Sirt1对高糖诱导的足细胞外泌体释放的影响  

Effect of Sirt1 on high glucose-induced exosome release from podocytes

作　　者：丁琳[1] 周燕[1] 刘珊珊[1] 刘南池 马瑞霞[1] DING Lin;ZHOU Yan;LIU Shanshan;LIU Nanchi;MA Ruixia(Department of Nephrology,The Affiliated Hospital of Qingdao Univesity,Qingdao 266003,China)

机构地区：[1]青岛大学附属医院肾病科,山东青岛266003

出　　处：《精准医学杂志》2024年第1期1-4,10,共5页Journal of Precision Medicine

基　　金：山东省自然科学基金项目(ZR2022MH161);青岛市医药卫生科研计划项目(2021-WJZD189);青岛市民生科技计划项目(16-6-2-20-snh);青岛大学附属医院临床医学+X项目(3390);青岛市医疗卫生重点学科建设项目。

摘　　要：目的探讨烟酰胺腺嘌呤二核苷酸依赖的去乙酰化酶1(Sirt1)对高糖诱导的足细胞外泌体释放的影响。方法将永生化小鼠足细胞MPC5分为正常糖组(5.5 mmol/L葡萄糖,A组)、高渗组(5.5 mmol/L葡萄糖+24.5 mmol/L甘露醇,B组)、高糖组(30.0 mmol/L葡萄糖,C组)、高糖+Sirt1过表达慢病毒转染组(Sirt1过表达慢病毒转染+30.0 mmol/L葡萄糖,D组)、高糖+阴性慢病毒转染组(阴性慢病毒转染+30.0 mmol/L葡萄糖,E组)、高糖+外泌体分泌抑制剂组(GW4869+30.0 mmol/L葡萄糖,F组)6组。采用免疫印迹法检测各组细胞Sirt1、足细胞裂孔膜蛋白(Nephrin、Podocin)及CD63、CD81、Alix的表达水平,采用实时荧光定量PCR(RT-qPCR)检测D、E组细胞Sirt 1 mRNA表达水平,使用透射电子显微镜观察足细胞外泌体形态,采用纳米粒子跟踪分析技术检测外泌体的粒径和浓度。结果RT-qPCR结果显示,D组足细胞Sirt1 mRNA相对表达量显著高于E组(t=14.580,P<0.01)。纳米粒子跟踪分析及免疫印迹结果显示,A~C组间足细胞Sirt1、Nephrin和Podocin蛋白相对表达量比较差异均具有显著性(F=49.84~106.40,P<0.01);与A组相比,C组足细胞外泌体分泌显著增加(t=14.550,P<0.01),Nephrin、Podocin、Sirt1相对表达量显著减少(t=7.446~15.110,P<0.01);与E组相比,D组足细胞外泌体分泌显著减少(t=74.610,P<0.01),Nephrin、Podocin、Sirt1相对表达量显著增加(t=4.657~32.860,P<0.05);与C组相比,F组足细胞外泌体分泌显著减少(t=16.300,P<0.05),Nephrin、Podocin相对表达量显著增加(t=3.790、8.151,P<0.01),Sirt1表达水平无统计学差异(P>0.05)。结论高糖诱导的足细胞Sirt1减少可促进外泌体分泌及足细胞损伤。Objective To investigate the effect of nicotinamide adenine dinucleotide-dependent deacetylase 1(Sirt1)on high glucose-induced exosome release from podocytes.Methods Immortalized mouse podocytes MPC5 were divided into six groups:normal glucose group(5.5 mmol/L glucose,group A),high mannitol group(5.5 mmol/L glucose+24.5 mmol/L mannitol,group B),high glucose group(30.0 mmol/L glucose,group C),high glucose+Sirt1-overexpressed lentivirus transfection group(Sirt1-overexpressed lentivirus transfection+30.0 mmol/L glucose,group D),high glucose+negative lentivirus transfection group(negative lentivirus transfection+30.0 mmol/L glucose,group E),and high glucose+exosome secretion inhibitor group(GW4869+30.0 mmol/L glucose,group F).Western blot was used to analyze the expression levels of Nephrin,Podocin,Sirt1,CD63,CD81,and Alix in each group.Real-time quantitative polymerase chain reaction was used to analyze the expression level of Sirt1 mRNA in D and E group.The morphology of podocyte exosomes was observed by a transmission electron microscope.The particle size and concentration of exosomes were determined by nanoparticle tracking analysis.Results The results of RT-qPCR showed that the relative expression of Sirt1 mRNA was significantly increased in group D compared with that in group E(t=14.580,P<0.01).The results of nanoparticle tracking analysis and Western blot showed that the relative expression of Sirt1,Nephrin,and Podocin proteins in podocytes among groups A to C was significantly different(F=49.84-106.40,P<0.01).Compared with group A,group C had significantly increased secretion of podocyte exosomes(t=14.550,P<0.01)and significantly reduced expression of Sirt1,Nephrin,and Podocin(t=7.446-15.110,P<0.01).Compared with group E,group D had significantly reduced release of podocyte exosomes(t=74.610,P<0.01)and significantly increased relative expression of Sirt1,Nephrin,and Podocin(t=4.657-32.860,P<0.05).Compared with group C,group F had significantly reduced release of podocyte exosomes(t=16.300,P<0.05)and significa

关 键 词：糖尿病肾病 血糖 足细胞 外泌体 抗衰老酶1 

分 类 号：R587.24[医药卫生—内分泌]