Mechanism Of Vitamin D Regulating Epithelial-mesenchymal Transition

Objective:To observe the effect of vitamin D on the mesenchymal transformation of alveolar epithelial cells induced by TGF-β1 in rats,and to explore the mechanism of vitamin D in the mesenchymal transformation of alveolar epithelial cells induced by TGF-β1in rats.Methods:Rat type II alveolar epithelial cells（RLE-6TN）were cultured in vitro,and were divided into the control group,the TGF-β1 group,the TGF-β1+1α,25（OH）₂D₃group,the TGF-β1+ICG001 group,the TGF-β1+1α,25（OH）₂D₃+ICG001 group.The cells were pretreated with 1α,25（OH）₂D₃,ICG001 or both,followed by stimulation with TGF-β1.The cell morphology was observed by inverted microscope after 48 h.Immunofluorescence staining was performed to detect the protein expression of E-cadherin,α-SMA,Fn andβ-catenin.Topflash/Fopflash dual-luciferase reporter system was performed to investigate the transcriptional activity ofβ-catenin.The MMP-9 activity was assessed by gelatin zymography.Results:Under an inverted microscope,the alveolar epithelial cell morphology showed paving stone-like in the control group,and the cells were tightly connected.The most cell morphology changed into spindle-like after TGF-β1 intervention,and the cell gaps increased significantly.The most cell morphology changed into paving stone-like after 1α,25（OH）₂D₃ or ICG001 intervention,and the cells gap decreased.Thecell morphology was similar to the control group by simultaneous treatmentwith1α,25（OH）₂D₃and ICG001.The immunofluorescence results showed theexpression levels ofα-SMA,Fn andβ-catenin were increased（P<0.05）,theexpression of E-cadherin was decreased after TGF-β1 intervention（P<0.05）,Following 1α,25（OH）₂D₃ or ICG001treatment,the expression levels ofα-SMA,Fnandβ-catenin were reduced（P<0.05）,while the expression of E-cadherin wasraised（P<0.05）.The expression levels ofα-SMA,Fn andβ-catenin were significantlyreduced（P<0.05）,while the expression of E-cadherin was markly increased after thecombination treatment with 1α,25（OH）₂D₃ and ICG001（P<0.05）.Topflash/Fopflashdual-luciferase reporter system indicated that the luciferase activity was up-regulatedafter TGF-β1 intervention（P<0.05）,the luciferase activity was down-regulated afteradministration of 1α,25（OH）₂D₃ or ICG001（P<0.05）,the luciferase activity wassignificantly reduced after the combination treatment with 1α,25（OH）₂D₃ andICG001（P<0.05）.The results of gelatin zymography showed the MMP-9 activity wasraised after TGF-β1 intervention（P<0.05）,the MMP-9 activity was reduced afteradministration of 1α,25（OH）₂D₃ or ICG001（P<0.05）,the MMP-9 activity wassignificantly reduced after the combination treatment with 1α,25（OH）₂D₃ andICG001（P<0.05）.Conclusion:1、1α,25（OH）₂D₃ inhibited TGF-β1-induced Epithelial Mesenchymal Transition of alveolar epithelial cells,which may be related to regulating theβ-catenin signaling pathway.2、1α,25（OH）₂D₃ or ICG-001 inhibited TGF-β1-induced EMT,and 1α,25（OH）₂D₃ has a synergistic effect with ICG001.