| Background and objective: Paraquat(PQ)poisoning remains a relatively high incidence of pesticide poisoning,especially in Asian countries and China.Paraquat will accumulated in the lungs due to the monoamine uptake system of the lungs.Acute lung injury(ALI)occurs soon in the early stage of paraquat poisoning,followed by irreversible pulmonary fibrosis,which eventually leads to death.Pulmonary fibrosis is a characteristic change after paraquat poisoning and is one of the main causes of death in PQ poisoning patients.However,the profound mechanism of paraquat poisoning leading to pulmonary fibrosis has not yet been fully elucidated.Current research suggests that epithelial-mesenchymal transition(EMT)may be an important pathway for pulmonary fibrosis.After lung injury,alveolar type II epithelial cells undergo a series of gene regulation,lose their epithelial cell phenotype,and then transform into mesenchymal cells to become myofibroblasts which is capable of expressing extracellular matrix such as type II collagen,eventually leading to interstitial collagen deposition,pulmonary fibrosis,oxygen diffusion dysfunction,and even death.Previous studies by our team have shown that hypoxia-inducible factor-1alpha(HIF-1α)plays an important role in epithelial-mesenchymal transition induced by paraquat poisoning.HIF-1α is a nuclear transcription factor,and its main function is that when hypoxia occurs in the body and tissues,HIF-1α can regulate the expression of target genes,which makes the body tolerant to ischemia and hypoxia.HIF-1α is involved in various cellular responses such as ischemia,hypoxia,inflammation,immune response,and tissue repair.A variety of studies have shown that HIF-1α can also induce epithelial-mesenchymal transition.Our previous studies have shown that HIF-1α promotes the expression of mesenchymal markers such as α-smooth muscle actin(α-SMA)in alveolar type II epithelial cells through positive feedback regulation,which promotes collagen deposition and leads to pulmonary fibrosis.Statins,a 3-hydroxy-3methylglutaryl coenzyme A(HMG-Co A)reductase inhibitor,are classic lipid-lowering drugs.At the same time,statins are the first-line preventive drugs for coronary heart disease and second-line preventive drugs for ischemic stroke.In addition to the effects of lowering blood fat and stabilizing vascular plaque,statins have many other physiological effects.These pleiotropic effects are the result of the action of statins as inhibitors of cholesterol synthesis and inhibitors of isoprene synthesis.These compounds are ultimately responsible for important pathways that regulate several diseases,including endothelin,nitric oxide synthase and decreased production of nicotinamide adenine dinucleotide phosphatase.Statins can increase endothelial function,reduce the amount of active oxidants,improving the pathology of certain diseases,reducing the expression of pre-inflammatory,immunomodulation,stabilizing the endothelial plate,regulating sympathetic excitability,reducing the activity of the clotting cascade,inhibiting platelet adhesion,and may physiological effects.There have been many studies showing that statins play a therapeutic role in many pulmonary diseases.Statins can be treated by inhibiting inflammatory responses by improving cellular function.Studies have shown that statins also regulate HIF-1α expression to exert biological effects.In previous studies,it was reported that the degree of pulmonary fibrosis caused by paraquat was reduced after treatment with the most common drug,atorvastatin(ATS),in rats.Therefore,we hypothese that atorvastatin may be able to regulate HIF-1α expression and theninfluence epithelial-mesenchymal transition and improve pulmonary fibrosis caused by paraquat poisoning.Methods 1.Atorvastatin attenuates lung injury and pulmonary fibrosis caused by paraquat poisoning.(1)SD rat in vivo model of PQ poisoning were established and divided into control group,PQ group,PQ+ATS 20 mg/kg group,and PQ+ATS 40 mg/kg group.The inflammatory cell infiltration of lung tissue was observed by HE staining.MASSON staining was used to observe the phenomenon of collagen deposition in lung tissue.The hydroxyproline content in the lung tissue was quantitatively determined to assess the collagen content and degree of fibrosis in the lung tissue.2.ATS inhibits PQ-induced epithelial-mesenchymal transition(1)SD rat in vivo model of PQ poisoning were established and divided into control group,PQ group,PQ+ATS 20 mg/kg group,and PQ+ATS 40 mg/kg group.Western Blot was used to detect the expression of epithelial marker ZO-1,E-Caderin,α-SMA and Vimentin in different groups of lung tissues.Immunohistochemistry was used to detect the expression of interstitial marker α-SMA in lung tissue of different groups.(2)To test the cytotoxic effect of different concentrations of ATS on A549 and RLE-6TN cell line to choose the appropriate ATS concentration as the in vitro test concentration.(3)The A549,RLE-6TN cell lines was used to establish in vitro PQ poisoning model,which were divided into PQ group,PQ+ATS low dose group and PQ+ATS high dose group.Western Blot was used to detect epithelial markers ZO-1,E-Caderin and mesenchymal markers α-SMA and Vimentin in different groups.(4)The A549,RLE-6TN cell lines was used to establish in vitro PQ poisoning models,which were divided into PQ group,PQ+ATS low dose group,PQ+ATS high dose group.Cell morphology under microscope was observed.(5)Using A549 cell lines to establish in vitro PQ poisoning models,which were divided into PQ group,PQ+ATS low dose group,PQ+ATS high dose group.Immunofluorescence was used to observe the expression of epithelial marker ZO-1 and interstitial marker α-SMA between groups.3.ATS inhibited epithelial-mesenchymal transition by regulating the expression of HIF-1α/β-catenin(1)The SD rats in vivo models of PQ poisoning were established and divided into control group,PQ group,PQ+ATS 20 mg/kg group,and PQ+ATS 40 mg/kg group.Western Blot was used to detect the expression of HIF-1α β-catenin in lung tissue of different groups.Immunohistochemistry was used to detect the expression of HIF-1α in lung tissue of different groups.(2)In vitro PQ poisoning models were established by A549,RLE-6TN cells,which were divided into PQ group,PQ+ATS low dose group and PQ+ATS high dose group.Western blot was used to detect the expression of HIF-1α and β-catenin.(3)In vitro PQ poisoning model was established by A549 cell lines,which was divided into PQ group,PQ+ATS low dose group,PQ+ATS high dose group,and immunofluorescence observation of cell line HIF-1α.Results 1 Atorvastatin attenuated lung injury and pulmonary fibrosis caused by paraquat poisoning(1)HE staining of SD rats in vivo showed that the degree of inflammatory cell infiltration was significantly reduced in the statin group compared with the paraquat group.Which was more obvious after 72 h.MASSON staining showed that collagen deposition was reduced at 24 h,72h and 168 h.(2)In the in vivo model of SD rats,quantitative determination of hydroxyproline showed that collagen deposition was significantly reduced at 24 h,72h and 168 h.At168h,the collagen deposition in the PQ+ATS40mg/kg group was significantly less than that in the PQ+ATS20mg/kg group.2.ATS inhibits PQ-induced epithelial-mesenchymal transition(1)In the in vivo model of SD rats,the statin group compared with the paraquat group,Western Blot showed that the epithelial marker E-Cadherin,ZO-1 expression in the statin group increased espicially in the high dose groups.Vimentin,α-SMA was decreased in the ATS treated group than in the PQ group which was more obvious in the high dose ATS group.(2)In the in vivo model of SD rats,immunohistochemistry showed a decreased α-SMA expression in the ATS treated group than in the PQ group.(3)Western Blot showed that the expression of E-Cadherin and ZO-1 in the ATS treated groups was increased in the in vitro model of A549 cells and RLE-6TN cell lines especially in the high dose ATS group.The mensenchymal marker Vimentin,α-SMA expression in the ATS treated group was decreased and the high dose ATS group was more obvious.(4)Immunofluorescence in an in vitro model of A549 cell line showed that the expression of the epithelial marker ZO-1 was increased in the ATS group,while the expression of the interstitial marker α-SMA was decreased.(5)In the in vitro model of A549 cell line and RLE-6TN cell line,ATS treatment attenuated PQ induced epithelial cell fusiform transition.3.ATS inhibits epithelial-mesenchymal transition by regulating the expression of HIF-1α/β-catenin(1)In the in vivo model of SD rats,compared with the PQ group,Western Blot method showed that the expression of HIF-1α,β-catenin in the ATS group was decreased.(2)In the in vitro model of A549,RLE-6TN cell line,compared with PQ group,Western Blot method showed that the expression of HIF-1α,β-catenin in ATS group was decreased.(3)In the in vivo model of SD rats,the immunohistochemical method showed that the expression of HIF-1α in the ATS group was decreased than in the PQ group.(4)In the in vitro model of A549 cell line,compared with the PQ group,immunofluorescence assay showed that the expressions of HIF-1α were decreased in the ATS groups.Conclusion Atorvastatin can alleviate pulmonary fibrosis caused by paraquat poisoning.The mechanism may be that atorvastatin inhibits epithelial-mesenchymal transition and reduces the degree of pulmonary fibrosis by reducing the expression of HIF-1α/β-catenin. |
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