| Objective:The addictive substance nicotine,found in cigarettes and some e-cigarettes,plays a vital role in pro-inflammatory and fibrotic processes.However,little is known about the role of nicotine in the progression of silica-induced pulmonary fibrosis.To observe the effect of nicotine on the progression of silicosis pulmonary fibrosis and to explore the effect of STAT3-BDNF-Trk B pathway on the progression of pulmonary fibrosis in mice exposed to nicotine combined with silica,as well as the impact and mechanism of blocking Trk B on the progression of pulmonary fibrosis.Methods:1.This study aimed to establish animal models for combined exposure to nicotine and silica.Thirty-two male C57/BL6 wild-type mice were randomly assigned to four groups: vehicle,nicotine,silica,nicotine combined with silica.The nicotine group was given a subcutaneous injection of nicotine(0.05 mg/m L,0.25 mg/kg)twice daily for 40 days and 50 μL saline via nasal administration for 15 days(from days 5 to 19).The silica group was injected with saline(0.25mg/kg)twice per day for 40 days,and silica particles was administered nasally at a dose of 20 mg/m L,50 μL for 15 days from days 5 to 19.The co-exposed mice were simultaneously exposed to nicotine and silica.They were injected with nicotine(0.25 mg/kg)for 40 days.From day 5 to 19,they were exposed to silica(20 mg/m L,50 μL)via nasal inhalation;The vehicle group was given a subcutaneous injection of saline twice a day from 1 to 40 days and had 50 μL sterile saline instilled via the nose from day 5 to day 19.On day 41,the mice were sacrificed to obtain the lung tissue.2.Pulmonary inflammation was evaluated utilizing Hematoxylin-eosin(H&E)staining,whereas pulmonary fibrosis score was assessed using the Ashcroft score.In addition,Masson staining was employed to evaluate the extent of fibrosis(collagen fibers).3.Immunohistochemical staining was utilized to determine the expression levels ofE-cadherin,fibroblast growth factor 7(Fgf7),prosurfactant protein C(SP-C),interleukin-33(IL-33),Ki67,brain-derived neurotrophic factor(BDNF),α-smooth muscle actin(α-SMA),and E-cadherin.Additionally,the colocalization and expression of double-positive cells for vimentin and E-cadherin/vimentin were also assessed.4.Western blot was used to detect Fibronectin,Collagen Ⅰ,SP-C,Fgf7,E-cadherin,N-cadherin,α-SMA,vimentin,STAT3,p-STAT3,BDNF,the tyrosine kinase receptor B(Trk B),p-Trk B,AKT,Snail,p-AKT and Twist in the lung tissue.5.The role of BDNF and its receptor inhibitors in type II alveolar epithelial cells(AT2)was scrutinized using A549 and MLE-12 cells in vitro experiments.The impact of BDNF(20 ng/m L),K252a(100 nmol/m L),BDNF(20 ng/m L)+ K252a(100 nmol/m L)on the STAT3-BDNF-Trk B signal was observed in A549 and MLE-12 cells.6.To further investigate the role of simultaneous exposure to nicotine and silica in activating STAT3-BDNF-Trk B signaling,cells were exposed to a medium containing nicotine(100 μmol/m L)and silica(50 μg/cm2)for 24 h or 48 h.K252 a was utilized to target the BDNF-specific receptor Trk B in the double-exposure group,with an introduction at 100 nmol/m L concentration.Results:1.A novel mouse model was created to investigate the effects of combined nicotine and silica exposure.Histological analysis via H&E staining revealed a marked increase in inflammatory cells in the lungs of mice belonging to the nicotine group,predominantly clustered around lymphatic and blood vessels.The lungs of mice treated with silica alone showed thickened alveolar septa and damaged alveolar structure,while those mice that were exposed to nicotine+silica showed more severe damage,with inflammatory cells infiltrating near lymphatic vessels.Furthermore,collagen fibers are deposited in fibrous clumps and individual alveoli.2.Immunohistochemical(IHC)staining observed proliferation of alveolar type II epithelial cells(AT2)in dual-exposed mice lungs(increased SP-C,Ki67;upregulated E-cadherin,Fgf7);increased pro-fibrotic factor IL-33;more severe EMT,with the number of α-SMA,vimentin-positive cells increased.Not only that,there were more E-cadherin/vimentin double positive cells compared to mice exposed to silica alone.3.Western blot and the quantification in dual-exposed mice lungs showed: the fibrosis markers(Fibronectin,Collagen I);increased expression of SP-C,E-cadherin,Fgf7;increased EMT-related proteins such as N-cadherin,vimentin,α-SMA;STAT3-BDNF-Trk B pathway related proteins such as p-STAT3,BDNF,p-Trk B were increased and the downstream pathway p-AKT was upregulated,p-AKT increased the EMT raleted transcription factor Twist;Twist was significantly upregulated and Snail was increased to a smaller extent compared with the silica group.4.The promotion of EMT in AT2 cells,which BDNF stimulated,was effectively suppressed upon the administration of K252 a.After experiencing scratches,the two types of cells supplemented with BDNF demonstrated a more rapid healing process after 24 hours.As the 72-hour mark approached,two kinds of cells transformed shape,resembling interstitial cells with reduced intercellular connections and greater cellular dispersion.The K252 a and BDNF+K252a groups demonstrated a notable inhibitory effect on the migration of both cell types at the 24-hour mark,resulting in a more interconnected cellular arrangement and the gradual formation of larger cell masses over a period of 72 hours.5.Upon exposure to nicotine and silica for 24 to 48 hours,an increase in the expression of p-Trk B,p-AKT,and Twist was noted.In addition,N-cadherin and Vimentin levels are heightened due to the pathway’s activation.However,the administration of K252 a caused a decrease in p-Trk B,p-AKT,Twist,N-cadherin,and Vimentin.Conclusion:1.The signaling pathway of STAT3-BDNF-Trk B is activated by nicotine,resulting in the induction of p-AKT expression by Trk B activation.This leads to an up-regulation of the transcription factor Twist,which is associated withepithelial-mesenchymal transition and accelerates the progression of pulmonary fibrosis in mice damaged by silica.2.The activation of the STAT3-BDNF-Trk B pathway increased Fgf7,facilitating the proliferation of AT2 cells.These proliferating cells exhibited an abnormal synthesis and secretion of the pro-fibrotic cytokine IL-33.3.The co-exposure of A549 and MLE-12 cells to nicotine and silica prompted the activation of the STAT3-BDNF-Trk B pathway,which led to the facilitation of EMT.However,the activation of the pathway and the resulting EMT were impeded by the Trk B inhibitor K252 a.Figure [18] Table [1] Reference [87]... |
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