| Objective By observing the effect of 1,25-（OH）2D3 on TGFβ1-induced mesenchymal transdifferentiation of human renal tubular epithelial cells（HK-2）and the effect of wnt/β-catenin signaling pathway,to explore the possible mechanisms of 1,25-（OH）2D3delays the progression of renal fibrosis.Methods Culture of human renal tubular epithelial cells in vitro with Keratinocyte Medium. First,divide HK-2 cells into five groups,（1）normal control group;（2）TGFβ1 group（10ng/ml）;（3）TGFβ1（10 ng/ml）+10-7mol/l 1,25-（OH）2D3;（4）TGFβ1（10 ng/ml）+10-8 mol/l1,25-（OH）2D3;（5）TGFβ1（10 ng/ml）+10-9 mol/l 1,25-（OH）2D3.After 48 hours of culture,the morphology of the cells was observed under a microscope.Real-time quantitative PCR was used to detect the expression ofα-SMA mRNA and E-cadherin mRNA in each group.Western Blot was used to detect the expression ofα-SMA protein in each group;the optimal effect dose of 1,25-（OH）2D3 was screened.The migration ability of three groups of cells in normal control group,TGFβ1 group and TGFβ1+10-7mol/l 1,25-（OH）2D3 group was determined by scratch test.The second part divided the cells into five groups:（1）normal control group;（2）TGFβ1 group（10ng/ml）;（3）TGFβ1（10ng/ml）+10-7mol/l1,25-（OH）2D3group;（4）TGFβ1（10ng/ml）+10-7mol/l1,25-（OH）2D3+Licl（20 mmol/l）group;（5）TGFβ1（10 ng/ml）+Licl（20 mmol/l）group.After 48 hours of culture,Real-time quantitative PCR was used to detect the expression of E-cadherin,α-SMA andβ-catenin mRNA in each group.The expression ofα-SMA,β-catenin,P-GSK3βand GSK3βprotein was detected by Western Blot.the expression ofβ-catenin in the normal control group,TGFβ1 group and TGFβ1+10-7mol/l 1,25-（OH）2D3group was detected by immunofluorescence.Results 1.In the normal control group,HK-2 cells were cobblestone-like,and the cells were closely arranged and distributed in a sheet shape.The cells in TGFβ1 group were long fusiform or irregular,and the intercellular space was enlarged.Most cells in TGFβ1+1,25-（OH）2D3group still had epithelium.Cell characteristics,most of the cell morphology was arranged in a cobblestone paving pattern.With the increase of 1,25-（OH）2D3 concentration,the degree of cell morphology was gradually reduced compared with the TGFβ1 stimulation group.2.Compared with the normal control group,the expression of E-cadherin mRNA and the expression ofα-SMA mRNA and protein in TGFβ1 group were significantly increased（P<0.05）.Compared with TGFβ1 group,the expression of E-cadherin mRNA andα-SMA mRNA and protein decreased in the 1,25-（OH）2D3 intervention group,the difference was statistically significant（P<0.05）,and it was dose-dependent.The higher the 1,25-（OH）2D3concentration was,the more obvious the effect was.Therefore,the subsequent experiment selected 10-7mol/l 1,25-（OH）2D3 group as the intervention dose.The results of the scratch test showed that the mobility of the normal control group was（15.3±1.52）%at 48h,and the difference was statistically significant（P<0.05）compared with the mobility of the TGFβ1group（P<0.05）,while the mobility of TGFβ1+10-7mol/l1,25-（OH）2D3 group was（25.6±2.08）%,significantly lower than that of TGFβ1 group,and the difference was statistically significant（P<0.05）.3.Compared with the normal control group,the expression of P-GSK3β/GSK3βprotein andβ-catenin mRNA and protein in TGFβ1 group increased,the difference was statistically significant（P<0.05）.The expression of P-GSK3β/GSK3βprotein andβ-catenin mRNA and protein in TGFβ1+10-7mol/l 1,25-（OH）2D3 group was lower than that in TGFβ1 group,the difference was statistically significant（P<0.05）.Compared with TGFβ1+10-7mol/l1,25-（OH）2D3 group,the expression of P-GSK3β/GSK3βprotein,β-catenin andα-SMA mRNA and protein in TGFβ1+1,25-（OH）2D3+licl group were up-regulated,and the expression of E-cadherin mRNA was down-regulated,the difference was statistically significant（P<0.05）.Compared with TGFβ1 group,the expression of P-GSK3β/GSK3βprotein,β-catenin andα-SMA mRNA and protein were up-regulated in TGFβ1+Licl group,and the expression of E-cadherin mRNA was down-regulated,the difference was statistically significant（P<0.05）.The results of immunofluorescence showed thatβ-catenin was mainly expressed in the membrane and cytoplasm of HK-2 in the normal control group,and the expression ofβ-catenin in the cytoplasm and nucleus of HK-2 in the TGFβ1 group was enhanced,andβ-catenin accumulated in the nucleus.The expression ofβ-catenin in the nucleus of TGFβ1+10-7mol/l 1,25-（OH）2D3 group was significantly decreased.Conclusion 1.1,25-（OH）2D3 may inhibit HK-2 cell transdifferentiation by down-regulating the wnt/β-catenin signaling pathway,suggesting that active vitamin D3 may delay the progression of renal fibrosis. |
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