Building Of An Animal Model Of Silicosis And The Effect And Mechanism Of Novel N-arylpyridone Compounds On Silicosis In Mice

Background:Silicosis is a fatal occupational disease caused by human beings inhaling a large amount of dust mainly composed of silica particles for a long time during occupational activities.Silicosis is the most important,common and harmful type of pneumoconiosis.In addition to pulmonary fibrosis,suffering from silicosis will also increase pulmonary tuberculosis,chronic obstructive pulmonary disease,lung cancer,cardiovascular disease and various autoimmune diseases.Nowdays,the number of people exposed to occupational dust and the number of newly reported cases of silicosis continue to increase despite aggressive control measures against silicosis worldwide.There is a lack of effective treatment for silicosis.In-depth exploration of the pathogenesis of silicosis and the development of effective therapeutic drugs are urgent problems to be solved in its diagnosis and treatment.But at the same time,the lack of standardized animal models and the unclear pathophysiological process of silicosis limit the development of silicosis research.AKEX0011 is an oral novel N-arylpyridone compound optimized for pirfenidone(PFD)-based phenylpyridone scaffolds with improved PFD pharmacokinetics,antifibrotic activity and tolerability.In preclinical studies,AKEX0011 exhibited better anti-fibrotic activity in animal models of lung and liver fibrosis,improved oral bioavailability,and decreased clearance compared to PFD.Objectives:1.To construct a mouse silicosis model with multiple doses and multiple time points,conduct comprehensive evaluation of the model,explore the most suitable modeling conditions and explore the pathophysiological changes of the silicosis model2.To evaluate the effectiveness of AKEX0011 for the early and late treatment of silicosis from various aspects and perspectives3.To explore the potential pharmacological mechanism and main targets of AKEX0011 in the treatment of silicosis4.Find the targets of silicosis treatmentMethods:C57BL/6J mice received the intratracheal instillation of the silica suspension with silica 0 mg/Kg 200 mg/Kg 400 mg/Kg 600 mg/Kg at 7 days,28 days and 42 days,respectively,through laryngoscope.Then at the corresponding time point,the lung function test was used to evaluate the deep inspiratory capacity(IC),respiratory system elasticity(Ers),respiratory system resistance(Rrs),static compliance(CST),tissue damping(G)and other lung functions of different models.Histopathological damage was assessed by HE staining and Masson staining.Real-time quantitative PCR and enzyme-linked immunosorbent assay(ELISA)were used to evaluate the fibrosis indicators in lung tissue and(or)bronchoalveolar lavage fluid(BALF)of each group--hydroxyproline content(HYP),TGF-?,Collagen-?,Fibronectin and inflammatory markers-TNF-?,IL-1?,IL-6.Focus on the use of flow cytometry to evaluate neutrophils,eosinophils,DC cells,macrophages,alveolar macrophages(AM)/interstitial macrophages(IM)typing,macrophage M1 in lung tissue /M2 subtype and other inflammatory cell infiltration to understand the dynamical pathophysiological characteristics of silicosis models.characteristics of silicosis models.In the same modeling method,600 mg/Kg silica was used to make mice model,and AKEX0011 was intragastrically administered on the day of modeling(early treatment)and the 14 th day(late treatment),and the drug was administered continuously for 4 weeks.Pulmonary function test was used to evaluate pulmonary function changes,Micro-CT to evaluate imaging changes,HE staining,Masson staining,Sirius Red to evaluate histopathological damage,and immunohistochemical staining and immunofluorescence staining to evaluate the morphology of Collagen-?,Fibronectin,and ?-SMA.The infiltration of neutrophils and macrophages in lung tissue was assessed by flow cytometry.Using real-time quantitative PCR,ELISA,and Western blot to evaluate the fibrosis indicators Collagen-?,Fibronectin,?-SMA,HYP,TGF-?,IL-4,IL-10 and inflammatory markers(TNF-?,IL-1?,IL-6)in lung tissue and/or BALF.Then we further explored the mechanism of drug action.Cell apoptosis was detected by tunel staining,ASK-1 P-ASK-1 P38 P-P38 protein expression was detected by Western blot(WB),immunohistochemical staining of M1/M2 markers i NOS and Arg1,real-time fluorescent quantitative PCR,and Western blot The protein and m RNA expressions of i NOS and Arg1 were detected.WB detection of P-NF-?B/NF-?B,I?B?,PPAR-?.In addition,the mouse macrophage cell line(RAW 264.7)was stimulated with silica to construct a silicosis cell model,and different concentrations of AKEX0011 were added at different time points for incubation.The m RNA and protein levels of TNF-?,IL-1?,IL-6 and TGF-? in cell lysates and/or cell supernatants were detected by real-time fluorescence quantitative PCR,ELISA and WB.Cell apoptosis and macrophage M1/M2 polarization were detected by flow cytometry.The changes of i NOS and Arg1 and the activation of ASK-1/P38 protein in each group were further detected by WB.Results:1.In different mouse models of silicosis,the degree of lesions caused by silica exposure is dose and time dependent Pulmonary function indicated that moderate to high concentration of silica exposure(400 mg/Kg and 600 mg/Kg)could induce significant damage to lung function at 28 days,and the damage was more pronounced at 42 days.HE staining showed that the middle and high doses of silica could cause significant pulmonary inflammatory damage within 7 days,and persisted throughout the course of the disease,while the changes were not obvious at any time at lower doses.In the same high-dose group,the secretion of inflammatory factors(TNF-a,IL-1B,IL-6)in BALF increased in the early stage.Flow cytometry of lung tissue showed that inflammatorycells such as neutrophils,eosinophils,DC cells,and macrophages were significantly increased in the model course,and the increase was most obvious on the 7th day.Unlike inflammation,pulmonary fibrosis progresses more slowly,silicic nodules with collagen deposition can be observed at D28 D42,and fibrosis markers(COLI,FN and HYP)levels are also elevated at D28 and at D42 more obvious.Low doses and short-term exposure to silica are difficult to cause significant fibrosis.In addition,the M1/M2 phenotype of macrophages in the silicosis model was significantly increased.M1 peaked on day 7 and M2 peaked on day 42.2.Exploration of the effectiveness of AKEX0011 in the treatment of silicosis mice Early treatment of AKEX0011 alleviated the imaging damage and pulmonary dysfunction in silicosis mice,alleviated the pathological damage of lung tissue by HE staining,Masson staining,Sirius Red,and reduced inflammatory and fibrotic factors(TNF-?,IL-1?,The secretion of IL-6,TGF-?,IL-4 and IL-10 at the protein level and m RNA level),reduced the infiltration of neutrophils and macrophages in lung tissue,and reduced the fibrosis-related protein levels and m RNA expression(Collagen-I,fibronectin and ?-SMA).AKEX0011 also attenuated lung function impairment,inflammation,and fibrosis in mice silicosis in late-stage treatment.3.To explore the potential pharmacological mechanism and main targets of AKEX0011 in the treatment of silicosis AKEX0011 can reduce the proportion of Tunel-positive cells,that is,inhibit apoptosis.It can reduce the phosphorylation of ASK-1/p38 protein,that is,inhibit the ASK-1/P38 signaling pathway,and can regulate the M1/M2 polarization of macrophages by reducing the protein phosphorylation of NF-?B/PPAR-?.4.Efficacy and mechanism exploration of AKEX0011 in the treatment of silicosis cell model In in vitro experiments,AKEX0011 can inhibit the secretion of cytokines(TNF-?,IL-1?,IL-6,TGF-?)by macrophages in both early and late treatment cell models of silicosis.Flow cytometry showed that both early and late treatment ofAKEX0011 inhibited apoptosis.At the same time,AKEX can also block the ASK-1/P38 pathway,and the use of P38 activators can partially reverse the therapeutic effect of AKEX0011.AKEX0011 and can inhibit M1 polarization by inhibiting the phosphorylation of NF-?B.Conclusion:1.600 mg/Kg silica exposure for 28-42 days can establish suitable mouse models of silicosis2.The progression of silicosis mice can be divided into inflammatory phase,advanced phase and fibrosis phase.The inflammatory phase is considered to be 0-7days,with obvious pulmonary inflammation;the advanced phase is considered to be7-28 days pulmonary dysfunction occurs,and the pulmonary inflammation and fibrosis continue to worsen;the fibrosis phase is considered to be 28-42 days,the degree of fibrosis and pulmonary insufficiency significantly worsened,but the inflammation did not continue to worsen.3.AKEX0011,as a novel N-arylpyridone compound,has protective effects against silica-induced lung inflammation and fibrosis in vivo and in vitro,so it may be a strong candidate for the treatment of silicosis.4.AKEX0011 can play a therapeutic role in silicosis by inhibiting ASK-1/P38 signaling pathway and regulating macrophage polarization.5.ASK-1/P38 signaling pathway and macrophage polarization are important mechanisms of silicosis,which is expected to be a potential therapeutic target for silicosis.