TRB3 Expression Affects EMT Of Mouse Type II Alveolar Epithelial Cells Stimulated By TGF-β Via MAPK/ERK-dependent Signaling Pathway

Objective:By observing the relationship between Tribbles homologous protein 3(TRB3) over expression or expression cut with mitogen-activated protein kinases/ extracellular-signal regulated protein kinase(MAPK/ERK) signaling pathway in adenovirus transfection mice II type lung epithelial cells MLE-12 epithelial mesenchymal transition(EMT), discussed the relationship between TRB3 and MAPK/ERK in pulmonary mutual effect.Methods:1. grouping,Blank control group(group C), Green fluorescent protein(GFP) gene tracer Ad-GFP infection group(group G),Ad-TRB3 infection group(group TRB3),Ad-TRB3-sh RNA infection group(group TRB3-sh RNA), Simple TGF-β1 irritate group(group T),TGF-β1+Ad-GFP group(group T+GFP),TGF-β1+Ad-TRB3 infection group(group T+TRB3),TGF-β1+Ad-TRB3-sh RNA infection group(group T+TRB3-sh RNA);2.The Ad-GTP,Ad-TRB3,Ad-TRB3-sh RNA carrier construction,authenticate andamplification;3.Infection of in different complex exponential phase of MLE-12,by fluorescence microscope and flow cytometry techniques to understand the besteficency of infection;4.Use CCK8 method to synthesis each cell DNA;5.FACS method testing each cell apoptosis;6.q PCR detection each cell m RNA expression,such as TRB3, Collagen I(Col1a I), Col1 a III, a smooth muscle actin(a-SMA)(48h for testing);7.Westernblot method detection each cell protein expression,such as TRB3,p-ERK1/2, ERK1/2,p-p38 MAPK, p38 MAPK, E-Cadherin,Vimentin, Fibrosis connection protein(Fibronectin),a-SMA(48h for testing);8. Use ELISA method to detect the content of each cell culture supernatant,such as Colla I,Colla III, interferon-gamma(IFN-r), tumor necrosis factor-a(TNF-a)(48h for testing);9. Spss17.0 statistical software for data analysis to the result of the experiment.Results:The TGF-β1 stimulate II type alveolar epithelial cells in mice MLE-12, the expression of TRB3 increase.TRB3 expression level, promote the MLE-12 Col1 a I, Col1 a III,and a-SMA m RNA expression, at the same time the p-ERK1/2, ERK1/2, p-p38 MAPK, p38 MAPK, Vimentin and Fibronectin, TNF-a protein expression increased, E-Cadherin, IFN-r protein expression decreased, promote cell apoptosis.When cut TRB3 expression, MLE-12 E-Cadherin, IFN-r protein expression increased, Col1 a I, Col1 a III cut, and aSMA m RNA expression and p-ERK1/2, ERK1/2, p-p38 MAPK, p38 MAPK, Vimentin and Fibronectin, TNF-a protein expression decreased, cell apoptosis at a slower pace.Conclusion: 1. TGF-β1 promote MLE-12 cells MET, lead to the occurrence of pulmonary fibrosis.2. TRB3 overexpression may through the MAPK/ERK signaling pathway to promote the MLE- 12 that through the TGF-β1 irritatived, increase the extracellular matrix(ECM) deposit, promote the pulmonary fibrosis.3. TRB3 expression cut may through the MAPK/ERK signaling pathway to inhibite the MLE- 12 that through the TGF-β1 irritatived, reduce the ECM deposit, inhibitation the pulmonary fibrosis.