| Objective:Paraquat(PQ)is one of the most widely used high-efficiency herbicides in the world.It has almost no residue in crops and soil,and high herbicidal efficiency.However,PQ is highly toxic to humans and animals,and the mechanism of poisoning is still unclear.There is no specific antidote in clinical practice,resulting in an increase in the mortality rate of poisoning.In China,PQ has become the pesticide product with the highest mortality rate and the incidence of poisoning in the acute poisoning case,which is second only to organophosphorus.At present,dozens of countries have banned the production and use of PQ.Since July 1,2014,China has cancelled the registration and production license for paraquat.On July 1,2016,the sales and use of water agents were stopped in China.Although pesticide ban and dosage form conversion have reduced the incidence of poisoning to some extent,cases of death from PQ poisoning still occur frequently.Therefore,in-depth study of the mechanism of PQ poisoning and the development of special-effect detoxification drugs are still important topics in toxicology.The lung is the main target organ for PQ poisoning.A polyamine transport system exists on the membrane of type I alveolar epithelium,type II alveolar epithelial cells and bronchial epithelial cells,which can rapidly concentrate PQ into the lungs,so that the concentration of PQ in the lung tissue reaches tens of times the plasma concentration.Acute alveolitis and rapid progression of irreversible pulmonary fibrosis are characteristic pathological manifestations of PQ poisoning.At present,the detailed mechanism of the origin and formation of pulmonary fibrosis caused by PQ poisoning is still unclear,and relevant research at home and abroad is scarce.The cytotoxic effect of PQ plays a role mainly through the production of hydrogen peroxide and peroxy radicals in the cell electron transport system,resulting in tissue cell damage.Recently,more and more studies have found that PQ exerts its toxic effects by impairing autophagy.Possible mechanisms are following: 1.PQ leads to classical mTOR activation,leading to inhibition of autophagy;2.Impairment of mitochondrial clearance injured autophagic flow;3.Inhibition of lysosomal hydrolase activity.Autophagy is an important protective mechanism for the maintenance of cellhomeostasis by degrading defective or excess macromolecular proteins and organelles in cells by lysosomes.Abnormal autophagy is involved in the development of multi-system diseases such as cancer,neurodegenerative diseases,infectious diseases,and autoimmune diseases.A number of studies have demonstrated that autophagy damage participates in the pathological process of pulmonary fibrosis by increasing inflammation,oxidative stress,and apoptosis.Recently,studies have found that autophagy protein expression in PQ-induced lung tissue changes,but no further systematic study of the relationship between autophagy and fibrosis and its molecular mechanism.To further reveal the role of autophagy in PQ-induced pulmonary fibrosis and its molecular mechanism,we designed this experiment.We used Masson staining and Sirius red(fibrosis dynamic observation),immunohistochemistry,immunofluorescence,western blot and other means to detect PQ poisoning human body materials,experimental animal lung autophagy related proteins Beclin 1,LC3 and p62 expression,study The state of autophagy in lung tissue of PQ poisoning,and the mechanism of autophagy abnormality in the formation of pulmonary fibrosis.We used in vitro cell experiments,using autophagy drug regulation and gene knockout methods to study abnormal changes in PQ-induced autophagy and participate in molecular pathways involved in pulmonary fibrosis.Methods:(1)Test of human body material.A sample of lung tissue from 8 cases of PQ poisoning deaths from July 2008 to March 2018 in the Judicial Appraisal Center of China Medical University was collected and selected,and 8 relatively normal lung tissue specimen were collected as a control.Pathological changes and fibrotic changes in lung tissue of poisoning cases and control cases were detected by HE staining,Masson,Sirius red and α-smooth muscle actin(α-SMA)immunohistochemistry.According to the results of microscopic pulmonary fibrosis staining,we divided the cases of poisoning into two groups: fibrosis group and non-fibrosis group.immunohistochemical staining of autophagy-related protein Beclin 1,microtubule-associated protein light chain 3(LC3)and SQSTM1/p62 was performed on each group,and the expression and distribution of autophagy-related protein in poisoned lung tissue were observed.Correlation with pulmonary fibrosis.At the same time,we also carried out immunofluorescence detection of LC3,indirectly observed changes in the number of autophagosomes.(2)Establishmentof a model of pulmonary fibrosis in mice with PQ poisoning.A poisoned mouse model was established by a one-time intraperitoneal injection.120 mice were randomly divided into the exposed group(PQ,n=60)and the control group(Cont,n=60).The exposed group was intraperitoneally injected with PQ(dose 40 mg/Kg),and the control group was intraperitoneally injected with the same volume.The normal saline was sacrificed on the1 st,3rd,7th,14 th,21st and 28 th day after exposure,and the experimental group and the control group were each 10,and the lung tissue samples were taken.The specimens were stained with hematoxylin-eosin(HE)and stained with Masson and Sirius red.The pathological changes of lung tissue and the morphological changes of pulmonary fibrosis were observed under light microscope.Hydroxyproline(HYP)in the lung tissue was detected.The level of autophagy and related channel protein expression in lung tissue of PQ-poisoned mice were detected,and the temporal changes of protein were observed.The expressions of autophagy-related proteins Beclin 1,LC3 and p62 in lung tissue of mice were detected by immunohistochemistry,immunofluorescence and Western blot.The expression of α-SMA in lung myofibroblasts was also detected.(3)Cell experiment:Human embryonic lung fibroblasts(IMR-90)were used,and PQ solution dissolved in sterile PBS was added to the medium to treat cells at a final concentration of 100 μM for24 h.The drug pretreatment group was given autophagy inducer RAPA(1 μM)or autophagic flow blocker CQ(25 μM)2 h before PQ administration.The Beclin 1 gene was silenced using RNAi.Cell viability was determined using the MTS kit.Immunofluorescence was used to detect changes in the number of fluorescent spots in LC3.The expression of Beclin 1,LC3,p62,α-SMA and LAMP-2 was detected by Western blot.Results: 1.The human body sample test results showed that the lung tissue of the control case showed a relatively normal tissue structure,and no fibrotic changes were observed.In the case of poisoning non-fibrosis group,the main pathological manifestations of lung tissue were pulmonary edema,pulmonary hemorrhage,acute alveolitis and other changes;in addition to the above performance,the case of the poisoning fibrosis group mainly showed pulmonary fibrosis and alveolar fibroblast proliferation,collagen.Deposition,even changes in lung consolidation,loss of tissue structure,etc.Immunohistochemical staining showed that the expression of Beclin 1,LC3 and p62 in the lung tissue of thepoisoning fibrosis group was significantly higher than that in the control case and the non-fibrotic group.There was a significant correlation with pulmonary fibrosis.These results suggest that autophagy inhibition may be involved in the formation of PQ-induced pulmonary fibrosis.Immunohistochemical staining also revealed elevated expression ofα-SMA in PQ-induced pulmonary fibrosis,suggesting that myofibroblastic differentiation may be involved in pulmonary fibrosis.2.The weight of mice in the PQ group showed a trend of decreasing first and then increasing;the main pathological manifestations of lung tissue in the PQ group were mainly pulmonary edema,acute alveolitis and pulmonary fibrosis.The fibrosis score showed a significant increase in the experimental group at 7d,14 d,21d,and the difference was statistically significant(P<0.05).The fibrosis of the 28 d group was decreased,but it was still significantly higher than the control group.The change trend of HYP content in each experimental group was almost the same as that of fibrosis.3.The contents of Beclin 1,LC3-II,p62 and α-SMA in the lung tissues of the PQ group at different time points were different,except for the 1d group,the other groups were statistically significant compared with the control group(P<0.05).There were significant differences between the groups(P<0.05).Beclin 1,LC3-II and p62 showed parallel expression,and there was a bimodal change with the time of exposure,and two peaks appeared at 3d and 21 d,respectively.The expression ofα-SMA increased continuously with the exposure time,peaked at 21 d,and decreased slightly at 28 d,but still higher than the control.4.RAPA reduced the expression of LC3-II and p62 protein and the number of LC3 fluorescent spots in PQ-induced fibroblasts,and decreased the expression of fibroblasts,α-SMA and Col-I protein,indicating that RAPA reduced PQ induction.Autophagy inhibits and reduces fibrotic changes.Beclin 1 siRNA silencing or autophagy inhibition by CQ increased PQ-induced expression of α-SMA and Col-I protein in fibroblasts,suggesting that autophagy inhibition can further aggravate fibroblast fibrosis.Conclusion: 1.PQ poisoning leads to blockage of autophagy in lung tissue cells.2.PQ poisoning may block autophagic flow by impairing lysosomal function.3.PQ poisoning-induced autophagic flow block participates in the formation of pulmonary fibrosis by promoting myogenic differentiation.4.Activation of autophagy may be a therapeutic target for lung fibers induced by PQ poisoning.5.The degree of pulmonaryfibrosis caused by different survival time after PQ poisoning may be used as an inferred basis for the time of poisoning. |
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