Effect of roxadustat on high glucose-induced fibrosis of mouse mesangial cells
Objective 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 and 5 μmol/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 containing 30 mmol/L glucose and 1 μmol/L roxadustat solution).After 24-h culture,ELISA was used to detect the levels of collagen Ⅳ(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-1α mRNA.Results After 24-h treatment,the cell survival rate decreased sequentially after being treated with 0,1,2,3 and 4 μmol/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 5 μmol/L roxadustat[(21.39±2.70)％]compared with that after being treated with 4 μmol/L roxadustat(P＞0.05).The cell survival rate was＞90％after being treated with 1 μmol/L roxadustat,therefor this concentration was chosen for the subsequent experiments.The levels of ColⅣ and FN in the supernatant showed significant differences among three groups(F=6.061,P=0.036;F=4.918,P=0.044),were higher in high glucose group[(2.72±1.08),(7.58±1.03)μg/L]than those in roxadustat group[(0.91±0.17),(5.79±0.99)μg/L]and control group[(1.37±0.35),(6.39±0.82)μg/L](P＜0.05),and showed no significant differences between roxadustat group and control group(P＞0.05).The relative expressions of TGF-β,Smad3,p-Smad3 and Smad7 proteins and HIF-1α mRNA showed significant differences among three groups(F=5.934,F=6.883,F=5.722,F=4.709,F=79.110;all P values＜0.05).The relative expressions of TGF-β,Smad3 and p-Smad3 proteins were higher in high glucose group(1.34±0.31,0.98±0.16,0.93±0.14)than those in roxadustat group(0.57±0.35,0.60±0.07,0.44±0.25)and control group(0.76±0.16,0.70±0.15,0.54±0.15)(P＜0.05),and showed no significant differences between roxadustat group and control group(P＞0.05).The relative expression of Smad7 protein was higher in roxadustat group than that in control group(P＜0.05),and showed no significant difference in high glucose group compared with that in roxadustat group and control group(P＞0.05).The relative expression of HIF-1α mRNA was lower in high glucose group(0.86±0.03)than that in roxadustat group(1.77±0.47)and control group(1.00±0.00)(P＜0.05),and higher in roxadustat group than that in control group(P＜0.05).Conclusion Roxadustat can inhibite the high glucose-induced hyperproliferation and fibrosis of SV40 MES13 cells,probably by upregulating HIF-1α and inhibiting TGF-β/Smads signaling pathway.
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