| Objective:To investigate the effect of Vitamin D(VD)on interstitial transformation of alveolar Ⅱ type epithelial cells(RLE-6TN)induced by TGF-β1 in rats.Methods:Cultured RLE-6TN cells in vitro,divided into normal control group(A),TGF-β1 group(B),TGF-β1+VD group(C),TGF-β1+ICG001 group(D),and TGF-β1+VD +ICG001 group(E).The doses were as follows: TGF-β1 10 ng/ml,vitamin D 1 μM,ICG001 5 μM.The cells were pre-treated with VD,ICGOOl or VD+ICGOOl,and TGF-β1 was added to each group after culturing for 1 day.Then 2days later,the morphological changes of the cells in each group were observed and recorded with microscope.Western blotting was used to detect the expression levels of E-cadherin,α-smooth muscle actin(α-SMA)and fibronectin(Fn).Image J software was used to analyze the gray level of each group of protein bands.Real-time PCR was used to determine the relative expression of Snail in each group.Results:Under microscope,the RLE-6TN cells in normal group showed typical paving stone-like changes,and the cells were closely connected.In group B,the cell morphology was obviously fusiform and the intercellular space increased significantly.Compared with group B,cells in group C and D showed obvious paving stone-like changes,and the cell-cell gap was reduced.The cells in group E were mostly paved stone-like changes with smallest gap and the morphology was similar to the normalgroup.Western blotting analysis showed,Compared with normal group α-SMA(0.27±o.04),Fn(0.36±o.02)and E-cadherin(0.93±o.07),the expression levels of group B α-SMA(0.95±o.05)and Fn(0.82±o.03)increased significantly,E-cadherin(0.32± o.08)is significantly reduced(p<0.05).The expression levels of α-SMA(0.81 ±O.02、0.80±O.04、0.69±O.06)and Fn(0.62±O.05、0.65±O.03、0.46±O.08)in C,D and E groups were lower than those in B group(higher than normal group),while E-cadherin(0.55±O.03、0.57±O.06、0.83±O.08)expression increased(lower than normal group)(P<0.05).Compared with group C and group D,the expression ofα-SMA and Fn decreased in group E,but the expression of E-cadherin increased(P<0.05).There were no significant difference in the expression levels of E-cadherin,α-SMA and Fn between group C and group D(P>0.05).Real-time PCR results display,the expression of Snail in group B(3.73 + O.08),C(2.66 + 0.07),D(2.68 +0.06),E(1.72 + 0.04)was higher than that in group A(1.00 ± 0.00).Snail was down-regulated in group C,D,E compared with group B,and Snail expression was also decreased in group E compared with group C and D(P<0.05).But there were no statistically significant difference between group C and group D(P>0.05).Conclusions:1、TGF-β1 can induce EMT in rat alveolar type Ⅱ epithelial cells.β-catenin is an important signaling molecule mediated by TGF-β1 in EMT pathway.2 、 VD can inhibit TGF-β1-induced alveolar type Ⅱ epithelial cells mesenchymal transition in rats.3 、 VD+ICGOO1 can more strongly inhibit the interstitial transformation of rat alveolar Ⅱ type epithelial cells stimulated by TGF-β1.4 、 VD inhibits TGF-β1-induced interstitial transformation of RLE-6TN.The mechanism may be that VD negatively regulates the Wnt/β-catenin signaling pathway and exerts a synergistic effect with ICG001,which plays an anti-fibrotic effect. |
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