| Objectives To investigate the effect and mechanism of conditioned medium(CM)obtained from miR-203a-3p transfected adipose stem cells(ASCs)on epithelialmesenchymal transitions(EMT)of idiopathic pulmonary fibrosis(IPF)in mice.Methods Mi R-203a-3p mimics were transfected into human adipose stem cells(h ASCs).The morphology of adipose stem cells(ASCs),which is before and after transfection,was observed by an inverted phase-contrast microscope.ASCs were incubated in serum-free media(SFM)for three days and centrifuged from culture supernatant to obtain the stem cells’ secretome(Sec).The IPF model in mice,4～6week-old C57BL/6J,was made by endotracheal infusion of bleomycin(BLM)soluble in normal saline at 5mg/kg.The control group was given the same amount of normal saline infusion.On the 14 d of model,the secretome released by ASCs(called the original secretome)and the secretome released by ASCs transfected with miR-203a-3p mimics(called the miR-203a-3p secretome)were treated with endotracheal infusion assisted by laryngoscopy,one time/d,for consecutive 7days,and euthanized on the 21 d model,Lung tissue was collected.To observe the pathological changes of lung tissue and evaluate the indexes of pulmonary fibrosis.MLE-12 cells were stimulated with 5μg/L transforming growth factor beta 1(TGF-β1)for 24 hours,and then added into the original secretome and the miR-203a-3p secretome coculture for 36 hours.The growth rate of MLE-12 cells was measured by the MTS method.The pathological changes and collagen distribution of lung tissues were assessed by hematoxylin-eosin staining and Masson trichromatic staining.The m RNA levels of miR-203a-3p in mouse lung tissues and MLE-12 cells were detected by quantitative reverse transcription PCR(q RT-PCR).The protein expression levels of IL-6,TGF-β,Collagen I,E-Cadherin,Vimentin,α-SMA and and the phosphorylated PI3K/Akt/m TOR(PAM)signaling pathway were detected by western blot.EMT marker proteins(E-Cadherin,Vimentin,α-SMA)in lung tissues and MLE-12 cells were detected by immunohistochemistry and immunofluorescence staining.Results 1 Compared with the control group,lung tissue of mice in model groups showed prominent inflammatory infiltration and increased collagen deposition,and protein levels of TGF-β and Collagen I increased in a time-dependent manner in the 7d,14 d,and 21 d in model groups(P<0.05).The protein level of Vimentin increased significantly in model groups;it peaked on the 14 th day of model(P<0.01),while the protein level of E-Cadherin decreased in a time-dependent manner in model groups(P<0.05).2 Compared with the control group,the m RNA levels of miR-203a-3p in lung tissues of mice induced by BLM for 21 days and MLE-12 cells stimulated by TGF-β1 for 24 hours were decreased(P<0.05).3 Compared with the control group,the m RNA level of miR-203a-3p was increased after miR-203a-3p mimics transfected ASCs 48 hours(P<0.05),and higher than that of transfected 24 hours and 36 hours.The cell morphology of ASCs transfected with miR-203a-3p was no change under an inverted phase-contrast microscope.Compared with the model group,inflammatory infiltration,collagen deposition,TGF-β,Collagen Ⅰ,and Vimentin levels decreased after treatment with the secretome of stem cells(P<0.05),while E-Cadherin levels were increased(P<0.01);compared with the Sec group(the original secretome),the levels of TGF-β,Collagen Ⅰ,and Vimentin in the t-Sec group(the miR-203a-3p secretome)were decreased(P<0.05),while the level of E-Cadherin were increased(P>0.05).Immunohistochemical staining showed that E-Cadherin protein staining was increased and α-SMA protein staining was decreased after treatment with the secretome of stem cells compared with the 21 d model group;compared with the Sec,ECadherin protein staining was increased,and α-SMA protein brown staining was decreased in the t-Sec group.4 Compared with the control group,the proliferation of MLE-12 cells was promoted in the secretome of stem cells;compared with the TGF-β1 induced group,the protein levels of IL-6,TGF-β,Collagen I,Vimentin,and α-SMA in cells were decreased after treatment with the secretome of stem cells(P<0.05),while the protein levels of E-Cadherin were increased(P<0.01).compared with the Sec group,the protein levels of IL-6,TGF-β,and Vimentin in the t-Sec group were decreased(P<0.05),while the protein levels of E-Cadherin were increased(P<0.05).5 Compared with the control group,the protein levels of p-PI3 K,p-Akt and,p-m TOR in MLE-12 cells induced by TGF-β1 and IPF mouse model induced by BLM were significantly increased(P<0.01);compared with the model group,the protein levels of p-PI3 K,p-Akt and p-m TOR in lung tissues and cells after treatment with the secretome of stem cells were decreased(P<0.05).Conclusions 1 The secretome of stem cells had the characteristics of inhibiting the degree of pulmonary fibrosis and epithelial-mesenchymal transformation of idiopathic pulmonary fibrosis in mice.2 Transfection of miR-203a-3p into ASCs can increase the anti-idiopathic pulmonary fibrosis and anti-EMT ability of the secretome of stem cells.3Conditioned medium after miR-203a-3p transfected adipose stem cells may inhibit the degree of pulmonary epithelial mesenchymal transformation and fibrosis in mice by reducing the activity of PI3K/Akt/m TOR signaling pathway.Figure18;Table5;Reference 197... |
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