The Role Of Epithelial To Mesenchymal Transition (EMT) In The Pulmonary Fibrosis Caused By Paraquat Poisoning In Rats

Paraquat dichloride (methyl viologen; PQ) is an effective and widely used herbicide all over the world. Over the last two decades, with the growing widespread application of PQ in China, the morbidity of PQ poisoning rapidly elevated and the number of reported cases in recent10years is15times that of the past10years from China Journal Full-text Database. PQ poisoning is an extremely frustrating condition to manage clinically, and the mortality was as high as60%～87.8%according to the domestic and abroad reports, due to the very lower fatal dose (ingestion of30～40mg/kg) and the lack of magic antidote. PQ poisoning has been becoming one of the focal questions in emergency medicine and even a severe social problem.Lung is the main target organ of PQ poisoning, and the pulmonary concentrations of PQ can be6to10times higher than those in the plasma, which can be explained by the participation of the polyamine transport system abundantly expressed in the membrane of alveolar cells type Ⅰ, Ⅱ, and Clara cells. The major cause of death in paraquat poisoning is a rapidly progressive respiratory failure due to an oxidative insult to the alveolar epithelium with subsequent fulminant obliterating fibrosis. Progressive and extensive pulmonary fibrosis is the distinctive pathological changes in PQ poisoning, but its source and molecular biological mechanism are still unknown so far and the correlative research is scanty.The activation and proliferation of myofibroblast (MFb) are the distinctive pathophysiologic changes of pulmonary fibrosis, and the accumulation of a large number of MFb is responsible for exaggerated and uncontrolled production of extracellular matrix during the development and progression of pathological fibrosis. But the traditionary opinion about the origin of myofibroblast which deemed that the myofibroblast comed from the resident mesenchymal cells in the lung is beening challenged by more and more new experiments. Epperly et al. discovered that there is a significant contribution of bone marrow-derived cells in the pathophysiology of murine lung irradiation fibrosis in2003, and the resent opinion concerning the contribution of alveolar epithelial cell (AEC) to the development of pulmonary fibrosis by Epithelial-Mesenchymal Transition (EMT) is been certificating. EMT is a graded response whereby epithelial cells reversibly acquire mesenchymal features and enhanced capacity of invasion, which has been considered a leading role in the cell transdifferentiation of embryonic development and malignant tumor for a long time. In recent10years, a large number of evidences have highlighted a link between EMT and the fibrosis. EMT plays a significant and even a key pole in fibrosis of renal interstitiumcancer, myocardium and liver. The importance of EMT as a factor of pulmonary fibrosis is also increasingly recognized. Kasai H et al. reported TGF-betal induces human alveoiar epithelial in vitro culture to mesenchymal cell transition in2005, and EMT was detected in airways of a bleomycin induced pulmonary fibrosis model derived from an a-smooth muscle actin-Cre Transgenic mouse in2009. Furthermore, EMT was observed even in patients with idiopathic pulmonary fibrosis (IPF).Well, PQ poisoning, a newly emerged disease20years ago, a specific one of pulmonary fibrosis diseases, is involved in EMT? Whether EMT participates in the pulmonary fibrosis of PQ poisoning or not? If it does, by which intra-cellular signal transduction pathway might EMT be regulated at molecular level?When EMT happened, epithelial cells would lose their features of epithelial phenotype, lose the gene and protein expressions of E-cadherin which is the marker of epithelial cells, and acquire the features of mesenchymal phenotype:the protein of expression of α-SMA in cytoplasm significantly enhances, the cell skeleton fracture changes, and the gene and protein expressions of markers of mesenchymal cells increase including Vimentin, Fibronectin and N-cadherin. Based on this theory, we designed this study to investigate the role of EMT in pulmonary fibrosis caused by PQ poisoning in rat and the possible mechanisms of EMT.At first, rat models of pulmonary fibrosis caused by PQ poisoning were established by intraperitoneal injection at the dose of15mg/Kg and the lung speciments at different time points were collected. And then, collagen specific picrosirius red staining was performed to observe the degree of pulmonary fibrosis. Subsequently, Gene and protein expression of the epithelial and mesenchymal phenotype markers were determined by real time reversed transcriptional polymerase chain reaction (Real time RT-PCR), immunohistochemistry and western blot analysis to investigate whether the alveolar epithelial cells participate the pulmonary fibrosis by EMT. At last, the roles of TGF-β1/Smads and Wnt/β-catenin signal transduction pathways in AECs EMT were explored to investigate the possible mechanism of AECs EMT primitively.Objective:To investigate the role of EMT and its possible mechanism in pulmonary fibrosis caused by paraquat poisoning in rats. Methods:Chapter one Establishment of rat models of pulmonary fibrosis caused by paraquat poisoning. Seventy-two adult male Sprague-Dawley (SD) rats were randomized to two groups:PQ poisoning group (n=48) and control group(n=24). PQ was administrated by intraperitoneal injection at a dose of15mg/Kg to the rats in PQ poisoning group to establish rat models of the pulmonary fibrosis, and equivalent volume of normal sodium to the rats in contral group by intraperitoneal injection, too. Rats were sacrificed at days1,3,7,10,14and21after the PQ treatment, each time8rats in PQ poisoning group and4rats in control group. Lungs were excised from rat bodies and lung speciments were processed and reserved for later histological evaluation, immunohistochemical analyses and the determination of gene and protein expression respectively by real-time RT-PCR assay and western blot analysis.In this chapter, H-E staining was performed to observe the pathological changes and collagen specific picrosirius red staining was performed to observe the degree of pulmonary fibrosis and to calculate the collagen volume fraction (CVF).Chapter two The role of EMT in pulmonary fibrosis caused by paraquat poisoning in rats. Gene expressions of the epithelial phenotype marker (E-cad) mesenchymal phenotype markers (α-SMA, FSP-1and Vimentin), fibrogenesis markers (collagens type Ⅰ and Ⅲ) and transcription factor (Slug and Twist) was determined by real time reversed transcriptional polymerase chain reaction (Real time RT-PCR). In addition, protein expression of E-cad, α-SMA and collagens type Ⅰ was determined by immunohistochemistry and western blot analysis.Chapter three The role of TGF-β1/Smads signal transduction pathway in AECs EMT in pulmonary fibrosis induced by paraquat poisoning in rats. Gene expression of TGF-β1,Smad2and Smad7in pulmonary tissues obtained at different time points was determined by Real time RT-PCR.Chapter four The role of Wnt/β3-catenin signal transduction pathway in AECs EMT in pulmonary fibrosis induced by paraquat poisoning in rats. Gene expression of TGF-β1, Wnt2and β-catenin in pulmonary tissues obtained at different time points was determined by Real time RT-PCR.Results:Chapter one Acute pulmonary alveolitis was the chief pathological change of paraquat poisoning in the first week, and collogen deposition was observed in alveolar septum in the second week which arrived peaking on day14but decreased on day21. Obvious pulmonary fibrosis marked the successful modeling of pulmonary fibrosis by paraquat poisoning in rat. In PQ poisoning group, the pulmonary tissue CVF began to rise on the3rd day and arrived peaking on the14st day, but down-regulated on the21st day, with significant difference (P<0.01). On day3,7,10,14and21, the values of CVF in PQ poisoning group were significantly higher than coinstantaneous that in control group (P<0.05or0.01).Chapter two There were significant difference among the gene and/or protein expressions of E-Cad, α-SMA, Vimentin, FSP-1, Col-Ⅰ, Col-Ⅲ, Slug and Twist at different time points in PQ poisoning group(P<0.05), but not in control group (P>0.05). In PQ poisoning group, the gene and protein expression of E-Cad down regulated on the3rd day and arrived valley bottom on the14st day, but up-regulated on the21st day. Also in PQ poisoning group, the expressions of α-SMA, Vimentin, FSP-1, Col-Ⅰ, Col-Ⅲ, Slug and Twist up-regulated during the first week and arrived peaking on the14st day, but showed up-regulation on21st day. Chapter three There was significant difference among the gene expressions of TGF-β1and Smad2in PQ poisoning group at different time points in PQ poisoning group(P<0.05), but not in control group (P>0.05). The expression of TGF-β1mRNA enhanced on the7th day and Smad2mRNA did on the10th day. Both of them arrived peaking on the14st day and down-regulated on the21st day. There was not significant difference among the gene expressions of Smad7at different time points either in PQ poisoning group or in control group (P>0.05).Chapter four There was significant difference among the gene expressions of Wnt2and β-catenin at different time points in PQ poisoning group(P<0.05), but not in control group (P>0.05). In PQ poisoning group, the expressions of Wnt2and β-catenin mRNA suddenly enhanced in a large argument on the day10, and arrived peaking on the21st day.Conclusions:Our study shows that some alveolar epithelial cells in pulmonary fibrosis model rat caused by paraquat poisoning underwent EMT. EMT may be an imporportant one of the sources where the myofibroblasts in lung tissue derived from and play a key role in pulmonary fibrosis caused by paraquat poisoning. TGF-betal, the critical cytokine in pulmonary fibrosis, showed a persistent homo-expression in our study and might induce AECs to occur EMT by TGF-betal/Smads pathway depending on Smad2and by Wnt/β-catenin pathway which not depending on Smads. If it is really like it, our observation of AECs EMT would brings a new breakthrough in the mechanisms of pulmonary fibrosis caused by paraquat poisoning and might provide base theoretic support for the search of magic antidote, exploitation of new drug and target gene treatment in clinical medcine of PQ poisoning. But as to the confirmation of AECs EMT, the percentage of MFb from AECs EMT and the indeed mechanism of EMT, much more studies should be done by transgenic animal model, flow cytometry, confocal microtechnique, gene chip and so on. In addition, our experiment lasted just for3weeks and the characteristics of pulmonary fibrosis by PQ poisoning over3weeks deserves our further observation and exploration.