The Role Of TGF-?1 In Pulmonary Fibrosis Induced By Paraquat Poisoning

[Research Background]"Pulmonary Fibrosis" is short for "Diffuse Pulmonary Interstitial Disease"(DPID).It is chronic progressive and destructive interstitial lung disease.Clinicopathological characteristics are shown as diffuse inflammation and interstitial fibrosis of alveolar wall(including respiratory bronchiole).Idiopathic pulmonary fibrosis is general type which covers 65%of all pulmonary fibrosis types.For image findings:subpleural lung tissue is shown as network or honeycomb changing;double lower lungs appeared significantly;ground glass opacity may be appeared[1].Metastasis mechanism of pulmonary fibrosis has been not clarified.According to research previously,migration,proliferation and activation of interstitial lung cell have been appeared during epithelium reconstruction of alveolus after pulmonary injury,thus to cause formation of fibroblastic foci;extracellular matrix secreted and accumulated greatly to cause interstitial lungs widened.All the processes are irreversible damage[2].As one of four respiratory diseases,pulmonary fibrosis has a few illness causes such as physical factor,infectious factor and vascular factor etc.With increasing Paraquat poisoning cases and treatment improvement during clinic works,patients of pulmonary fibrosis caused by Paraquat poisoning are increasing.Paraquat(PQ)is the most widely used organic heterocyclic herbicides with product name of parqual.Its chemical composition is 1,1'-Dimethyl-4,4'-bipyridinium Dichloride Hydrate.Absorption rate of paraquat is 5-15%by the gastrointestinal tract in clinical.It distributed the whole body after absorption.It is the highest concentration in lung tissue because of active uptake function of alveolar epithelial cells,the concentration will be 10-90 times more than blood.Acute pulmonary edema can appear witnin One week of paraquat poisioning,can appear fibrosis change after 1 week,2 to 3 weeks to achieve high obvious fibrosis.As main target organ for Paraquat poisoning,lung lesion is the main reason for Paraquat dying.Most of the patients died because of ARDS of early lung injury.Later dying reasons are mostly pulmonary fibrosis caused by Paraquat poisoning[3].Previous research of pulmonary fibrosis caused by Paraquat poisoning:epithelium cell is the main target of lung injury;EMT is one of the important mechanisms for pulmonary fibrosis;EMT may cause number of interstitial fibroblasts increased significantly;promote interaction among interstitial cells;thus to cause lung tissue fibrosis[4].Certain research mentioned that imbalance of CK network is important factor of lung injury caused by Gramoxon poisoning.In the CK network of pulmonary fibrosis caused by Paraquat,TGF-?1 is commonly recognized initiation factor.Research found that:use Paraquat and TGF-?1 to stimulate A549 cell respectively,the cells appeared formation of mesenchymal cells.It further proves that TGF-?1 is key factor for Paraquat to cause pulmonary fibrosis[5]During EMT occurrence and development,lung tissue cell protein expression changed significantly;lung epithelium cell protein E-Cadherin expression decreased significantly;interstitial marked protein ?-SMA expression increased gradually[6-9].The protein expression profile changing in fibrosis has been widely used in vitro and in vivo studies of fibrosis occurrence and development mechanisms.For Part I:According to CT of lungs,screening of patients with pulmonary fibrosis as the research object,according to the time point to return blood samples,get to know relationship between pulmonary fibrosis and inflammatory factor changes by discovering inflammatory factor changes in serum of Paraquat poisoning patients.For part II:select A549 cells as the research object.use exogenous TGF-?1 and Paraquat to stimulate A549 cell for EMT changing to build cell model,further to verify pulmonary fibrosis mechanism caused by TGF-?1?and Paraquat.For Part ?:choose rat as the research object.use Paraquat to induce rat pulmonary fibrosis to build animal model;predict possible pulmonary fibrosis mechanisms through pathomorphology,protein expression levels and detecting expression changing of TGF-?1?in pulmonary fibrosis occurrence.So far,still no effective treatment of acute paraquat poisoning,clinical mortality remains high,cause great economic loss.Therefore,to explore the mechanism of acute paraquat poisoning,clear therapeutic targets,has extremely important social effect and practical significance[Experimental Methods and Research]Part ?:Inflammatory factor changes in serum of Paraquat poisoning patients.Pick Paraquat poisoning patients in emergency department of Binzhou Medical University Hospital during 2015.01-2016.01.Taking time is less than 24 hours with 18-60 years' old,detect concentration changing of E-cadhern,a-SMA,TGF-?1 for plasma in 3,51 and 147 hours.Part ?:verify effect of TGF-?1?in pulmonary fibrosis caused by acute Paraquat poisoning through cell level.TGF-?1?Paraquat and paraquat+blocker of TGF-?1?induced A549 cells to occur epithelium interstitial transdifferentiation(EMT)to construct cell model of fibrosis,observe morphological changes of A549 cells.Immunofluorescence assay and Western Blotting displays expressions of cell surface epithelium marked protein E-Cadherin and interstitial marked protein a-SMA.Part ?:Individual level research of effects for TGF-?1?in big ratpulmonary fibrosis induced by Paraquat poisoning.Build acute Paraquat poisoning induced big ratpulmonary fibrosis model.The 60 male rat were divided into experimental group(P group)and saline control group(N group).Give animal of group P with 80mg/kg Paraquat;dilute to 1ml by saline and gavage at one time;gavage group N by 80mg/kg Paraquat.From gavage modelling day,the time is prescribed as the 0th day of experiment.Kill 6 animals on the 1th day,3th day,7th day,14th day and 21th day;observe formation changes of lung tissues for rat by HE staining.Observe collagen expression of lung tissue by electric mirror;detect collagen contents in lung tissue by HYP;detect expressions of TGF-?1,a-SMA and E-cadherin by Immunohistochemistry.Results:with the time prolong of poisoning,pulmonary interstitium per hour of group P widened gradually;alveolus structure disordered gradually;collagen deposition of interstitial tissue becomes obviously gradually;contents increased without obvious changes in lung tissue of group N.According to verification,the model of pulmonary fibrosis caused by acute Paraquat poisoning has been successful.[Experimental Results]1.Image changing of acute Paraquat poisoning patient and contents of TGF-?1,E-cadhern,a-SMA in plasmaStudies have shown that acute paraquat poisoning cause pulmonary fibrosis patients compared with control group.After 3 hours,TGF-?1 of Paraquat poisoning patients has no significant changes;TGF-?1?contents in plasma of 51 hours,147 hours increased significantly;TGF-?1?increased in early acute poisoning in consistent with double CT pulmonary fibrosis process.It means that TGF-?1?might promote pulmonary fibrosis process.Compared with 3 hours,51 hours and control group,E-Cadherin contents of acute Paraquat poisoning patient has not reduced significantly;it reduced significantly compared with 147th hour.It means:the longer time,the lower epithelium expression.Contents of a-SMA has not changed significantly.It means:the longer time,the more interstitial cell.2.Verify effect of TGF-?1?in pulmonary fibrosis caused by acute Paraquat poisoning through cell level.According to research:add TGF-?1?Paraquat?Paraquat and blocker of TGF-?1 into A549 cell culture medium respectively;formation of A549 cells changed gradually from cubes(triangles)into long spindle,Cells are distributed in line strips loose,that means EMT phenomenon happened;The change in the group of Paraquat and blocker of TGF-?1?is line the middle of the former groups?In the three groups,Immunofluorescence assay and Western Blotting showed epithelium marked protein E-Cadherin of cell surface decreased.In turn,interstitiafl marked protein a-SMA expression increased.TGF-?1?and Paraquat may induce A549 cell to occur EMT.The blocker of TGF-?1?can attenue the progress of EMT.It proved important effect of TGF-?1?in pulmonary fibrosis process caused by acute Paraquat poisoning.3.Individual level research of effects for TGF-?1?in pulmonary fibrosis induced by Paraquat poisoning in rat(1)According to research:TGF-?1?contents of acute Paraquat poisoning rat in the 1th,3th,7th,14th day rose significantly;No comparative significance in the 21th day.Compared with control group,the E-Cadherin contents in plasma of acute Paraquat rat patients on the 14th,21th day lowered significantly.It means:the longer time,the lower epithelium expression.Compared with the 7th,14th and 21th day,the contents of a-SMA protein rose significantly.It means:the longer time,the more interstitial cells.(2)HE staining expression:interval of part areas for acute Paraquat poisoning rat breaks and fusion;alveolus interval of part area become wider;a little exudate and red blood cells can be seen from alveolus cavity.(3)Electron microscope observation:alveolus epithelium of acute Paraquat poisoning rat damaged significantly;pulmonary interstitium become wider significantly;collagen expression increased.(4)Compared with control group,hydroxyproline contents in lungs of the acute Paraquat poisoning rat become higher.(5)With longer poisoning time,TGF-?1?expression in lung tissue of acute Paraquat poisoning rat become higher;the E-Cadherin expression become lower;a-SMA expression become higher.[Conclusion]Through CT changes of lungs in patients with acute paraquat poisoning and giving rat paraquat lavage confirmed acute paraquat poisoning can lead to pulmonary fibrosis.Acute paraquat poisoning can cause the decline in E-Cadherin expression in lung tissue and blood plasma,a-SMA expression increased,hydro xypro line contents in lungs of the acute Paraquat poisoning rat become higher confirmed the diagnosis of pulmonary fibrosis.Cell experiments confirm paraquat can cause EMT of A549 cells by TGF-?1,TGF-?1?block group can inhibit the progress of EMT.Comprehensive the above conclusions confirm paraquat can lead to pulmonary fibrosis by stimulating the body to produce TGF-?1.