The Expression Of MiR146a In Different Lung Fibrosis Models

Objective:（1）By establishing a pulmonary fibrosis model of C57 mice using different methods,comparing the differences of each model,we may find the most ideal modeling method for pulmonary fibrosis.（2）To investigate the transcriptional levels of TRAF6,IRAK-1,miR146a and the expression of TNF-αand IL-6 in different degrees of pulmonary fibrosis models,and to explore the different lungs mediated by miR146a in TNF-αand IL-6.The role and significance of the fibrosis model,inferred that miR146a is a potential therapeutic target for pulmonary fibrosis.Method:（1）120 C57BL/6 mice were randomly divided into 6 groups:IN（tracheal control）group,IA（low dose,2mg/kg）,IB（medium dose,3.5 mg/kg）,IC（high dose,5 mg/kg）,PN（abdominal control）group and PA（35 mg/kg）group,with 20 mice in each group.In IA,IB and IC groups,different doses of BLM were injected into trachea at one time,while in IN group,normal saline was injected into abdominal cavity of PA group,35 mg/kg,twice a week for 4 weeks,and in PN group,normal saline was injected.The mice in each group were sacrificed 28 days after modeling and the lung tissues and serum were collected.（2）The changes of mental,body mass,lung coefficient,number of flexing noses,surviral rate were observed and record.（3）The pathological changes and collagen contents of lung tissue in each group were examined through H＆E stain and Masson trichrome stain.（4）TGF-β,α-SMA,PICP,PIIINP,TNF-α,IL-6 were detected by ELISA.（5）The expression of TGF-β,Col-I,Col-III andα-SMA in lung tissue was detected by immunohistochemistry.（6）Col I m RNA,Col III m RNA,α-SMA,miR-146a expression in lung tissue was examined by RT-PCR.Results:（1）In the control group,mental and diet are normal,and the body weight is increasing at different levels.BLM group showed different degrees of reduction in food intake,weight,and activity after modeling.The highest mortality rate of the model was I Cgroup,and P A group was the most stable among them;The peak mortality rate arrived from 7 to 10 days,and the weight loss tends to be stable from the 24^th to the 28^th.（2）In the control group,the lung were bright,pink,with a clear outline and without swelling or bleeding.With increasing dose of bleomycin,the tracheal model has different degrees of lung tissue deformation and patch hemorrhage.The lung injury is the most severe but uniform in PA group.（3）According to the lung coefficient,the lung coefficient of the control group was significantly lower than that of the BLM model,and the degree of pulmonary fibrosis was positively correlated with the lung coefficient.（4）H＆E staining results:After instillation of different doses of BLM in the trachea on the 28th day,the alveolar structure of the mice was significantly damaged compared with the normal group,and the alveolar wall was significantly thickened,accompanied with inflammatory cell infiltration and fibroblastic foci.The PA group is the most obvious.（5）The results of ELISA:the serum levels of TGF-β,Col-I,Col-III andα-SMA in BLM group were significantly higher than those in the control group,and PA group was the highest.（6）Immunohistochemical results:The expression of TGF-β,Col-I,Col-III andα-SMA in the tracheal BLM group increased with the increase of BLM dose,but the expression of TGF-β,Col-III,α-SMA was decreased in the tracheal BLM group,but still more than N groups（P<0.05）.（7）The results of RT-PCR:（1）TRAF6,IRAK-1,α-SMA,Col Im RNA and Col III m RNA in lung tissue of BLM were significantly higher than those in control group.The expression of PA group was the most significant.（2）miR-146a was the least expressed in PA model,which was significantly lowest than the tracheal model,and the control group had the highest expression.Conclusion:（1）Intraperitoneal injection of BLM 35mg/Kg 2 weeks per week for 4 weeks modeling the most obvious degree of pulmonary fibrosis,low mortality,simple operation,is a suitable method for pulmonary fibrosis modeling（2）miR146a induced by bleomycin Low expression in the pulmonary fibrosis model,its expression trend is opposite to IRAK-1,TRAF-6,α-SMA,Col-I,Col-III,may be involved in the development of pulmonary fibrosis.