罗沙司他对高糖诱导的小鼠肾小球系膜细胞纤维化的影响  

Effect of roxadustat on high glucose-induced fibrosis of mouse mesangial cells

作　　者：闫桐 马东红[1] 师晶晶 秦吉林 周慧娜 李雨静 郭明好[1] 贾新燕 YAN Tong;MA Donghong;SHI Jingjing;QIN Jilin;ZHOU Huina;LI Yujing;GUO Minghao;JIA Xinyan(Nephrology Hospital,the First Affiliated Hospital of Xinxiang Medical University,Weihui,Henan 453100,China)

机构地区：[1]新乡医学院第一附属医院肾脏病医院,河南卫辉453100

出　　处：《中华实用诊断与治疗杂志》2024年第10期992-996,共5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(LHGJ20200494,LHGJ20230517);河南省科技攻关计划项目(212102310779)。

摘　　要：目的观察高糖诱导的小鼠肾小球系膜细胞(SV40 MES13)低氧诱导因子-1α(HIF-1α)表达及转化生长因子-β(TGF-β)/Smads信号通路变化,探讨罗沙司他对高糖诱导的SV40 MES13细胞增殖和纤维化的影响及可能机制。方法对数生长期SV40 MES13细胞饥饿化处理24 h后采用含30 mmol/L葡萄糖的高糖完全培养基及0、1、2、3、4、5μmol/L罗沙司他溶液培养24 h,采用CCK-8法检测细胞存活率,筛选最佳罗沙司他浓度进行后续实验。对数生长期SV40 MES13细胞分为对照组(采用含5.5 mmol/L葡萄糖的低糖完全培养基培养)、高糖组(采用含30 mmol/L葡萄糖的高糖完全培养基培养)、罗沙司他组(采用含30 mmol/L葡萄糖的高糖完全培养基培养+罗沙司他1μmol/L处理),培养24 h,采用ELISA法检测细胞上清液Ⅳ型胶原(ColⅣ)、纤维连接蛋白(FN)水平,采用Western blot法检测细胞TGF-β、Smad3、p-Smad3、Smad7蛋白相对表达量,采用实时荧光定量PCR法检测细胞HIF-1αmRNA相对表达量。结果0、1、2、3、4μmol/L罗沙司他处理24 h时细胞存活率[(107.93±3.24)%、(92.44±1.20)%、(75.55±5.98)%、(40.96±3.32)%、(17.32±1.95)%]依次降低(P<0.05);5μmol/L罗沙司他处理时细胞存活率[(21.39±2.70)%]与4μmol/L处理时比较差异无统计学意义(P>0.05);1μmol/L罗沙司他处理时细胞存活率>90%,采用该浓度进行后续实验。3组细胞上清液ColⅣ、FN水平比较差异均有统计学意义(F=6.061,P=0.036;F=4.918,P=0.044);高糖组细胞上清液ColⅣ、FN水平[(2.72±1.08)、(7.58±1.03)μg/L]均高于罗沙司他组[(0.91±0.17)、(5.79±0.99)μg/L]、对照组[(1.37±0.35)、(6.39±0.82)μg/L](P<0.05),罗沙司他组与对照组比较差异均无统计学意义(P>0.05)。3组细胞TGF-β、Smad3、p-Smad3、Smad7蛋白及HIF-1αmRNA相对表达量比较差异均有统计学意义(F=5.934、6.883、5.722、4.709、79.110,P均<0.05);高糖组细胞TGF-β、Smad3、p-Smad3蛋白相对表达量(1.34±0.31、0.98±0.16、0.93Objective To observe the expression of hypoxia-inducible factor-1α(HIF-1α)and the change of transforming growth factor-β(TGF-β)/Smads signaling pathway of high glucose-induced mouse mesangial cells(SV40 MES13),and to investigate the influence of roxadustat on the high glucose-induced proliferation and fibrosis of SV40 MES13 cells and its potential mechanisms.Methods After 24-h starvation treatment,the SV40 MES13 cells in logarithmic growth phase were cultured for 24 h with high glucose complete medium(containing 30 mmol/L glucose)and 0,1,2,3,4 and5μmol/L roxadustat solution.CCK-8 assay was used to detect the cell survival rate,and the optimal concentration of roxadustat was screened for subsequent experiments.The SV40 MES13 cells in logarithmic growth phase were divided into control group(cultured with complete medium containing 5.5 mmol/L glucose),high glucose group(cultured with complete medium containing 30 mmol/L glucose),and roxadustat group(cultured with complete medium containing30 mmol/L glucose and 1μmol/L roxadustat solution).After 24-h culture,ELISA was used to detect the levels of collagenⅣ(ColⅣ)and fibronectin(FN)in the supernatant.Western blot was used to detect the relative expressions of TGF-β,Smad3,p-Smad3 and Smad7 proteins.Real-time fluorescence quantitative PCR was used to detect the relative expression of HIF-la mRNA.Results After 24-h treatment,the cell survival rate decreased sequentially after being treated with 0,1,2,3 and 4μmol/L roxadustat solution respectively[(107.93±3.24)%,(92.44±1.20)%,(75.55±5.98)%,(40.96±3.32)%,(17.32±1.95)%](P<0.05),but it showed no significant difference after being treated with 5μmol/L roxadustat[(21.39±2.70)%]compared with that after being treated with 4μmol/L roxadustat(P>0.05).The cell survival rate was>90%after being treated with 1μmol/L roxadustat,therefor this concentration was chosen for the subsequent experiments.The levels of ColⅣand FN in the supernatant showed significant differences among three groups(F=6.061,P=0.036;F=4.918,P

关 键 词：肾纤维化 高糖 罗沙司他 小鼠肾小球系膜细胞 低氧诱导因子-1Α TGF-β/Smads信号通路 

分 类 号：R692.9[医药卫生—泌尿科学] R587.2[医药卫生—外科学]