高糖干预对小鼠肾小球系膜细胞自噬和BIRC2的影响  

Impacts of high glucose intervention on autophagy in mouse glomerular mesangial cells and BIRC2

作　　者：周慧娜 李雨静 秦吉林 闫桐 郭明好[1] 赵晓露 ZHOU Huina;LI Yujing;QIN Jilin;YAN Tong;GUO Minghao;ZHAO Xiaolu(Nephrology Hospital,the First Affiliated Hospital of Xinriang Medical College,Xinriang,Henan 4531000,China)

机构地区：[1]新乡医学院第一附属医院肾脏病医院,河南新乡453100

出　　处：《中华实用诊断与治疗杂志》2025年第3期231-237,共7页Journal of Chinese Practical Diagnosis and Therapy

基　　金：河南省医学科技攻关计划联合共建项目(LHGJ20200493)。

摘　　要：目的观察高糖对小鼠肾小球系膜细胞SV40 MES13自噬及BIRC2表达的影响,探讨BIRC2在高糖干预后SV40 MES13细胞自噬中的作用及机制。方法取对数生长期SV40 MES13细胞,应用30 mmol/L葡萄糖进行干预。分别于干预0、24、48、72 h时,采用Western blot法检测BIRC2、LC3Ⅱ、LC3Ⅰ、Beclin-1、P62、Bax蛋白相对表达量,计算LC3Ⅱ/Ⅰ;采用实时荧光定量PCR法检测BIRC2 mRNA相对表达量。选择自噬水平最高的高糖干预24 h时的细胞进行后续实验。取高糖干预24 h时的细胞,分为高糖阴性对照组(正常培养)和高糖BIRC2敲低组(转染BIRC2-shRNA),取对数生长期SV40 MES13细胞为阴性对照组(正常培养)。3组细胞转染24 h,采用Western blot法检测BIRC2、LC3Ⅱ、LC3Ⅰ、Beclin-1、P62、Bax、mTOR、p-mTOR蛋白相对表达量,计算p-mTOR/mTOR;采用实时荧光定量PCR法检测BIRC2 mRNA相对表达量。结果(1)高糖干预0、24、48、72 h时,SV40 MES13细胞LC3Ⅱ/Ⅰ及BIRC2、Beclin-1、P62、Bax蛋白和BIRC2 mRNA相对表达量比较差异均有统计学意义(F=16.280~29.710,P均<0.05)。高糖干预24 h时SV40 MES13细胞LC3Ⅱ/Ⅰ(2.28±0.30)及BIRC2(1.30±0.09)、Beclin-1(1.55±0.24)蛋白和BIRC2 mRNA相对表达量(1.63±0.07)均高于高糖干预0 h(1.13±0.25、0.92±0.19、1.00±0.17、1.00±0.07)、48 h(1.14±0.24、0.66±0.12、0.99±0.13、1.10±0.02)、72 h(0.52±0.08、0.49±0.09、0.53±0.15、0.67±0.02)时(P<0.05),P62(0.48±0.08)、Bax(0.72±0.02)蛋白相对表达量均低于高糖干预0 h(0.74±0.14、0.93±0.05)、48 h(0.83±0.11、0.91±0.09)、72 h(1.13±0.03、1.12±0.09)时(P<0.05)。(2)高糖阴性对照组、高糖BIRC2敲低组、阴性对照组LC3Ⅱ/Ⅰ及p-mTOR/mTOR、BIRC2、Beclin-1、P62、Bax蛋白和BIRC2 mRNA相对表达量比较差异均有统计学意义(F=11.000~40.200,P均<0.05)。高糖阴性对照组LC3Ⅱ/Ⅰ(2.81±0.44)及Beclin-1(1.13±0.19)、BIRC2(1.31±0.15)蛋白和BIRC2 mRNA(1.84±0.24)相对表达量均高于阴性对照组(1.38±0.26、0.7Objective To observe the impacts of high glucose on the autophagy of mouse glomerular mesangial cells SV40 MES13 and BIRC2 expression,and to investigate the role and mechanism of BIRC2 in SV40 MES13 autophagy after high glucose intervention.Methods SV40 MES13 cells in logarithmic growth phase were treated with 30 mmol/L glucose.At 0,24,48 and 72 h after intervention,Western blot was performed to detect the relative expressions of BIRC2,LC3Ⅱ,LC3Ⅰ,Beclin-1,P62 and Bax proteins,and the LC3Ⅱ/Ⅰratio was calculated.Real-time fluorescence quantitative PCR was employed to determine the relative expression of BIRC2 mRNA.Cells with the highest autophagy level at 24-h high glucose intervention were selected for subsequent experiments.The 24-h high glucose-treated cells were divided into the high glucose negative control group(cultured under normal conditions)and the high glucose BIRC2 knockdown group(transfected with BIRC2-shRNA).Log-phase SV40 MES13 cells served as the negative control group(cultured under normal conditions).After 24-h transfection,Western blot analysis was conducted to determine the relative expressions of BIRC2,LC3Ⅱ,LC3Ⅰ,Beclin-1,P62,Bax,mTOR and p-mTOR proteins,with calculation of the p-mTOR/mTOR ratio.The relative expression of BIRC2 mRNA was detected with real-time fluorescence quantitative PCR.Results(1)Significant differences were observed in the LC3Ⅱ/Ⅰratio and the relative expressions of BIRC2,Beclin-1,P62 and Bax proteins as well as BIRC2 mRNA in SV40 MES13 cells across 0,24,48 and 72 h of high glucose intervention(F=16.280-29.710,all P values<0.05).At 24 h of high glucose intervention,the LC3Ⅱ/Ⅰratio(2.28±0.30)and the relative expressions of BIRC2 protein(1.30±0.09),Beclin-1 protein(1.55±0.24),and BIRC2 mRNA(1.63±0.07)in SV40 MES13 cells were significantly higher than those at 0 h(1.13±0.25,0.92±0.19,1.00±0.17,1.00±0.07),48 h(1.14±0.24,0.66±0.12,0.99±0.13,1.10±0.02),and 72 h(0.52±0.08,0.49±0.09,0.53±0.15,0.67±0.02)(P<0.05),while the relative expressions of P62

关 键 词：糖尿病肾病 BIRC2 自噬 小鼠 肾小球系膜细胞SV40 MES13 

分 类 号：R587.2[医药卫生—内分泌]