| Background Silicosis caused by long-term inhalation of crystalline silica（CS）in occupational activities seriously threatens the health of the occupational population and is a major global public health problem.Lack of effective treatment and measures to reverse silicosis,patients with silicosis suffer from respiratory damage and even die from respiratory failure.Therefore,the potential mechanism and therapeutic targets of silicosis need to be further clarified.Silicosis manifests as progressive and irreversible pulmonary fibrosis,which is characterized by the proliferation and differentiation of fibroblasts,the accumulation of a large amount of extracellular matrix（ECM）.According to the development characteristics of silicosis,it can be divided into early inflammation stage and late pulmonary fibrosis stage.Alveolar macrophages（AMs）are the main immune cells in the alveolar space and play a regulatory role in both stages.The polarization direction and phenotype of AMs depend on the environment.In the microenvironment of different cytokines,AMs can be polarized into the M1 subtype by classically activated and the M2 subtype via alternatively activated.Studies have shown that the polarization changes of AMs may be involved in the development of pulmonary fibrosis,but the exact role and mechanism have not been fully elucidated.Objective1.To investigate the time-dependent relationship between CS-induced pulmonary fibrosis and AMs polarization in silicosis model mice by detecting changes of AMs polarization at different times.2.To detect the related molecules regulating the polarization of AMs toward M2subtype in lung tissue of silicosis model mice,and to explore the molecular mechanism of promoting the polarization of AMs to M2 subtype in CS-induced pulmonary fibrosis.3.Use IL-4 Neutralizing antibody（IL-4 NA）to pretreat silicosis model mice to explore the intervention effect of IL-4 NA on CS-induced pulmonary fibrosis.MethodsPart I Time-dependent relationship between CS-induced pulmonary fibrosis and AMs polarization8-week-old C57BL/6J WT mice were randomly divided into CS group and normal saline（NS）group,with 48 mice in each group.Mice in CS group and NS group received 100μL CS suspension of 20mg/m L or NS by intra-tracheal instillation,and were sacrificed at 3,7,28 and 56 days,respectively.Flow Cytometry（FCM）were used to detect the proportion of M1（F4/80+CD11c+）subtype and M2（F4/80+CD206+）subtype AMs after collect bronchoalveolar lavage fluid（BALF）.q RT-PCR was used to detect the m RNA levels of i NOS,Arg-1,IL-1β,TNF-α,IL-6,IL-10 and TGF-βin AMs.ELISA was used to detect the activities of i NOS,Arg-1 and IL-1β,TNF-α,IL-6,IL-10,TGF-β,IL-4 contents in BLAF.The linear correlation between the proportion of M2subtypes in AMs and the contents of IL-4 by Person Correlation analysis.HE staining and Masson staining were used to assess lung tissue inflammation and fibrosis,and Hydroxyproline（HYP）detection kit to detect HYP level after lung tissue was separated.The expression levels of lung tissue EMT-related protein E-cadherin（E-cad）,Vimentin（Vim）and FMT-related proteinα-smooth muscle actin（α-SMA）,collagen I（COL-I）were assessed by Western Blotting（WB）and immunohistochemistry（IHC）.Part II Mechanism of CS-induced AMs polarization towards M2 subtypeThe AMs were separated from mice exposed to CS or NS on 28 days,and q RT-PCR was used to detect m RNA expression levels of Fox O-1,BMP-4,CEBP-β,AMPK-α,KLF-4,B7-H3,STAT-3,PPAR-γ,PTP1B,STAT-6,TIPE-2,IRF-4.Immunofluorescence（IF）detection KLF-4,p-STAT-6,PPAR-γprotein expression.PartⅢIntervention effect of IL-4 NA on CS-induced pulmonary fibrosis Eight-week-old C57BL/6J WT mice were randomly divided into NS group,CS group,CS+Ig G group,CS+IL-4 NA group,with 12 mice in each group.The treatment way of NS group and CS group as previously described.Mice in CS+Ig G group and CS+IL-4 NA group were injected with 100μL CS suspension of 20 mg/m L and intraperitoneal injection of Ig G or IL-4 NA 100μL of 10mg/m L per week.Mice in four treatment groups were sacrificed at 28 days.ELISA was used to detect IL-4 content in BALF and FCM was used to detect the proportion of M1 subtype and M2 subtype AMs.The m RNA levels of Arg-1,IL-10,TGF-β,PPAR-γ,STAT-6,and KLF-4 in AMs were detected by q RT-PCR and Arg-1 activity and IL-10,TGF-βcontent in BLAF were assessed by ELISA,respectively.Separate the lung tissue,HE staining and Masson staining to observe lung tissue structure and fibrosis.HYP level were assessed by HYP detection kit.The protein expression levels of E-cad,Vim,α-SMA and COL-I were assessed by WB and IHC.Results1.CS induced lung tissue inflammation in the early stage and lung fibrosis in the late stageCompared with the NS group,the normal alveoli structure was destroyed,and inflammatory cell infiltration appeared in the pulmonary interstitium after day 3exposure to CS.On day 7,the lung tissue inflammation subsided,and on day 56 the lung parenchyma increased and silicosis nodules formed.Compared with the NS group,the expression levels of collagen fibers and HYP in the lung tissue of the mice exposure to CS 28 days or 56 days were significantly increased（P<0.05）.Compared with the NS group,the protein level of E-cad was significantly decreased and Vim,COL-I,α-SMA was significantly increased on day 28 and 56 in the lung tissue of mice exposure to CS（P<0.05）.2.CS induces AMs polarization toward M1 subtype in the early stage,and M2 subtype in the late stageCompared with the NS group,the proportion of M1 subtype AMs in the CS group was significantly increased on day 3 and reduced from day 7 to 56（P<0.05）.Compared with NS group,the m RNA expression level and activity of i NOS,m RNA level and content of IL-1β,TNF-α,IL-6 significantly increased on day 3 and decreased on day 7,28 or 56 exposure to CS（P<0.05）.Compared with the NS group,the proportion of M2 subtype AMs in the CS group significantly increased on day 28 or day 56（P<0.05）.Compared with the NS group,the m RNA expression level and activity of Arg-1,m RNA level and content of TGF-βand IL-10 significantly increased on day 28 and day 56 exposure to CS（P<0.05）.3.CS activates STAT-6/KLF-4/PPAR-γsignaling pathwayCompared with the NS group,the m RNA and protein levels of STAT-6,KLF-4,and PPAR-γin AMs were significantly increased in the CS group on day 28（P<0.05）.4.The proportion of M2 subtype in AMs is linearly correlated with the IL-4 cytokine contentCompared with the NS group,the IL-4 content in the CS group was significantly increased on 28 and 56 days（P<0.05）.Person correlation analysis showed that the proportion of M2 subtypes in AMs was positively correlated with the content of IL-4 in BFLA on day 28 and 56 exposure to CS（r=0.963,P<0.05;r=0.956,P<0.05）.5 Intervention effect of IL-4 NA on CS-induced pulmonary fibrosis5.1 IL-4 NA inhibits CS-induced AMs polarization towards M2 subtypeCompared with NS group,the proportion of M1 subtype in AMs was significantly reduced in CS group,CS+Ig G,CS+IL-4 NA group on day 28 post administration（P<0.05）.Compared with the NS group,the proportion of M2 subtype in AMs was significantly increased in CS group and CS+Ig G group on day 28 post administration（P<0.05）.Compared with the CS group and CS+Ig G group,the proportion of M2subtype AMs in CS+IL-4 NA group was significantly decreased on day 28 post administration（P<0.05）.The m RNA level and activity of Arg-1 and the m RNA level and content of TGF-βand IL-10 were in the same trend as the results of FCM.5.2 IL-4 NA attenuates pulmonary fibrosis levelsCompared with the NS group,the lung tissue structure was disordered,the parenchyma increased,the collagen fiber deposition was obvious,and extensive fibrosis appeared in CS group and the CS+Ig G group on day 28 post administration.After using IL-4 NA,it can effectively protect CS mediated disorder of lung tissue structure and reduce the deposition of collagen fibers.Compared with the NS group,the protein levels of Vim,COL-I andα-SMA in the lung tissues of mice in the CS group and CS+Ig G group were significantly increased on day 28 post administration（P<0.05）.Compared with the CS group and CS+Ig G group,the protein levels of Vim,COL-I andα-SMA in the lung tissues of mice in the CS+IL-4NA group were significantly decreased on day 28 post administration（P<0.05）.In contrast,compared with the NS group,the protein levels of E-cad in the lung tissue of mice in the CS group and CS+Ig G group were significantly decreased on day 28 post administration（P<0.05）.Compared with the CS group and CS+Ig G group,the protein level of E-cad in lung tissue of mice in CS+IL-4 NA group increased significantly on day 28 post administration（P<0.05）.5.3 IL-4 NA inhibits STAT-6/KLF-4/PPAR-γsignaling pathway activated by CSCompared with NS group,the STAT-6,KLF-4 and PPAR-γm RNA expression levels in AMs in CS group and CS+Ig G group were significantly increased on day 28post administration（P<0.05）,compared with CS group,the STAT-6,KLF-4 and PPAR-γm RNA expression levels in AMs in CS+IL-4 NA group were significantly decreased（P<0.05）.Conclusions1.CS promotes AMs polarization toward M1 subtype in the early stage to induce lungtissue acute inflammation,and promotes AMs polarization toward the M2 subtypein the late stage to mediate pulmonary EMT and FMT to induce pulmonary fibrosis.2.Mechanistically,CS may activate the STAT-6/KLF-4/PPAR-γsignaling pathway topromote AMs polarization toward the M2 subtype.3.IL-4NA inhibits CS-induced AMs polarization toward M2 subtype to relievepulmonary EMT and FMT,and alleviates pulmonary fibrosis.Mechanistically,IL-4NA may inhibit CS activation of KLF-4/STAT-6/PPAR-γsignaling pathway toinhibit AMs polarization towards the M2 subtype. |
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