| Paraquat is high-toxic herbicide can be absorbed' through the skin,respiratory tract,digestive tract.Paraquat can cause acute poisoning to human and animal.Paraquat poisoning is common in China,which complicated with pulmonary fibrosis and multiple organ damage.Paraquat poisoning without specific antidote and treatment effect is poor,so the mortality is high.Paraquat is widely dispersed in the.body,lung and skeletal muscle has the highest paraquat concentration.Paraquat can still be detected in the patient's heart,lung,kidney and liver tissues even after poisoning 6 days later.Paraquat induced cellular mitochondria produce ultra oxygen anion through NADH voltage dependent anion channel 1 and resulted in cell toxicity.Alveolar type II cells makes paraquat gathered in lung alveolar epithelial cells by polyamine intake system in the paraquat poisoning early stage.The mortality of paraquat poisoning caused pulmonary fibrosis is high,there is no specific method to treat paraquat-induced pulmonary damage.Paraquat can promote lung cell apoptosis and extracellular matrix degradation,increase inflammation factor expression and reduce antioxidant gene expression to cause lung damage.Paraquat can promote inflammation cell apoptosis,increased extracellular matrix degradation cause acute lung injury in the poisoning early stage.In the late poisoning stage,paraquat activate fibroblast activity,reduce collagen degradation so that lead to pulmonary fibrosis.Extracellular matrix metabolism imbalance is extremely important in the process of paraquat induced pulmonary fibrosis.Matrix metalloproteinases included a variety of protease.MMP-9 is one of the Matrix metalloproteinases.The main function of MMP-9(2)is to degrade ?,?,?,?,? collagen,the core protein of proteoglycan,gelatin,elastin,fiber adhesion protein,etc.MMP-9 gene is on chromosome 20 qll.1?13.1,the size is 26?27 kbp,and with 13 exons and 9 introns.Chinese scholars found that MMP-2 and MMP-9 and TIMP-1 gene expression increased in acute paraquat poisoning rat lung tissue,the imbalance expression of MMP/TIMP-1 participated in the process of paraquat induced pulmonary fibrosis.Multiple matrix metalloproteinases gene transcription and protein expression increased in pulmonary fibrosis patients.Matrix metalloproteinases participated in the remodeling of pulmonary structure of pulmonary fibrosis patients.There is a interactive talk between alveolar epithelial cells and pulmonary interstitial.The injured alveolar epithelial cells can promote extracellular matrix hyperplasia through the interactive dialogue form to cause pulmonary fibrosis.WNT1 induction signal protein 1(WISP1)is a newly discovered secreted signaling molecules belong to CCN family.WISP1 by autocrine and paracrine way to increased MMP-9 gene expression in the animal model of pulmonary fibrosis and fibrosis patients alveolar cells.WISP1 resulted in pulmonary fibrosis through promote fibroblast proliferation,activate alveolar epithelial cells and extracellular matrix collagen deposition.To search literatures in recent years at home and abroad,we did not see the reports about the role of WISP1 in pulmonary fibrosis caused by paraquat poisoning.Through the reports of WISP1 in the EMT(epithelial mesenchyma transition)and pulmonary fibrosis,we speculated that WISP1 can change the MMP-9 gene expression,which resulted in lung extracellular matrix metabolism imbalance.WISP1 involved in the process of pulmonary fibrosis caused by paraquat poisoning.We proposed examining the expression of WISP1 genes and MMP-9 gene in paraquat poisoning patients and study the effect of gene expression of WISP1 and MMP-9 in A549 cell induced by paraquat,to study the role of WISP-1 in the possible mechanisms of pulmonary fibrosis caused by paraquat poisoning.There are many reports about risk factors lead to death after paraquat poisoning.Studies had shown that the risk factors of patients with paraquat poisoning include plasma paraquat concentration,amount of poison,APACHE II score and the like can reflect the prognosis of patients with acute paraquat poisoning.We studied paraquat poisoning inpatients of our hospital and followed up them for 3 years to investigate risk factors related to prognosis of paraquat poisoning.Paraquat complicated with a high incidence of pulmonary fibrosis,but there is less reports of relevant risk factors for pulmonary fibrosis resulted from paraquat poisoning.We observed the lung image change of paraquat poisoning patients,analysis of the laboratory indexes and APAGHE? score during hospitalization of those patients,to determine the risk factors for paraquat poisoning complicated by pulmonary fibrosis.Objective:To investigate the gene expression of MMP-9 and WISP1 in peripheral blood mononuclear cell and the MMP-9 and WISP1 protein concentration in serum.Observe the effect of PQ on the EMT,the expression of MMP-9 and WISPI in A549 cell.Study the effect of WISP1 on EMT and gene expression of MMP-9,to investigate the influence of PQ on EMT,gene expression of MMP-9 and WISP1 in vivo and in vitro.Follow up the incidence of death and pulmonary fibrosis of the paraquat poisoning patients.We detected paraquat serum concentration,record the laboratory indexes and APACHE II scores of paraquat poisoning patients,observe the relationship among clinical index,incidence of death and the pulmonary fibrosis in those paraquat poisoning patients.To search the reliable index that can help determine the prognosis and incidence of pulmonary fibrosis in paraquat poisoning patients.Methods:In vivo studiesl.Observe and follow up 120 cases of patients with paraquat poisoning from January 2012 to December 2014,analysis the predictor of prognosis and pulmonary fibrosis.According to the enter criterion,a total of 37 cases of patients with paraquat poisoning were included into the study from January 2013 to June 2014.2.Test group:Follow-up paraquat poisoning patients,according to the result of treatment of paraquat poisoning,patients were divided into death group,survival group,pulmonary fibrosis group and no pulmonary fibrosis group;The healthy people as healthy control group.3.Rt-PCR method was used to determining MMP-9 and WISP1 gene expression.4.Using ELISA method to determining MMP-9,WISP1 protein concentrations in the serum.5.Using high performance liquid chromatography(HPLC)method to determinate paraquat concentration in serum.6.Evaluate APACHE II score of patients on the poisoning first day and third day,follow-up the survival patients and those who complicated pulmonary fibrosis in paraquat poisoning patients;collect the laboratory index after patients admitted to hospital,analysis the relationship of these indicators and the prognosis of patients.In vitro test1.The A549 cells were cultured and treated with paraquat.The cells were divided into PO group,P50 group,P100 group,P200 group and P400 group according the concentration of paraquat.The morphology of A549 cells was observed under light microscope.2.Western Blot method was used to determine the protein concentration of MMP-9,WISP1 and E-cadherin and Vimentin in the cells.3.The gene expression of MMP-9 and WISP1 was determined by RT-PCR.4.A549 cells were treated with WISP1siRNA,which were divided into group PO group,P200 group,NCgroup and Wgroup.Western Blot method was used to detect protein changes of WISP1,MMP-9,E-cadherin and Vimentin.Statistical MethodUse SPSS 19.0 statistical software,using analysis of ANOVA,t test,Spearman correlation and Logistics regression method for statistical analysis.Results:In vivo studies1.MMP-9 gene expression and serum protein of paraquat poisoning patients were higher than those of normal control group(p<0.01).MMP-9 gene expression and serum protein of the death group is higher than those of the survival group(p<0.01).MMP-9 gene expression and serum protein of pulmonary fibrosis group is higher than those of no pulmonary fibrosis group(p<0.01).2.MMP-9 gene expression of patients with pulmonary fibrosis group on 1st day(t = 5.269,p = 0.000)and 3rd day(t = 5.435,p = 0.000)was higher than those without pulmonary fibrosis group.3.WISP1 gene expression and serum protein concentration of paraquat poisoning patients is higher than the normal control group(p<0.01).WISP1 gene expression and serum protein concentration of the death group is higher than those of the survival group(p<0.01).WISPl gene expression and serum protein concentration of pulmonary fibrosis group is higher than those of no pulmonary fibrosis group(p<0.01).4.WISP1 gene expression on the poisoning 1st day was positive correlation to the 1st day MMP-9 gene expression(r=0.822,p<0.01)and the 3rd day MMP-9 gene expression(r=0.852,p<0.01).WISP1 gene expression on the poisoning 3rd day also positively correlated to the 3rd day MMP-9 gene expression(r=0.664,p<0.01).5.The paraquat poison quantity of the death group is greater than that of survival group(p<0.01).The serum concentrations of paraquat of the death group is greater than those of survival group(p<0.01).Serum paraquat of pulmonary fibrosis patients is higher than those of no pulmonary fibrosis group(p<0.01).6.Serum concentration of paraquat was positively correlated to MMP-9 gene expression and serum MMP-9 concentration.Serum concentrations of paraquat was positively correlated to thelst day(r=0.672,p=0.000)and the 3rd day(r=0.703,p-0.000)MMP-9 gene expression and MMP-9 serum protein concentration(r=0.596,r=0.705,all P<0.01).7.Seru.concentration of paraquat was positively correlated to WISP1 gene expression on the 1st day(r=0.621,p=0.000),but had no correlation with the 3rd day WISP 1 gene expression.Serum concentrations of paraquat was positively correlated to the 1st day(r=0.596,p=0.000)and the 3rd day(r=0.447,p=0.006)WISP1 serum protein concentration.8.The APACHE ? score of paraquat poisoning death group higher than those of the survival group(p<0.01).Pulmonary fibrosis patients with APACHE II score higher than no pulmonary fibrosis group(p<0.01).APACHE II score on the poisoning third day is higher than those of the first day(p<0.01).9.We found that APACHEII score,BUN,Cr,amylase,AST and merge pulmonary fibrosis is the risk factors for paraquat poisoning death through Logistic analysis.10.APACHEII score,MMP-9 gene and protein,BUN,Cr,blood sugar,AST,ALT,poison quantity,number of white blood cells,and neutrophils number is the risk factor for paraquat poisoning complicated by pulmonary fibrosis.In vitro tests1.The morphology of A549 cells can be observed from epithelial morphology to interstitial cell morphology.400ug PQ treatment led to cell apoptosis significantly.2.E-cadherin protein significantly decreased in P100 group,P200 group and P400 group,they were all significantly lower than that of P0 group and P50 group(p<0.01),and the decrease was most obvious in P200 group.3.Vimentin protein increased with the concentration of.PQ,and the expression of Vimentin in P50 group was higher than than those of the other groups(p<0.01).4.The E-cadherin protein in the cells treated with 100ug PQ on the 2nd day was higher than that on the 1st day(p<0.05),and the decrease of E-cadherin on 3rd day was close to that of 1st day.Vimentin gene expression decreased on the 2nd day,but it raised on the 3rd day and close to the level of 1 st day.5.Western Blot method showed that the protein concentration and gene expression of MMP-9 and WISP1 increased with the increase of PQ concentration(p<0.01).Treated with 200ug PQ,the gene expression of MMP-9 and WISP1 increased with time,and the differences were significant(p<0.01).6.WISP1 siRNA could inhibit the EMT induced by PQ.E-cadherin in W group was higher than that of P200 group and NC group,Vimentin in W group was less than that of P200 group and NC group,the differences were significant(p<0.01).7.WISP1 siRNA inhibited the expression of MMP-9 induced by PQ,the MMP-9 expression in W group was lower than that of P200 group and NC group,the differences were significant(p<0.01).Conclusion:1.PQ could increase the expression of MMP-9 and WISP1 in vivo and invitro.2.PQ induced EMT in alveolar cells.3.WISP1 promoted the occurrence of PQ-induced EMT and increased the expression of MMP-9.4.The serum paraquat concentration,MMP-9 gene expression level,APACHE? score,BUN,Cr quantitative value can be used to predict the occurrence of pulmonary fibrosis in paraquat poisoning patients.5.The expression of MMP-9,results of paraquat poisoning patients'white blood cells,neutrophils,liver and kidney function index,blood sugar and amylase were the indicators of paraquat poisoning patients' poor prognosis.6.WISP1 played an importent role in PQ-induced pulmonary fibrosis by promoting EMT and upregulation of MMP-9 expression. |
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