In Vitro And Vivo Study About The Effect Of Vitamin D On Pulmonary Fibrosis

Objective:Investigate the effects and molecular mechanism of Vitamin D on pulmonary fibrosis, and give some suggestions in the treatment and prevention of lung fibrosis for the future.Methods:Our experiment objective to investigate the effect of Vitamin D on pulmonary fibrosis in vivo and in vitro. There are two aspects in vitro study. At first, we used TGF-β stimulated cells to induce epithelial-mesenchymal transition(EMT) establish cell fibrosis model and check the model by morphological and western blot. Second, we investigated the influence of 1,25-(OH)2D3 on TGF-β induced EMT. As the result of part one, TGF-β dose of 5 ng/ml and 10 ng/ml can induced A549 cells and BEAS-2B cells EMT. Vitamin D dose of 10 n M and 50 n M have been used by CCK-8 and preliminary results. Our experiment have 4 groups: control, TGF-β, VD and TGF-β + VD group. We used wound healing to check ability of migration and cell invasion by Transwell analysis. Apoptosis was detected by flow cytometry and the location and expression of E-cad, Vimentin, VDR were detected by Immunofluoresecence(IF) and Western blot. The expression of N-cad, Snail, Slug, β-catenin were detected by Western blot. The m RNA expression of Collagen I and Fibronectin were determined by RT-PCR. In vivo experiment, we injected with a single dose of paraquat(PQ) intraperitoneal(ip.) in mice to create the model of pulmonary fibrosis. The mice were injected 1000 IU/w Vitamin D simultaneously to observed the vitamin D effect on PQ induced pulmonary fibrosis. HE stain was been used to inspect the degree of pulmonary fibrosis. Hydroxyproline content in lung tissue was detected by Hydroxyproline assay kit. Use western blot analyzed the expression of E-cad, Vimentin, β-catenin, Snail, Slug, VDR, TGF-β and MCP-1.Result:(1)5 ng/ml TGF-β can induce morphological changes in A549 cells and 10 ng/ml TGF-β can induce morphological changes in BEAS-2B cells. However, 10 ng/ml TGF-β can’t induce morphological changes in HBE cells. The expression of E-cad was significantly reduced and expression of N-cad, Vimentin, Snail, β-catenin were increased after TGF-β stimulated. According to the result of morphological changes and western blot, we have successfully established cell fibrosis model though TGF-β inducing EMT in A549 cells and BEAS-2B cells.(2)Processing A549 cells with 5ng/ml TGF-β, morphological changes from polygonal to spindle-shaped and lose cells contact were observed. The morphological changes was time dependent. Wound healing analysis showed that TGF-β can increase the migration activity of A549 cells and Vitamin D can reduce this effect. Transwells experiment indicated that TGF-β can increase cells metastasis in A549 cells. However, vitamin D can decrease this effect. The results of IF and western blot indicated that TGF-β can inhibit E-cad expression and increase Vimentin, β-catenin and Snail expression. Vitamin D can increase expression of E-cad and VDR, inhibited Snail expression. But, it has no influence on Vimentin, N-cad and β-catenin expression. However, compared with TGF-β, treatment with TGF-β and vitamin D at same time can increase E-cad expression, decrease Vimentin, N-cad, β-catenin, Snail expression. So, we hypothesized Vitamin D may through inhibited the expression of Snail and β-catenin to increase expression of E-cad, decrease N-cad and Vimentin expression.(3) Treatement with 10 ng/ml TGF-β induced morphological changes from polygonal to spindle-shaped and lose cells contact in BEAS-2B cells. The times longer, and the morphological changes more obvious. But, the morphological changes was lighten when stimulated with TGF-β and Vitamin D. Comparing with TGF-β, treated with TGF-β and Vitamin D can inhibit expression of β-catenin and Snail. But, the expression of Vimentin and E-cad were inconspicuous. However, Vitamin D can increase E-cad expression and inhibit Snail expression.(4) Single injection with one dose of PQ ip. can successfully established the model of pulmonary fibrosis in mice. Our results showed that 10 mg/kg PQ had not obvious lung injury, 20 mg/kg PQ can induce interstitial edema and congestion, inflammatory cells infiltration, 30 mg/kg can induce interstitial edema and congestion, inflammatory cells infiltration, alveolar hemorrhage, fibroblast proliferation, increased collagen deposition and lower mortality. 40 mg/kg can induce interstitial edema and congestion, inflammatory cells infiltration, alveolar hemorrhage, fibroblast proliferation, increased collagen deposition. And death after PQ injected. So, we selected a single dose of 30 mg/kg PQ ip. to establish a model of pulmonary fibrosis.(5) Establish a model of pulmonary fibrosis use single dose of 30 mg/kg PQ ip. in mice. Vitamin D was injected im. with dose of 1000 IU per week before and after beginning PQ injection. Our data showed that Vitamin D can attenuate PQ induced Hydroxyproline increased as same as HE staining. The result showed Vitamin D can attenuated PQ induced pulmonary fibrosis.Conclusion:(1) TGF-β can induced EMT in A549 cells and BEAS-2B cells, which successfully establishes cell fibrosis model, TGF-β can’t induced EMT in HBE cells.(2) 1,25-(OH)2D3 can inhibit TGF-β induced EMT in A549 cells and BEAS-2B cells. The molecular mechanism probably is that in EMT process 1,25-(OH)2D3 inhibits the expression of Snail and β-catenin, decreases Vimentin and N-cad expression, increases E-cad expression.(3) We have successfully established pulmonary fibrosis model in mice through single dose of 30 mg/kg PQ ip. The lung injury was obvious but mortality is low.(4) Vitamin D can reduce PQ induced pulmonary fibrosis.