Study On The Role Of TFEB In Experimental Silicosis And The Mechanism Of The Protective Effect Of Trehalose On Experimental Silicosis Fibrosis

Objective: Silicosis is an occupational lung disease caused by the long-term inhalation of respirable Crystalline silica(CS),characterized by persistent inflammation and irreversible fibrosis.It is the most common,fastest-growing and most serious type of pneumoconiosis.The pathogenesis of silicosis has not been fully defined,and the lack of specific clinical treatment measures is still one of the public health issues worldwide.Our previous studies have demonstrated that autophagy plays an important role in the development of silicosis.Although the existing pulmonary fibrosis is irreversible and difficult to eliminate,the pathogenesis of pulmonary fibrosis is a chronic and progressive pathological reaction process.It is still of great practical significance to find the target of treatment from the perspective of improving the function of autophagy lysosome system and to delay the development of pulmonary fibrosis.In this study,we explored the possible mechanism of transcription factor EB in CS-induced pulmonary inflammation and fibrosis,and the possible mechanism of trehalose in the alleviation of experimental silicosis fibrosis to provide a potential target for the delay and treatment of silicosis fibrosis.Methods: In this study,we established C57 BL / 6 experiment mouse model of silicosis as research object.Experiment mouse model of pulmonary fibrosis was induced by intratracheal instillation of CS suspension.We employed Western Blot,q RT-PCR and immunofluorescence to detect the transformation of TFEB in lung tissue and alveolar macrophages(AMs)of experimental silicosis model mice in early(7 days)and late(56 days)after exposure to CS.It was found that TFEB entered the nucleus of lung tissue and AMs of mice exposed to CS.In vitro,we established TFEB silence and overexpression MH-S cells line,to verify the possible role and mechanism of TFEB in silicosis model.Western Blot,TUNEL and ELISA were used to detect the effects of TFEB silencing and overexpression on apoptosis and inflammation of silica-exposed MH-S cells in vitro.It was concluded that TFEB silencing can aggravate apoptosis and inflammation but TFEB overexpression relieve these symptoms.Autophagy inhibitors were administrated to confirm the inhibition of autophagy of silica-exposed MH-S cells in vitro.Western Blot,acridine orange staining,Lysotracker staining and immunofluorescence detection were used to further confirm the effect of TFEB overexpression on autophagic lysosomal system. TUNEL and ELISA were conducted to further confirm that TFEB overexpression reduces apoptosis and inflammation in CS group were related to autophagy lysosomal system.The above experiments were used to explain the role of TFEB in silica-exposed MH-S cells.In vivo,2g / kg trehalose was injected to CS-treated mice.Western Blot,q RT-PCR and immunofluorescence were used to detect TFEB in lung tissue and AMs.The effect of Tre on autophagy lysosomal system in silicosis model was detected by Western Blot and immunofluorescence.Western Blot,ELISA and HE staining were used to detect the effect of Tre on lung apoptosis and inflammation caused by silica dust.In CS exposure models in vitro,50 mmol / L Tre was administrated to silica-exposed MH-S cells in vitro.Western Blot,q RT-PCR and immunofluorescence were used to detect the changes of TFEB in MHS cells.LC3-GFP-m RFP transfected macrophages were constructed to observe the dynamic regulation of Tre and CS on autophagy flux.Western Blot,AO,Lysotracker staining and immunofluorescence detection were used to confirm that Tre regulates autophagy lysosomal system in CS-treated MH-S cells through TFEB.Western Blot,TUNEL and ELISA were used to detect the effect of Tre on the apoptosis and inflammation in CS exposure models in vitro.The effects of Tre on pulmonary fibrosis induced by silica dust were determined by hydroxyproline assay,Western Blot,immunohistochemistry(COL-1,Fn)and Masson staining.The above experiments were used to explain the protective mechanism of TRE on experimental silicosis fibrosis.Results: 1.CS induces TFEB nuclear localization and increases TFEB expression in vivo Western Blot,q RT-PCR,immunohistochemistry and immunofluorescence were used to detect the transformation of TFEB on 7 and 56 days after CS treated.The results of Western Blot showed the expression of TFEB in lung tissue of CS group was significantly higher than that of the control group(P < 0.05);The results of q RT-PCR showed that the expression of TFEB in lung tissue of CS group was significantly higher than that of the control group(P < 0.05);The results of immunohistochemistry and immunofluorescence showed that the expression of TFEB in AMs of CS group was higher than that of the control group.Compared with the Ctrl group,the TFEB gene expression of AMs in CS group was significantly higher(P < 0.05).2.TFEB silencing aggravates CS induced apoptosis and secretion of inflammatory factors in MH-S cells Compared with the Ctrl group,CS exposure increased the expression of TFEB protein in nuclear and the gene expression in MH-S cells(P < 0.05).Western Blot,TUNEL and ELISA analysis were used to detect the apoptosis and the secretion of inflammatory factors.Compared with the Ctrl group,CS exposure resulted in the increase of apoptosis and the secretion of inflammatory factors,TFEB silenceing aggravated the apoptosis and the secretion of inflammatory factor in CS-treated MH-S cells(P < 0.05).3.TFEB overexpression reduces CS induced apoptosis and secretion of inflammatory factors in MH-S cells Western Blot,TUNEL and ELISA were used to detect the apoptosis and the secretion of inflammatory factors.Compared with the Ctrl group,CS exposure resulted in the increase of apoptosis and the secretion of inflammatory factors in MH-S cells.TFEB overexpression reduced the apoptosis and the secretion of inflammatory factor in CStreated MH-S cells(P < 0.05).4.TFEB overexpression relieves CS-induced macrophages apoptosis and secretion of inflammatory cytokines via regulation of autophagy-lysosome pathway Western Blot results showed that the level of LC3 II,Beclin1 and Atg5 protein in CS group was significantly higher than that in the Ctrl group(P < 0.05);While TFEB overexpression further increased the level of Beclin1 and Atg5 induced by CS,but decreased the expression of LC3 II protein level(P < 0.05).The expression of LAMP1 in MH-S cells of CS group were significantly reduced,TFEB overexpression alleviated CS induced LAMP1 decrease(P < 0.05).In addition,Lysotracker Red and AO staining also showed that CS induced the red fluorescence of lysosomal decreased,and TFEB overexpression reduced the basification of CS-treated MH-S cells.The results of immunofluorescence showed that CS decreased the expression of LAMP1 and LC3 II in MH-S cells,meanwhile decreased the colocalization of LAMP1 and LC3 II,suggesting that the combination of these two proteins decreased.Compared with CS group,TFEB overexpression increased the expression of LAMP1,LC3 II and the colocalization of these two proteins in CS treated MH-S cells;Western Blot showed that TFEB overexpression decreased the increased p62 in CS treated MH-S cells(P < 0.05); Immunofluorescence colocalization also showed that TFEB overexpression decreased the overexpression and the colocalization of p62 and Ubiquitin proteins in CS treated MH-S cells.The autophagy inhibitors BAF,CQ and 3MA were used to interfere the autophagy flux.The results of TUNEL and ELISA showed that TFEB overexpression reduced the apoptosis and the secretion of inflammatory cytokines through autophagy lysosomal pathway in CS-treated macrophages(P < 0.05).5.Tre activates TFEB and regulates the expression of autophagy related proteins in lung tissue and AMS cells of silicosis mice Western Blot results showed that the expression of TFEB in lung tissue of CS + tre group was significantly higher than that of CS group(P < 0.05).The results of q RT-PCR showed that the expression of TFEB m RNA in lung tissue of CS + tre group was significantly higher than that of CS group(P < 0.05).The results of immunofluorescence showed that TFEB in AMS cells increased in CS + tre group compared with CS group.The results of q RT-PCR showed that TFEB m RNA in AMs cells of CS + tre group was significantly higher than that of CS group(P < 0.05).The results of Western Blot showed that the expression of LC3 II,Atg5 and Beclin1 in CS group was significantly higher than that in Ctrl group;Compared with CS group,the expression of Beclin1 and Atg5 in CS + Tre group was significantly higher,while that of LC3 II was significantly lower(P < 0.05).The results of immunofluorescence showed that the expression of LC3 II in CS group was higher than that in Ctrl group and the expression of LC3 II in CS + Tre group was lower than that in CS group.Western Blot showed that the expression of LC3 II and Atg5 in CS group was significantly higher than that in Ctrl group,and the expression of Atg5 in MH-S cells in CS + Tre group was significantly higher,and the expression of LC3 II was significantly lower than that in CS group(P < 0.05).The results of Western Blot showed that the expression of LAMP1 was decreased in lung tissue and AMs cells of CS group compared with group Ctrl;The expression of LAMP1 in CS + Tre group was significantly higher than that in CS group(P < 0.05).Western Blot analysis of CTSB protein in BALF showed that the secretion of CTSB protein in CS group increased significantly than that in Ctrl group,and Tre in CS + Tre group effectively reduced the secretion of CTSB protein in BALF.In the lung tissue of silicosis model mice,Western Blot results showed that the expression of p62 and Ubiquitin in CS group was significantly higher than that in Ctrl group;Compared with the CS group,CS + Tre group effectively reduced the deposition of p62 and Ubiquitin(P < 0.05).In the AMs of silicosis model mice,compared with the control group,CS significantly increased the expression of p62 protein;Compared with the CS group,CS + Tre group effectively reduced the deposition of p62 protein(P < 0.05).Immunofluorescence also found that Tre effectively reduce the deposition of p62 protein in model mice AMS cells.6.Tre reduces lung apoptosis and inflammation in silicosis model mice Western Blot results showed that CS exposure increased the apoptosis of lung tissue and AMS cells in silicosis model mice,and Tre intervention reduced this apoptosis(P < 0.05).The results of ELISA showed that the secretion of IL-6,MCP-1,TNF-? and IL-1? protein in the alveolar lavage fluid of CS group was significantly higher than that of Ctrl group(except for IL-6 of 56 day);Compared with the CS group,CS + Tre significantly reduced the secretion of inflammatory factors in the alveolar lavage fluid(except for IL-6 of 56 day)(P < 0.05).The results of HE staining showed that the lung tissue structure of CS group mice was destroyed,many inflammatory cells infiltration was observed in the lung tissue after 7day CS exposed,and obvious cell nodules were observed in the lung tissue after 56 day CS exposed.In the silicosis model mice treated with Tre,the inflammatory cell infiltration was reduced and the size of fibrocyte node was significantly reduced.7.Tre activates TFEB and regulates the expression of autophagy related proteins in silicaexposed MH-S cells The results of Western Blot showed that CS exposure increased the expression of TFEB in nuclear,and Tre further promoted the expression of TFEB(P < 0.05);The results of q RT-PCR showed that the expression of TFEB m RNA in CS group was higher than that in control group,and the expression of TFEB in CS + Tre group was higher than that in CS group(P < 0.05).We used m RFP-GFP-LC3 adenovirus-infected MH-S cells to monitor autophagy flux. The counts of both fluorescent dots increased significantly after CS treatment,while Tre treatment decreased the yellow fluorescent dots,suggesting Tre alleviate CS-induced accumulation of autophagy substrates;Western Blot results showed that the level of Beclin1 and Atg5 protein in CS + Tre group was significantly higher than that in the CS group,while the level of LC3 II was lower(P < 0.05);The level of LC3 II,Beclin1 and Atg5 protein in CS + Tre + sh-TFEB group were all significantly lower than that in CS or CS + Tre group(P < 0.05).Western Blot results showed that the level of LAMP1 in MH-S cells was significantly higher and the level of CTSB in cell supernatant was significantly lower of CS + Tre group than that of the CS group,(P < 0.05);sh-TFEB reversed the effects of Tre on LAMP1 and CTSB of CS-treated MH-S cells(P < 0.05).In addition,Lysotracker Red and AO staining also showed that Tre reduced the damage of CS on the acid structure of lysosome while TFEB silencing made Tre lose this function.Immunofluorescence results showed that the expression of LAMP1 was significantly higher and the expression of LC3 II was significantly lower,but the colocalization of LAMP1 and LC3 II was significantly higher of CS + Tre group than that of CS group.Compared with CS + Tre group,the expression of LAMP1 and LC3 II in CS + Tre + shTFEB group are lower;Western Blot results showed that Tre reduced the expression of p62 protein in CS-treated MH-S cells,but in CS + Tre + sh-TFEB group,sh-TFEB counteracted the effect of Tre on the degradation of p62(P < 0.05).Immunofluorescence results showed that Tre reduced the expression and the colocalization of p62 and Ubiquitin in CS-treated MH-S cells.The results above suggested that Tre reduce the deposition of autophagic substrates in CS-treated MH-S cells through TFEB.8.Tre reduces apoptosis and the secretion of inflammatory factors in CS exposure models in vitro by TFEB Western Blot,TUNEL and ELISA were used to detect apoptosis and the secretion of inflammatory factors.Compared with CS group,CS + Tre group reduced apoptosis and the secretion of IL-6,TNF?,MCP-1 and IL-1?,while CS + Tre + sh-TFEB group lost the anti-apoptosis and anti-inflammatory effects of Tre(P < 0.05).9.Tre reduces pulmonary fibrosis in silicosis model mice Hydroxyproline(HYP),COL-1 and Fn protein were detected in lung tissue of mice after exposure to CS for 56 days,Immunohistochemistry of COL-1 and Fn protein and Masson trichrome staining were performed on paraffin sections.The results of HYP showed that the content of HYP in lung tissue of CS exposed mice increased significantly,and Tre treatment significantly decreased the content of hydroxyproline in lung(P < 0.05).Western Blot and immunohistochemistry showed that Tre effectively reduced the expression of COL-1 and Fn protein in lung tissue of CS exposed mice(P < 0.05).The results of Masson showed that obvious collagen deposition was found in the fibronodular lesions of CS group lung tissues.After Tre treatment,the fibronodular lesions and the collagen deposition in the lung were significantly reduced(P < 0.05).Conclusion: 1.CS induces TFEB nuclear localization and increases TFEB expression in vivo in lung tissue and AMs of model mice.2.TFEB silencing aggravates CS induced apoptosis and secretion of inflammatory factors in MH-S cells,and TFEB overexpression reduces these damages.3.TFEB overexpression relieves CS-induced macrophages apoptosis and secretion of inflammatory cytokines via regulation of autophagy-lysosome pathway.4.Tre promotes the recovery of autophagy lysosomal system in silicosis model through TFEB.5.Tre reduces the apoptosis and the secretion of inflammatory factors in silicosis model by TFEB.6.Tre alleviates pulmonary fibrosis of CS exposed model mice.