Study On The Roles And Mechanisms Of Crystalline Silica-induced Alveolar Macrophage Pyroptosis Interacting With Mitophagy Modulating The Progression Of Silicosis Fibrosis

Objective:Alveolar macrophages play a crucial role in the pathogenesis of silicosis.Previously,we demonstrated that enhancing alveolar macrophage mitophagy exerted protective effects on silicosis with a restrained inflammatory response.However,the exact molecular mechanisms are elusive.Pyroptosis and mitophagy are two different biological processes that determine cell fate.Exploring whether there were interactions or balances between these two processes in alveolar macrophages would provide new insight into the pathogenesis of silicosis.Methods:In this study,a non-tracheal exposure method was used to construct an experimental model of silicosis in mice by perfusing crystalline silica suspension.Western blot,q PCR,and immunofluorescence staining were used to analyze the changes in the level of pyroptosis in the lung tissue of mice in the silicosis model at the fibrosis stage.Primary alveolar macrophages were extracted from alveolar lavage fluid,and the pyroptosis level of alveolar macrophages in each group of mice was further examined by immunofluorescence staining and q PCR.An in vitro silica dust exposure model was constructed using a mouse alveolar macrophage line MH-S.Western blot and immunofluorescence staining were used to verify the alveolar macrophage pyroptosis induced by silica dust in vitro.Mitochondrial damage was detected by transmission electron microscopy and q PCR.The effect of silica dust on mitochondrial ROS was observed using mito SOX^TM fluorescence probe.The effect of silica dust on mitochondrial membrane potential was observed using a JC-1 fluorescence probe.A model of PINK1 overexpression in MH-S alveolar macrophages exposed to silica dust in vitro was constructed,and the effect of PINK1 overexpression on mitophagy in MH-S cells was detected by Western blot and immunofluorescence staining.The synthesis of mitochondrial DNA was detected by q PCR.The effect of overexpression of PINK1 on pyroptosis was detected by immunofluorescence staining and Western blot.An in vitro silica dust exposure model of MH-S alveolar macrophages with Atg5 or PINK1knockdown was constructed,and the effect of Atg5 knockdown on mitophagy of MH-S cells was detected by Western blot and immunofluorescence staining.The synthesis of mitochondrial DNA was detected by q PCR.The effect of Atg5 or PINK1 knockdown on pyroptosis was detected by immunofluorescence staining and Western blot.An in vitro silica dust exposure model was constructed and the NLRP3 inhibitor MCC950,Caspase1inhibitor VX-765 and GSDMD oligomerization inhibitor Disulfiram were administered to detect the effect of inhibition of pyroptosis cascades on cellular mitophagy from upstream to downstream respectively by Western blot as well as immunofluorescence co-localization of mito Tacker and LC3.The mito SOX^TM fluorescent probe was used to detect the effect of pyroptosis cascades inhibition on mitochondrial ROS content by regulating mitophagy.The effect of pyroptosis cascades inhibition on mitochondrial membrane potential through the regulation of mitophagy was detected by JC-1 fluorescent probe staining.The regulation effect of pyroptosis cascades inhibition to mitochondrial damage through regulating mitophagy was confirmed by transmission electron microscopy and q PCR assay.A mouse model of PINK1 overexpression in silicosis was constructed by tracheal perfusion of adeno-associated virus,and the effect of PINK1 overexpression on CS-induced pyroptosis by promoting mitophagy was examined by Western blot and immunofluorescence staining.Finally,the effect of inhibition of CS-induced pyroptosis on silicofibrosis in vivo was explored by therapeutically administering Disulfiram,an inhibitor downstream of pyroptosis.The inhibitory effect of Disulfiram on pyroptosis was determined by immunofluorescence staining of primary alveolar macrophages in alveolar lavage fluid of mice.Pulmonary function analysis was used to detect the modulation of pulmonary function in silicotic mice by Disfiram.Real-time lung tissue photography was used to observe the effect of Disulfiram on lung tissue injury.H&E staining was used to observe the pathological damage of inflammatory infiltration in mouse lung tissue.Masson staining,immunohistochemical staining for Col-1,q PCR to detect the expression of fibrosis-related genes,and hydroxyproline content were used to examine the effect of Disulfiram on silicosis fibrosis through inhibition of CS-induced pyroptosis.Result:1.Pyroptosis was increased in AMs of silicosis mice.In the present study,we used Western blot and q PCR to detect the expression levels of proteins and genes related to pyroptosis in the lung tissue of silicosis model mice,and found that the levels of pyroptosis-related proteins NLRP3,Caspase1 p20,and GSDMD NT were significantly upregulated in the lung tissue of silicosis model mice（P<0.05）;the expression levels of pyroptosis-related genes Nlrp3,Casp1,Gsdmd,Il1b,and Il18 also showed significantly increased.Immunofluorescence staining to the lung tissue sections observed a significant increase in the number of F4/80⁺GDSMD⁺double-positive pyroptosis macrophages in the lung tissue of silicosis mice.Immunofluorescence staining by GSDMD of primary alveolar macrophages extracted from alveolar lavage showed a significant increase in the level of pyroptosis macrophages in silicosis model mice,and the expression level of pyroptosis-related genes was also significantly upregulated by q PCR on the extracted alveolar macrophages（P<0.05）.2.CS particles damaged mitochondria and lead to pyroptosis in AMs.The pyroptosis-related proteins levels of NLRP3,Caspase1 p20,and GSDMD NT were significantly upregulated in silica dust-exposed MH-S cells（P<0.05）,and immunofluorescence staining of GSDMD showed similar results.Transmission electron microscopy of the ultrastructure of MH-S cells revealed significant mitochondrial damage in cells stimulated by silica dust.q PCR for mitochondrial DNA content showed a significant increase in the synthesis of mitochondrial DNA in silica-treated cells（P<0.05）.Fluorescent probe mito SOX staining for mitochondrial reactive oxygen species showed increased mitochondrial ROS production in silica-treated cells.Fluorescent probe JC-1staining of mitochondrial membrane potential showed a significant depolarization of mitochondrial membrane potential after silica treatment.3.Enhancing PINK1-mediated mitophagy alleviated CS-induced pyroptosis in AMs.The expression levels of autophagy-related proteins such as P62 and LC3II in total cellular proteins and PINK1 and Parkin in mitochondrial proteins were examined by Western blot.PINK1 overexpressing cells received silica stimulation showed reduced P62and increased LC3II expression compared to non-overexpressing cells,and PINK1 and Parkin proteins in mitochondrial proteins expression was also significantly increased（P<0.05）.Immunofluorescence staining of mito Tracker and LC3 co-localization showed a significant increase of mitophagy in PINK1 overexpressing cells.q PCR assay of mitochondrial DNA synthesis showed a significant decrease in mitochondrial DNA synthesis in the overexpressing group（P<0.05）.GSDMD was detected by immunofluorescence staining,and the GSDMD fluorescence intensity and spot formation were reduced in the PINK1 overexpression group.Western blot assay detected pyroptosis-related proteins NLRP3,Caspase1 p20,and GSDMD NT expression were significantly reduced in the overexpression group（P<0.05）.4.AMs defected in mitophagy aggravated CS-induced pyroptosis.The expression levels of P62 and LC3II autophagy-related proteins in total cellular proteins were measured by Western blot.P62 was increased and LC3II was decreased in Atg5 knockdown alveolar macrophages（P<0.05）.Immunofluorescence mito Tracker and LC3 co-localization showed a significant decrease of mitophagy in Atg5 knockdown cells.q PCR detected for mitochondrial DNA synthesis showed a significant increase in mitochondrial DNA synthesis in Atg5 knockdown group cells（P<0.05）.GSDMD was detected by immunofluorescence staining,and the fluorescence intensity of GSDMD was enhanced in the Atg5 or PINK1 knockdown group.Western blot assay showed that the expression of NLRP3,Caspase1 p20,and GSDMD NT was significantly increased in the knockdown group（P<0.05）.5.Restraining CS-induced pyroptosis promoted PINK1-mediated mitophagy and helped maintain cellular mitochondrial homeostasis.The expression levels of autophagy-related proteins such as P62 and LC3II in total cellular proteins and PINK1 and Parkin in mitochondrial proteins were examined by Western blot.Cells with pyroptosis cascades inhibition showed a decrease in P62 and an increase in LC3II expression when receiving silica stimulation compared to cells treated silica only,and the expression of PINK1 and Parkin in mitochondrial proteins was also significantly increased（P<0.05）.Immunofluorescence staining showed a significant increase in mito Tracker and LC3 co-localization after treament of pyroptosis cascades inhibitors.mito SOX fluorescent probe staining showed a decrease in mitochondrial ROS production after inhibition of pyroptosis cascades.JC-1 probe staining showed enhanced fluorescence of JC-1 aggregates（red）and decreased fluorescence of JC-1 monomers（green）in pyroptosis cascades inhibited cells compared to CS treatment group,indicating decreased mitochondrial membrane depolarization was detected.Transmission electron microscopy showed that the morphology of mitochondria improved and mitochondrial damage was reduced after the treatment of pyroptosis cascades inhibition.q PCR showed that mitochondrial DNA synthesis was reduced（P<0.05）.6.Promoting PINK1-dependent mitophagy constrained macrophages pyroptosis in silicotic mice.Western blot results showed that PINK1 overexpression in silicosis mice expressed lower levels of pyroptosis-related proteins NLRP3,Caspase1 p20,and GSDMD NT than in without overexpressing silicotic mice.Immunofluorescence staining of F4/80⁺GSDMD⁺double-positive pyroptosis macrophages in lung tissue sections were significantly reduced after overexpression of PINK1.7.Abolishing GSDMD-dependent pyroptosis attenuated CS-induced fibrosis in mice.Immunofluorescence staining was used to detect the pyroptosis level of alveolar macrophage in the alveolar lavage fluid,and the fluorescence of GSDMD was attenuated after administration of disulfiram,a cell pyroptosis inhibitor,compared with that of silicosis mice.Pulmonary function analysis of the changes in minute volume（MV）,tidal volume（TV）,and breathing frequency（f）in mice showed that the lung function of silicotic mice was improved after disulfiram treatment.H&E staining showed a reduction in inflammatory cell infiltration and silica nodule formation in silicosis mice after disulfiram administration.Masson staining was performed to examine collagen deposition,which was significantly reduced in disulfiram-treated silicosis mice.Immunohistochemical results were similar to Masson staining,with a significant reduction in Col-1-positive areas in lung tissue.q PCR results also showed a significant reduction in fibrosis-related gene expression（P<0.05）.The hydroxyproline content was measured by spectrophotometrically and was significantly reduced in the lung tissue of disulfiram-treated silicotic mice（P<0.05）.Conclusion:1.Crystalline silica-injured mitochondria and induced pyroptosis in alveolar macrophage.2.Crystalline silica-induced alveolar macrophage pyroptosis reciprocal inhibits mitophagy modulating mitochondrial homeostasis.3.Inhibited alveolar macrophage pyroptosis attenuates inflammatory and fibrosis insilicotic mice.