The Study Of The Mechanism Of DDAH In Pulmonary Fibrosis Induced By Long-term Smoking

Pulmonary fibrosis(PF)is a severe pulmonary interstitial disease that occurs in advanced stages of many chronic lung diseases and eventually causes pulmonary failure.Currently,the development of pulmonary fibrosis cannot be reversed and there is no effective clinical treatments for it.Cigarette smoke contains a variety of harmful chemical substances and is usually considered as one of the major causes of chronic lung diseases.It is widespread considered as an important risk factor for pulmonary interstitial fibrosis.Researching the mechanism of smoking-induced pulmonary fibrosis has important clinical value and is significant for searching prevention measures and new drug targets of PF.This thesis is divided into two parts:Part ? The effects of long-term smoking on lung injury and fibrosis in ratsObjectiveWe want to observe the effect of long-term smoking on the structure of lung tissue in rats and ascertain the fact that smoking can promote the development of pulmonary fibrosis in rats.We want to confirm the effect of smoking on DDAH activity in rat lung tissue,and localize the DDAH high expression effector cells.Methods1.We constructed a rat model of long-term smoking-induced pulmonary fibrosis,and take pictures of the rats' lung tissues for recording.2.We fixed lung tissue samples and made paraffin sections for H&E,Masson and Sirius red staining to observe the pathological changes,the fibroblast foci and collagen deposition of lung tissue.3.We extracted protein and RNA from lung tissue samples,and detected the expression levels of fibrosis markers such as ?-SMA,TGF-? and Collagen by Western blot and RT-PCR.4.We used Immunohistochemistry,immunofluorescence,Western blot and RT-PCR to detect the expression changes of DDAH1/DDAH 1/iNOS in rats'lung tissues and localized DDAH high expression effector cells.Results1.The results of H&E,Masson,and Sirius red staining showed that after exposure to cigarette smoke for 3 months,the lung tissues of rats showed diffuse interstitial inflammation,and a large number of lung macrophages were observed in the alveolar space.The alveolar septum was generally thickened,alveolar type ?epithelial cells were abnormally hyperplastic,fibroblast foci and myofibroblast foci formed,and collagen fiber deposition increased significantly.2.Real-time PCR and Western blot results showed that fibroblast markers were upregulated,especially TGF-?,after long time smoking.3.Real-time PCR,Western blot and immunofluorescence results show that DDAH2 and iNOS protein and RNA levels was up-regulated.Immunohistochemistry results confirmed that iNOS was over-expressed in airway epithelial cells and macrophages,DDAH 1 expressed in vascular endothelial cells,and DDAH2 over-expressed in alveolar type ? epithelial cells.Conclusion1.It was found that long-term smoking caused chronic pulmonary inflammation,interstitial hyperplasia of type ? alveolar epithelial cells,and eventually caused pulmonary fibrosis.The extent of disease was positively correlated with smoking time.2.The expression of TGF-? and DDAH-related proteins was up-regulated by smoking,and DDAH2 was over-expressed in alveolar type ? epithelial cells.Part ? The exploration of effects of cigarette smoke extract(CSE)on alveolar type ? epithelial cells(MLE-12)and its mechanismObjectiveAll experiments would base on MLE-12 cells which was derived from alveolar type ? epithelial cell.We want to clarify that CSE caused oxidative stress in MLE-12 cells and explore the effect of CSE on proliferation,migration and differentiation of MLE-12 cells.To confirm that CSE can cause up-expression of DDAH and TGF-? in MLE-12 cells.And through siRNA of DDAH2 to explore its relationship with the TGF-? signaling pathway.Methods1.MLE-12 cell line was cultured in vitro with different concentrations of CSE between 0.5%-20%.We used CCK8 to assay cell viability,and selected the appropriate concentration of CSE for subsequent experiments.2.Use CCK8 method and Wound-Healing assay to detect the effect of CSE and antioxidant NAC on the proliferation and migration of MLE-12 cells.In the NAC group,the MLE-12 cells was pre-incubated with 10 mM NAC for 24h and were detected by the ROS detection kit.3.Western blot and RT-PCR were used to detect the expression changes of ?-SMA,vimentin,fibronectin,DDAH2 and TGF-?,and compared the NAC group with the control group.4.We designed and transfected DDAH2 targeting siRNA,and detected the siRNA inhibition effect by Western blot and RT-PCR.5.After been transfected with siRNA,MLE-12 cells were cultured with CSE for 24 hours.Western blot and RT-PCR were used to detect the expression levels of DDAH2,TGF-? and phosphorylated Smad2 and Smad3.Results1.We confirmed 5%CSE could promote cell proliferation and migration of MLE-12 cells,clearing ROS could resist the apoptosis and migration of CSE on cells.2.Culturing MLE-12 cells with CSE could up-regulate the EMT-related markers,such as ?-SMA,vimentin and fibronectin.3.Culturing MLE-12 cells with CSE could up-regulate DDAH2 and active TGF-?,and DDAH2 and TGF-?1 expression was positively correlated,NAC can reduce the DDAH2 and TGF-? upregulation caused by CSE.4.DDAH2-siRNA transfection could effectively down-regulate the expression of DDAH2 without significant changes in TGF-? expression.5.Culturing MLE-12 cells with CSE after down-regulation of DDAH2,comparing with CSE group,down-regulated TGF-?,p-Smad2 and p-Smad3.Conclusion1.Cigarette smoke extract could cause oxidative stress,promote epithelial cell proliferation,migration and epithelial mesenchymal transformation by activating TGF-? pathway in MLE-12 cells.2.CSE could up-regulate the expression of DDAH2,and the specific down-regulation of DDAH2 could inhibit the activation of TGF-?/Smads signaling pathway by smoke extracts in MLE-12 cells.