The Protective Effect And Underlying Mechanism Of Amitriptyline On Paraquat-Induced Pulmonary Fibrosis In Mice

Background:Paraquat（PQ）is still widely used as a fast-acting contact quaternary amineherbicide with high toxicity to humans and animals.Ingestion is the main mode ofhuman paraquat poisoning,and some patients suffer from severe depression.Themortality rate of paraquat poisoning is high.Even if some patients survive fromhemoperfusion,glucocorticoid,antioxidant,immunosuppression and other treatments,they are generally left with sequelae such as severe pulmonary fibrosis and otherorgan dysfunction.Paraquat poisoning has become the first pesticide poisoning typewith absolute number of deaths.The lung is the main target organ of injury after acute paraquat poisoning（APP）,and the concentration of paraquat in the lung tissue can reach 10-90 times the plasmaconcentration.Paraquat poisoning can promote the release of a large number ofinflammatory factors and oxidative stress,which in turn can further stimulate theproduction of inflammatory cytokines through various signaling pathways.Thus,theearly manifestation of paraquat pulmonary toxicity is acute alveolitis.As the diseasein progresses,myofibroblasts penetrate into the alveolar space and septum,along withthe differentiation of myofibroblasts,which progressively worsens and rapidlyprogresses to irreversible pulmonary fibrosis.Severe hypoxemia which due to thepulmonary fibrosis is the most important cause of death in patients with paraquatpoisoning.At present,there is no specific antidote or treatment in clinical practice,theexact mechanism of lung injury and fibrosis in paraquat poisoning has not been fullyelucidated.The diagnosis and treatment of paraquat poisoning is still a topic studywhich is difficult to conquer at present.Therefore,it is very important to deeplyinvestigate the pathophysiological mechanism of acute paraquat poisoning and findnew,safe and effective therapeutic strategies to improve the treatment and prognosisof paraquat poisoning.The lung is also a reservoir for antidepressants.As a classic representative oftricyclic antidepressants（TCAs）,amitriptyline（AMT）not only has antidepressanteffects,but also is considered to have multiple effects in current studies.Studies havefound that amitriptyline has an effective effect by altering cytokine activity,inhibiting the release of proinflammatory cytokines such as interleukin-1（IL-1）,nuclearfactor-κB（NF-κB）and tumor necrosis factor-α（TNF-α）,increasingimmunosuppression and the release of interleukin-10（IL-10）.By inhibiting theactivity of natural killer cells and the production of nitric oxide（NO）,prostaglandinE2（PGE2）and hyaluronic acid,amitriptyline also showed strong antioxidantproperties.In recent years,with the development of TCAs in anti-fibrosis,the resultsclearly show that amitriptyline has anti-fibrosis effect.Amitriptyline candown-regulate tubular myofibroblast differentiation,reduce hepatic collagendeposition,decrease fibrotic mediators,TGF-β1 and TNF-αmRNA expression,andprotect the development of renal and hepatic fibrosis through a variety of mechanisms.Further studies also found that amitriptyline could down-regulate pulmonary arterialpressure and improve pulmonary function in a mouse model of fibrosis（CF）,while itcould significantly inhibit bleomycin-induced pulmonary fibrosis in mice.The organprotection and anti-fibrosis effects of amitriptyline based on oxidation are basicallyclear,but whether it can treat pulmonary fibrosis caused by paraquat poisoning isunknown.Our topic was to establish an animal model of paraquat poisoning by single doseintragastric administration and intervene the pulmonary fibrosis model in paraquatpoisoned mice with amitriptyline drugs to study whether it has a protective effect onparaquat-induced pulmonary fibrosis in mice,which by observing thehistopathological changes in the lung,the degree of pulmonary edema,inflammatoryfactors,fibrosis-related factors,and the expression of epithelial-mesenchymaltransition（EMT）-related proteins and caveolin-1 proteins.In addition,weinvestigated the effect of amitriptyline on blood gas analysis and survival in fibroticmice.In order to investigate the possible mechanism of amitriptyline inhibits EMT inA549 cells,Caveolin-1 gene was silenced by in vitro cell assay and siRNAtransfection technique was used to study the effects of amitriptyline onparaquat-induced cell apoptosis rate and EMT-related protein expression.That in all,the study was to provide a strong experimental basis for the clinical application ofamitriptyline in the treatment of paraquat-induced pulmonary fibrosis.Part I:Establishment of paraquat-induced pulmonary fibrosis model in miceObjective:To establish a stable mouse model of pulmonary fibrosis caused by acuteparaquat poisoning by intragastric administration,and to explore the appropriateexposure dose.Methods:Forty C57BL/6 mice were randomly divided into four groups of 10 miceeach:control group（Ctrl group）;low-dose paraquat exposure group（L-PQ group）;medium-dose paraquat exposure group（M-PQ group）and the high-dose paraquatexposure group（H-PQ group）.The general status and survival of mice in the fourgroups were recorded for 21 days after intragastric administration with 1 ml normalsaline or diluted with 10mg/kg,50mg/kg,150mg/kg paraquat plus normal saline tolml respectively.By the end of the experiment,the lung tissues of surviving micewere taken for HE staining and Masson staining to observe the pathological changesof lung tissues,and the Ashcroft score of each group was calculated to evaluate thedegree of fibrosis.Results:1.General status and survival rate:Paraquat-exposed mice have obviousgastrointestinal symptoms in the early stage,mainly respiratory symptoms and signsin the middle and late stages,and the more exposure dose,the more significant of theclinical manifestations.The trough of weight loss in paraquat-exposed mice was aboutin the 7th day,and then gradually increased,with a more significant increase in the M-PQ than H-PQ group.By day 21,there was a significant difference in the bodyweight of mice among the four groups（p<0.05）.Compared with that before exposure,the body weight decreased more significantly in the H-PQ than M-PQ group（p<0.01）.The dead mice in the H-PQ group were within 14 days of the experiment,and therewere no deaths in the other groups.2.Lung histopathology:mice in the Control group had normal lung volume,clear andintact tissue structure,and no inflammatory or fibrotic changes.In the L-PQ group,inflammation were predominant in the pulmonary interstitium,collagen fiberproliferation and fibroblasts were also observed.In the M-PQ and H-PQ groups,thelung volume was reduced,alveolar exudation,septal edema,inflammatory cellinfiltration,fibroblast proliferation,disorganized and thick collagen fiber proliferationwere observed.Lung tissue consolidation and alveolar structure collapse were also observed in the H-PQ group.The Ashcroft scores of the Control,L-PQ,M-PQ andH-PQ groups were 0.3±0.13,3.09±0.28,5.38±0.44 and 6.74±0.35 respectively,whichwere significant differences between the groups（p<0.01）.Conclusion:The C57BL/6 mouse model of pulmonary fibrosis was successfullyestablished on day 21 after intragastric administration of 50mg/kg paraquat.Thehigh-dose gavage administration method of 150mg/kg paraquat can be used to studythe survival rate of modeling mice.Part II:Protective effect of amitriptyline on paraquat-inducedpulmonary fibrosis in miceObjective:To investigate whether amitriptyline has a protective effect onparaquat-induced pulmonary fibrosis in mice.Methods:Fifly-eight C57BL/6 mice were randomly divided into six groups.8 miceper group in the control group（Ctrl group）,paraquat exposure group（PQ group）,paraquat exposure and amitriptyline intervention group（PQ+AMT group）and theamitriptyline group（AMT group）.There were 13 mice each in the high-dose paraquatexposure group（H-PQ group）and the high-dose paraquat exposure and amitriptylineintervention group（H-PQ+AMT group）.PQ-exposed and H-PQ-exposed mice wererespectively modeled with 50mg/kg and 150mg/kg paraquat in the same way as in thepart I.The control and amitriptyline intervention groups were intragastricallyadministered with 1 ml saline or 20mg/kg amitriptyline once daily until day 21 as theend point of observation,respectively.The protective effect of amitriptyline onpulmonary fibrosis in mice was comprehensively evaluated by observing the generalstatus;the survival rate of mice exposed to high-dose paraquat;histopathological andultrastructural observation of lung tissues;detecting the levels of TNF-α,DL-1β TGF-β1 and IL-17 in lung homogenates by ELISA,together with the TGF-pi andIL-17 in serum;the correlation analysis between Ashcroft score and TGF-pi as wellas IL-17 levels in lung tissues;study the effect of amitriptyline on EMT relatedproteins such as E-cadherin,N-cadherin,a-SMA and Caveolin-1 expression in lungtissues using immunohistochemistry and Western-blot.In addition,we also studied theeffects of amitriptyline on the internal environment such as oxygenation andmetabolism in flbrotic mice by drawing abdominal aortic blood for blood gas analysis.Results:1.General state.The coat color,reaction and respiration of mice in the Control or AMT groupwere unremarkable.The digestive tract and respiratory symptoms of mice in the PQ+AMT group,who with luster coat color,were milder than those in the PQ group.The body weight on day 21 in the PQ group and PQ+AMT group were 20.28±1.23g and 21.41±1.51g,whose difference was not statistically significant（p=0.12）,but theywere significantly lower than those in the Control group,respectively（p<0.01&p<0.0l）.Survival analysis:There were no deaths in experimental mice at a paraquat dose of 50mg/kg.The 21-day survival rates of H-PQ and H-PQ+AMT mice were 53.85% and 84,62%,with no significant difference between them（p=0.095）.Amitriptylineintervention could not improve their short-term survival rates.2.Gross morphology and lung coefficient.Mice in the PQ+AMT group had slightly larger lung volume,less congestion onthe lung surface,and moderate humidity than those in the PQ group.The lungcoefficients in PQ and PQ+AMT groups were 0.79±0.10 and 1.07±0.15 respectively,which were higher than those in Control group of 0.60±0.07（p=0.017&p<0.01）.Thelung coefficients in PQ+AMT group were lower than those in PQ group（p<0.01）.And there was no significant difference between the other groups,which suggestingthat amitriptyline gavage could reduce the lung coefficients of fibrotic mice inducedby PQ.3.TEMM.itochondrial swelling,loss of mitochondrial ridges,widening of cell matrixspaces,thickening of alveolar septa and proliferation of elastic fibers,collagen fibers,reticular fibers in alveolar septa were significantly typical findings of ultrastructuralchanges in lung histiocytes in the PQ group.The PQ+AMT group were milder thanthose in the PQ group,and the Control and AMT groups were intact.4.Blood gas analysis results.One-way ANOVA showed no significant difference in PH values among the fourgroups（p=0A5）,PaC〇2 and Lactic acid levels were slightly higher in the PQ groupthan in the Control and PQ+AMT groups,but the differences were not significant （p=0.093&p=0.97）,SaO₂（86.71%±4.87%）and PaO₂（87.84±9.99mmHg）levels were significantly decreased in the PQ group compared with those in the Control group of 93.4%±1.99%and 110.53士8.37mmHg（/?=0.003&p=0.0002）.SaO₂（91.15%±1.71%）and Pa〇2（105.15±7.61 mmHg）levels were higher in thePQ+AMTgroup than in the PQ group,and the difference were also statisticallysignificant（p=0.03&/?=0.002）.5.Histopathology of lung tissues.In the PQ group,most of the fibrotic lesions were located in the subpleural andperivascular areas,fibroblasts proliferated significantly and collagen fibers weredisorganized and thick;the pathology in the PQ+AMT group were milder than those in the PQ group,and the pulmonary interstitium was still dominated by inflammation,with collagen fiber and fibroblast proliferation.The Ashcroft scores in the PQ andPQ+AMT groups were 5.53±0.57 and 3.91±0.24,and the difference was statisticallysignificant（p<0.05）.6.Amitriptyline inhibited PQ-induced lung inflammation in mice.In the lung tissue of mice,the TNF-a level in the PQ group was 194.49±22.57ng/L,which significantly higher than that in the Control group（91.98±8.79ng/L）;the PQ+AMT group and AMT group were 111.70±8.25ng/L and 82.78±8.52ng/L,whose differences were significant（p<0.01）.The IL-1β level in the PQ group was also higher than that in the other groups respectively（p<0.01）.Thelevel of IL-1β in PQ+AMT group was 8.98±1.42ng/L,which was significantly lowerthan that in PQ group of 10.70±1.71ng/L（p=0.046）.7.Amitriptyline down-regulates hydroxyproline content in paraquat-induced lungtissue of mice.The hydroxyproline content in lung tissue of PQ group was 1171.68士142.19(μg/g,which was higher than that of Control group（746.36±18.20|ig/g）,PQ+AMT group（839.3土40.99|ig/g）and AMT group(747.2±39.32（μg/g）.There was significantdifference between PQ and PQ+AMT group（p<0.01）.8.Amitriptyline downregulated TGF-β1 and IL-17 levels in lung tissue anddownregulated TGF-β1 levels in serum.Detection of TGF-β1 level in lung tissue by ELISA showed:PQ+AMT groupwas 263.48±8.57ng/L,which significantly lower than PQ group of 327.91±4.59ng/L（p<0.01）;serum level of TGF-pi in PQ+AMT group was 131.76±6.31ng/L,lower than PQ group of 140.69±6.83ng/L the difference was also significant（p=0.047）.However,there were no significant differences between the other groups.The IL-17 level in lung tissue of the PQ+AMT group was 27.70±2.78ng/L,which lower than the PQ group of 43.08±0.85ng/L（p<0.01）.The serum level of IL-17in the PQ+AMT and PQ group were 19.32±0.53ng/L and 19.71±0.59ng/Ls with nosignificantly difference（p=0.19）.Besides,there were no significant differencebetween the other groups of the serum IL-17 level（p>0.05）.Pearson correlationanalysis showed a positive correlation,that to say the higher the Ashcroft score,thehigher the levels of TGF-β1 and IL-17 in lung tissue.9.Amitriptyline inhibits EMT and up-regulates caveolin-l protein in lung tissue ofmice with paraquat-induced pulmonary fibrosis.Immunohistochemical results showed that the expression of E-cadherin proteinin the lung tissue of mice in the PQ group was decreased and the N-cadherin and α-SMA proteins were increased,with the pulmonary interstitial fibers proliferatedsignificantly.E-cadherin protein in the PQ+AMT group was higher than that in the PQ group,while α-SMA and N-cadherin protein expression were decreased,with pulmonary interstitial fibroplasia was relieved.Caveolin-1 protein was positivelyexpressed in the alveolar membrane and cytoplasm of the Control and AMT groups,and weakly positive or negative expression in the PQ group.Caveolin-1 proteinexpression in the PQ+AMT group was stronger than that of the PQ group,whilepulmonary interstitial fibrosis was weaker than that of the PQ group.The western-blot results showed that the level of E-cadherin protein in lungtissue of mice in the Control and AMT groups was high,and the PQ+AMT group wassignificantly down-regulated compared with the Control group（p<0.01）;theexpression of E-cadherin protein in the PQ+AMT group was higher than that in the PQ group,but the difference was not significant（p=0.85）.The expression of α-SMA and N-cadherin protein in lung tissue of PQ group were higher than that of Controlgroup（p<0.01） and the level of both proteins in PQ+AMT group was significantlydown-regulated compared with PQ group（p<0.0l&p=0.0l2）.The expression ofCaveolin-1 protein in PQ group was significantly lower than that of Control group（p<0.01）and also significantly lower than that of PQ+AMT group（p=0.047&p=0.004）.Conclusion:1.Amitriptyline improves the general status and lung histopathology of paraquat-induced fibrotic mice,but does not improve 21-day survival.2.Amitriptyline improves PaO₂ and SaO₂ levels in fibrotic mice,which may berelated to the fact that amitriptyline protects the structural and fiinctional integrity ofcellular mitochondria.3.Amitriptyline inhibits paraquat-induced pulmonary fibrosis in mice,and caveolin-1protein expression was modulated by amitriptyline intervention.Amitriptyline alsocan inhibit lung inflammation and improve lung fibrosis.Part III:Amitriptyline inhibits paraquat-inducedepithelial-mesenchymal transition in A549 cells by up-regulatingcaveolin-1 Objective:To investigate the effect of amitriptyline on paraquat-induced EMT andapoptosis in A549 cells,preliminarily explore its possible mechanism of action.Methods:A549 cells were cultured.1.CCK-8 assay was used to detect the cell viability of A549 cells,which treated withdifferent doses of AMT（1.0μmol/L,5.0μmol/L and 25μmol/L）for 48h.After A549 cells were treated with different doses of AMT or PQ（50μmol/L,lOOμmol/L and ?200μmol/L）for different time length,total cellular RNA was extracted,E,cadherinand N-cadherin levels of intracellular were determined by RT-PCR.2.A549 cells who induced by paraquat（100μmol/L）were treated with different dosesof amitriptyline（l.Oμmol/L,5.0μmol/L,25μmol/L）for 48h.Total cellular proteinswere extracted and intracellular levels of E-cadherin,N-cadherin,α-SMA and Caveolin-1 proteins were determined by western blot;the effect of different doses of amitriptyline on the apoptosis rate of paraquat-induced A549 cells was detected by flow cytometry too.3.A549 cells were pretransfected with Caveolin-1 siRNA,then induced by 100μmol/L paraquat and simultaneous interved by 25μmol/L amitriptyline for 48h.Total cellular protein was extracted and intracellular levels of E-cadherin,N-cadherinand α-SMA proteins were determined by western blot;the apoptosis rate of A549 cellsin each group was also detected by flow cytometry.Results:1.Different doses of amitriptyline had no significant effect on A549 cell viability（p=0.123）as well as E-cadherin and N-cadherin levels.Compared with the controlgroup,the apoptosis rate of A549 cells was significantly increased after paraquattreatment（15.02±0.44%vs 3.76±0.29%,p<0.01）.Meanwhile,paraquat significantlydecreased E-cadherin and increased N-cadherin expression in A549 cells,both of which were significantly regulated by increasing paraquat concentration.However,there were no significant differences in E-cadherin and N-cadherin expression between M-PQ and H-PQ groups（p=0.07&p=0.34）.2.Compared with the PQ group,different concentrations of amitriptylinesignificantly decreased the apoptosis rate,the expression of N-cadherin and α-SMA protein,while increased E-cadherin protein level in A549 cells,who were inducted by paraquat.AMT can increas Caveolin-1 protein expression in A549 cells indose-dependent.3.Compared with the PQ+AMT+siControl group,A549 cells pretransfected with Caveolin-1 siRNA had increased α-SMA and N-cadherin protein expression but decreased E-cadherin protein expression,which indicating that Caveolin-1 is involvedin the EMT process and mediates the expression of EMT-related proteins.Theapoptotic rate of A549 cells,which pretransfected with Caveolin-1 siRNA,induced by paraquat and amitriptyline（i.e.,PQ+AMT+siCaveolin-1 group）was not significantly different from the PQ+AMT+siControl group（p=0.604）.Conclusion:1.Amitriptyline at 25μmol/L was not significantly toxic to A549 cells.2.Amitriptyline dose-dependently upregulated caveolin-1 to inhibit the EMT processinduced by paraquat in A549 cells.3.Paraquat induced apoptosis in A549 cells in a concentration-dependent manner,and amitriptyline could down-regulate their apoptosis rate,but it was not related tocaveolin-1.