Effects Of TRB3 Expression On Idiopathic Pulmonary Fibrosis By MAPK Signaling Pathway

Objective 1.To clarify the role of TRB3 in the C57BL/6 mice.2.To clarify the role of exogenous regulation of TRB3 on C57BL/6 pulmonary fibrosis mice induced by bleomycin(BLM)and to explore the relationship between the three in the MAPK signaling pathway and to presume the possible regulatory mechanism in MAPK signal pathway.Methods 1.Experimental groups: animal experimental groups:(1)the control group and saline(control group).(2)fibrosis group and saline(F group).(3)fibrosis+GFP group(F+GFP group).(4)fibrosis+TRB3 overexpression group(F+TRB3group).(5)fibrosis+TRB3-sh RNA interference(F+Sh-TRB3 group).2.Experimental modeling.Animal model: After constructing bleomycin(3.5mg/kg)tracheal injection molding,we can make the intravenous injection of adenovirus on the next day.Respectively in D7,D14 and D28 time points,a group of rats were put to death in order to collect lung tissue samples and BALF for the experiment;3.HE/Masson staining of lung tissue morphological observation and fibrosis standards of grading.In order to further clarify the content of collagen,we detect the lung tissue of hydroxyproline content.Bronchoalveolar lavage fluid(BALF)cells were classified and counted,and ELASA was used to detect the expression of Colla?and Colla?in BALF.Immunohistochemical method was used to identify the expression of stromal and epithelial related proteins.4.Using Western blot on the protein lever to detect the expression of TRB3,p-ERK1/2,ERK1/2,p-p38 MAPK,p38MAPK and EMT-related protein(such as E-cadherin?Fibronectin?Vimentin??-SMA);and using RT-q PCR methods to detect TRB3 and pro Colla?/?m RNA level.Results 1.Successed to construct Bleomycin induced pulmonary fibrosis model in mice.2.HE/Masson staining of lung tissue morphological observation and fibrosis standards of grading,lung tissue hydroxyproline measurement,the results showed thatlung tissue of TRB3 overexpression,the fibrosis is more serious.The down-regulation of TRB3 expression significantly reduced the degree of fibrosis.In BALF,over expression of TRB3 showed a significant increase in the number of total cells and neutrophils,but macrophages.The down-regulation of TRB3 expression has the opposite result.3.Western blot and RT-PCR methods detect the expression of TRB3,p-ERK1/2,ERK1/2,p-p38 MAPK,p38MAPK and EMT-related protein and m RNA showed that the TRB3,p-ERK1/2,ERK1/2,p-p38 MAPK,p38MAPK and EMT-related gene m RNAs and proteins expression levels were upregulated when overexpression of TRB3;While,the down-regulation of TRB3 expression has the opposite result.Conclusion 1.Overexpression of TRB3 can increase the expression of the key protein.The down-regulation of TRB3 expression has the opposite result.It is suggested that TRB3 may regulate TGF-?1 induced EMT.2.The expression of TRB3 and the key proteins in MAPK signaling pathway was up-regulated in bleomycin induced pulmonary fibrosis in mice,suggesting that TRB3 may be involved in the process of fibrosis through MAPK signaling pathway.Overexpression of TRB3 can aggravate the bleomycin induced pulmonary fibrosis,and downregulation of TRB3 can alleviate the fibrosis.It means that exogenous regulation of TRB3 can have an effect on bleomycin induced pulmonary fibrosis.