The Preliminary Study On The Role Of CDKN2B Methylation In The Development Of Pulmonary Fibrosis

Background:pulmonary fibrosis is a serious interstitial pulmonary disease,characterized by early inflammation of lung epithelial cells,a large number of lung fibroblasts/myofibroblasts proliferation and extracellular matrix deposition in the late stage,thereby disrupting the normal lung structure.Although the exact cause is unknown and the treatment is few,there is some evidence that genetic and epigenetic changes such as DNA methylation,plays a crucial role in the pulmonary fibrosis.A large number of studies have shown that pulmonary fibroblasts are the key effector cells in the development of pulmonary fibrosis.Recent studies confirm that CDKN2B has differential methylation status in lung fibroblasts in patients with pulmonary fibrosis,but there is no report about its mechanism in the pathogenesis of pulmonary fibrosis.Objective:In this study,we constructed the animal model of pulmonary fibrosis to detect the methylation status of CDKN2B promoter in primary cultured lung fibroblasts of rats,and to preliminarily study its mechanism in the development of pulmonary fibrosis.Methods:1.36 male Wistar rats were randomly divided into six groups:control group,bleomycin group(model group)and non-treatment control group,model group of pneum-oconiosis(sand group),5’-azacytidine intervention group(intervention group of dust),5-azacytidine intervention group of bleomycin(bleomycin intervention group).The control group received intratracheal instillation of saline;model group received intratracheal instillation of bleomycin solution(2.2mg/kg,0.3ml);sand group were exposed to the wind tunnel simulation environment(the total suspended particulate matter concentration(TSP)is about 9000ug/m~3)5days a week,5h per day,and the rest time of the living environment are the same as the non-treatment control group;non-treatment control group were placed outside of the wind tunnel;intervention group of dust was exposed to wind tunnel environment as dust group),and after one month,received intraperitoneal injection of 5’-azacytidine(1mg/kg 1ml);bleomycin interventi-on group received intraperitoneal injection of 5’-azacytidine(1mg/kg,1ml)after 4 days’intratracheal injection(as dose method of model group);Respectively,in postoperative third week,third weeks,twenty-fourth weeks,twenty-fourth weeks,twenty-fourth weeks and third weeks,rats were sacrificed.2.Hematoxylin eosin staininglung,Masson trichrome staining and related gene detection were used to verify whether the fibrosis model was successfully constructed.Total cellular DNA was extracted from primary fibroblasts.Using the bisulphite pyrosequencing,methylation status of CDKN2B promoter was detected.4.we use the bioinformatics web site(KEGG PATHWAY Database)and biomedical search website PubMed to predict the related pathway and target gene of CDKN2B fibrosis.Western-blot was used to analyze the expression of related target genes.Result:1.Rats were sacrificed and lungs were dissected,fixed and stained.we found that the sand and bleomycin can cause lung damage in different degree,including inflammation,fibrous tissue proliferation,lung nodule formation.In dust intervention group,inflammation and hyperp-lasia of fibrous tissue decreased compared with dust group.However,no obvious change was found in bleomycin intervention group.2.The methylation status of CDKN2B promoter in lung fibroblasts in the pulmonary fibrosis group was significantly higher than that in the non-treatment control group.3.No significant difference was found in the methylation status of the CDKN2B promoter between the control group and the model group.4.In sand dust group,the a-SMA expression of fibroblasts increased.5.The expression of p-Smads protein in the sand dust group was higher than that in the non treatment control group.Conclusion:1.Primary culture of rat lung fibroblasts showed that hypermethylation of CDKN2B gene promoter region in rat lung fibroblasts was caused by dust particles.2.CDKN2B methylation may be one of the mechanisms of the formation of pulmonary fibrosis induced by sand dust.That is,CDKN2B methylation activates TGF-β/Smads signaling pathway,which promotes the EMT process and participates in the pathogenesis of pulmonary fibrosis.3.The occurrence of bleomycin-induced pulmonary fibrosis is not related to CDKN2B methylation.