaved Caspase-1、NLRP3和ASC的蛋白表达水平升高(P<0.05)。与可溶性尿酸组比较,可溶性尿酸+438079组HK细胞焦亡相关因子的m RNA和蛋白表达水平下降(P<0.05)。与对照组比较,可溶性尿酸组光镜下出现较多数量的细胞焦亡(P<0.01);与可溶性尿酸组比较,可溶性尿酸+A43Objective To investigate the role of purinergic receptor P2X7(P2X7R)in pyroptosis induced by soluble uric acid in renal tubular epithelial HK-2 cells.Methods HK-2 cells were treated with different concentrations of soluble uric acid(0,1,5,10,20 mg/dl)and different action time(0,12,24,48,72 h),and cell viability was measured by CCK-8 method.HK-2 cells were stimulated with soluble uric acid and/or A438079(P2X7R inhibitor)and divided into control group,soluble uric acid group,A438079 group,and soluble uric acid+A438079 group.Cell viability was measured by CCK-8 method.The expression of P2X7R mRNA was detected by RT-qPCR and immunofluorescence.The expression levels of Cleaved IL-18,Cleaved IL-1β,Cleaved Caspase-1,and GSDMD-N were detected by Western blot.RT-qPCR was used to detect the mRNA expression levels of inflammatory factors(interleukin[IL]-1β,IL-18),NLRP3 inflammasome(including pattern recognition receptors NLRP3,Caspase-1,and apoptosis-related spot-like protein(ASC),GSDMD);the morphology of pyroptosis was observed under light microscope.Results Compared with 0 mg/dl soluble uric acid,the cell viability of 10 and 20 mg/dl soluble uric acid decreased(P<0.05).Compared with 0 h,the cell viability decreased at 24,48,and 72 h after 10 mg/dl soluble uric acid treatment(P<0.05).Compared with the control group,the expression intensity of P2X7R on the cell membrane of the soluble uric acid group increased(P<0.01);compared with the soluble uric acid group,the expression intensity of P2X7R in the soluble uric acid+A438079 group decreased(P<0.01).The results of RT-qPCR showed that the expression of P2X7 R mRNA in the soluble uric acid group was higher than that in the control group(P<0.01).Compared with the soluble uric acid group,the expression of P2X7R mRNA in the soluble uric acid+A438079 group decreased(P<0.01).Compared with the control group,the cell viability of the soluble uric acid group decreased(P<0.01).Compared with the soluble uric acid group,the cell viability of the soluble uric acid+A438079 group was incr

关 键 词：P2X7受体 可溶性尿酸 肾小管上皮细胞 细胞焦亡 

分 类 号：R691.3[医药卫生—泌尿科学]