高糖腹膜透析液对人腹膜间皮细胞NLRP3-IL-1β的影响  

Effect of high glucose-based peritoneal dialysis fluids on NLRP3-IL-1β in human peritoneal mesothelial cells

作　　者：李相友[1] 伍军[1] 罗丹[1] 陈晚先 朱戈丽[1] 张艳霞[1] 毕智敏 封宝红 

机构地区：[1]武汉大学附属同仁医院武汉市第三医院肾内科,武汉430068

出　　处：《北京大学学报（医学版）》2017年第6期954-960,共7页Journal of Peking University:Health Sciences

基　　金：国家自然科学基金(81200555);湖北省自然科学基金(2016CFB590);武汉市卫计委科研项目(WX16B09)资助~~

摘　　要：目的:观察高糖腹膜透析液对人腹膜间皮细胞NLRP3-IL-1β的影响。方法:体外培养人腹膜间皮细胞株第5~10代[HMr SV5,DMEM/F12培养基含10%(体积分数)胎牛血清],观察1.5%、2.5%、4.25%(质量分数)葡萄糖腹膜透析液(dextrose)及线粒体呼吸链复合酶Ⅰ抑制剂鱼藤酮(rotenone),线粒体呼吸链复合酶Ⅱ抑制剂噻吩甲酰三氟丙酮(thenoyltrifluoroacetone,TTFA),线粒体呼吸链复合酶Ⅲ抑制剂抗霉素A(antimycin A)对细胞IL-1β表达的影响(免疫印迹);通过小RNA干扰技术下调NLRP3的表达,免疫印迹分析4.25%高糖腹膜透析液作用下细胞IL-1β的表达;通过白藜芦醇(resveratrol,RSV)诱导自噬,3-甲基腺嘌呤(3-methyladenine,3-MA)、自噬相关蛋白5(autophagy related gene 5,ATG5)siRNA、自噬相关蛋白(Beclin1)siRNA抑制自噬,流式细胞仪分析细胞活性氧(reactive oxygen species,ROS),免疫印迹分析IL-1β的表达。结果:对照组、1.5%高糖腹膜透析液、2.5%高糖腹膜透析液、4.25%高糖腹膜透析液、鱼藤酮(10μmol/L)、抗霉素A(50 mg/L)、噻吩甲酰三氟丙酮(10μmol/L)各组细胞上清IL-1β的相对表达依次为0、0.175±0.082、0.418±0.163、2.357±0.288、2.642±0.358、3.271±0.462、0.123±0.091,提示2.5%高糖腹膜透析液、4.25%高糖腹膜透析液、鱼藤酮、抗霉素A作用细胞IL-1β表达明显升高,应用NLRP3 siRNA可阻断上述效应;此外,对照组、阴性对照siRNA(negative control,NC siRNA)、RSV(50μmol/L,诱导自噬)、3-MA(10 mmol/L,抑制自噬)、ATG5 siRNA(抑制自噬)、Beclin1 siRNA(抑制自噬)各组总线粒体荧光强度分别为1.76±0.42、1.83±0.55、1.85±0.62、7.36±0.92、5.35±0.77、5.06±0.62,超氧化线粒体荧光强度分别为821.68±95.12、868.15±102.82、723.39±92.56、1 660.08±113.65、1 433.01±107.24、1 562.36±112.88,结合免疫印迹结果,提示抑制自噬可增强线粒体ROS产生和提高IL-1β表达,诱导自噬对ROS、IL-1β无明显影响。结论:长期应用高糖腹膜透析液,Objective： To explore the effect of high glucose-based peritoneal dialysis fluids on NLRP3-IL-1β in human peritoneal mesothelial cells. Methods： HMr SV5 cells（ SV40 immortalized human peritoneal mesothelial cell line） were grown in type Ⅰ collagen-coated dishes in DMEM/F12 containing 10%fetal calf serum（ FCS）. All experiments on HMr SV5 cells were performed between passages 5 and 10.The cells were divided into 7 groups： control,1. 5% dextrose,2. 5% dextrose,4. 25% dextrose,rotenone,thenoyltrifluoroacetone（ TTFA）,and antimycin A. Immunoblotting was used to evaluate the expression of IL-1β. Small interfering RNA（ siRNA） targeting NLRP3 was used to downregulate the expression of NLRP3 and Western blot was used to evaluate the expression of IL-1β in human peritoneal mesothelial cells exposed to 4. 25% dextrose. In the meanwhile,resveratrol（ RSV） was used to induce autophagy,3-methyladenine（ 3-MA） and siRNA against Beclin 1 or ATG5 were used to block autophagy,flow cytometric was used to analyze the respiring（ mitotracker deep red）,total（ mitotracker green） and reactive oxygen species（ ROS）-generating mitochondria（ mito SOX）; Western blot was used to evaluate the expression of IL-1β. Results： The IL-1β relative expressions were 0,0. 175 ± 0. 082,0. 418 ±0. 163,2. 357 ± 0. 288,2. 642 ± 0. 358,3. 271 ± 0. 462,and 0. 123 ± 0. 091,indicating that the cells exposed to high glucose-based peritoneal dialysis fluids and cells treated with mitochondria respiratory chain key enzyme complex Ⅰ,and complex Ⅲ inhibitors increased the IL-1β expression. And we found that NLRP3 knock-down significantly blocked the upregulation of IL-1β. In addition,the fluorescence intensity of total mitochondria and ROS-generating mitochondria in the following groups： control,negative control,RSV,3-MA,ATG5 siRNA,Beclin1 siRNA were 1. 76 ± 0. 42,1. 83 ± 0. 55,1. 85 ± 0. 62,7. 36 ± 0. 92,5. 35 ± 0. 77,5. 06 ± 0. 62 and 821. 68 ± 95. 12,868. 15 ± 102. 82,723. 39 ± 92. 56,1 660

关 键 词：高糖腹膜透析液 人腹膜间皮细胞 NLRP3 IL-1Β 

分 类 号：R692[医药卫生—泌尿科学]