| BackgroundPneumoconiosis is caused by long-term inhalation of different pathogenic dust during occupational activities.Silicosis is irreversible pulmonary fibrosis caused by long-term inhalation of silica dust particles,and is a kind of pneumoconiosis with the highest morbidity and mortality in our country and even in the world.The main pathological features of silicosis are pulmonary inflammation and fibrosis.When the concentration of silica exceeds the clearance capacity of the lung,it will be deposited in the lung.Alveolar macrophages are macrophages that reside in the lung tissue and maintain immune homeostasis and host defense in the lung.Continuous activation of alveolar macrophages through chronic inhalation of silica particles can trigger pulmonary inflammatory response.Much evidence showed that many proinflammatory cytokines produced by alveolar macrophages after phagocytosis of silica participate in silicosis inflammation,which then upregulate the expression of profibrotic factros.These profibrotic factors subsequently activate the proliferation and differentiation of fibroblasts into myofibroblasts.The excessive depostion of extracellular matrix eventually leads to pulmonary fibrosis.Therefore,inflammation mediated by alveolar macrophages plays a key role in the development of silicosis.However,these clinical drugs against the existing inflammatory pathogenesis have limited effects and can only alleviate the pain of patients to some extent.Moreover,targeted drugs such as pirfenidone and Nintedanib have achieved preliminary efficacy in the clinical treatment of patients with idiopathic pulmonary fibrosis,but currently there is no useful targeted drugs in the clinical treatment of silicosis.Therefore,based on the research breakthrough in the pathogenesis of inflammation,it is urgent to explore the key molecules that regulate the inflammation of silicosis and targeted treatment strategies.Progranulin（Pgrn）is an important cytokine involved in the regulation of tissue repair,inflammation and host defense response.Studies have shown that Pgrn deficiency can reduce the inflow of neutrophils and monocytes and the permeability of alveolar epithelial barrier after influenza infection,thus reducing lung tissue inflammation and injury.Pgrn has also been reported to induce airway inflammation by increasing the number of natural killer（NK）T cells.However,the role of Pgrn in silicosis inflammation remains unclear.Inflammation is one of the main pathological manifestations of silicosis and a key factor in the occurrence of fibrosis.However,whether Pgrn is involved in the inflammatory response in silicosis remains unclear,which is worth further study.Exploring the role and mechanism of Pgrn in silica-induced lung tissue inflammation may provide new ideas for the diagnosis and treatment of silicosis.ObjectivesIn this paper,the role of Pgrn in lung tissue inflammation induced by silica will be studied from the following four aspects:（1）To study the expression of Pgrn in lung tissues of mice treated with silica at different time points.（2）To analyze the effect of Pgrn on lung tissue inflammation in mice treated with silica.（3）Based on transcriptome sequencing,the mechanism of Pgrn regulating lung tissue inflammation induced by silica was revealed and the animal model was used for inflammation.（4）To explore the effect and mechanism of Pgrn on the inflammatory response of alveolar macrophages.MethodsThis paper is divided into four parts.In the first part,C57BL/6J mice were used to construct silica-treated models at different time points.Based on molecular biology experiments and omics studies,the expression of Pgrn in mouse lung tissues and the types of cells with the highest expression of Pgrn were analyzed.In the second part,the effect of Pgrn deficiency on lung tissue inflammation in mice treated with silica was analyzed by pathological methods.In the third part,the transcriptome sequencing was used to analyze the key molecules of Pgrn deficiency in regulating lung tissue inflammation induced by silica.In the fourth part,the mechanism of Pgrn on the inflammatory response of alveolar macrophages was analyzed by molecular biology experiments and omics studies.Specific methods for each part are as follows:（1）Mice were given 2.5 mg silica by disposable tracheal intubation and dissected at 1 week,4 weeks,and 24 weeks after modeling.Transcriptomic sequencing and q PCR were used to analyze the gene expression of Pgrn in the lung tissues of mice.The protein level of Pgrn in mouse lung tissue was detected by immunohistochemistry and Western blotting.Single-cell sequencing,immunohistochemistry,and immunofluorescence were used to identify the cell types with high expression of Pgrn,and the expression of Pgrn in these cells was verified using in vitro cell models.（2）Pgrn-/-mice were bred by Pgrnwt/-mice,and the mouse genotypes were identified by PCR and agarose nucleic acid gel electrophoresis.The expression of Pgrn gene and protein in Pgrn-/-mice were detected by q PCR and Western blotting.Based on Pgrn knockout mice,mice models were established for 1 and 4 weeks after silica instillation.H&E staining,immunohistochemistry,q PCR and inflammatory factor microarray were used to evaluate the effects of Pgrn deficiency on inflammation,number of inflammatory cells and expression of inflammatory factors in lung tissue of mice exposed to silica.Masson staining was used to evaluate the effect of Pgrn deficiency on pulmonary fibrosis in mice treated with silica.（3）Transcriptome sequencing was used to identify differentially expressed genes（DEGs）in lung tissue of Pgrn-/-mice treated with silica.Gene Ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）were used to analyze the functions of DEGs and identify the downstream key molecules of Pgrn regulating the inflammation of silica-exposed mice model（the results showed that interleukin（Il）-6 might be related to the regulation of Pgrn on silica-induced lung inflammation）.q PCR and ELISA were used to detect the expression of Il-6 in Pgrn-knockout mice.Subquently,Il-6 knockout mice were used to construct a silica-treated mouse model.H&E staining and q PCR were used to evaluate the effects of Il-6 deficiency on the inflammation and inflammatory factors expression in lung tissue of mice treated with silica.（4）MH-S cells were treated with different concentrations of Pgrn for 24 h and48 h,and the expression of inflammatory factors in MH-S cells was detected by q PCR and Western blotting.MH-S cells with Pgrn knockdown were constructed and treated with 250μg/m L silica for 24 h to detect the expression of inflammatory factors.c AMP response element-binding protein 1（Creb1）is a transcription factor that regulates the expression of several genes,such as inflammatory cytokine Il-6.In this study,666-15（Creb1 phosphorylation inhibitor）was used to explore whether Pgrn regulates the expression of inflammatory factors in MH-S cells by regulating Creb1 phosphorylation.Transcriptomics and immunocoprecipitation-mass spectrometry were used to reveal the underlying molecular mechanism by which Pgrn regulates the function of alveolar macrophages.Results（1）The inflammatory cell infiltration and thickening of alveolar septa were observed in the lung tissue of mice after 1 week of silica instillation（inflammation stage）.After silica exposure for 4 weeks,the lung tissue inflammation of mice continued to develop,and collagen fiber deposition was observed（progression stage）,and the lung tissue inflammation of mice after 24 weeks of silica poisoning was no longer aggravated.Extensive collagen deposition and fibrous nodules was observed in 24 weeks（fibrosis stage）.Transcriptomic sequencing and q PCR showed that the level of Pgrn gene was increased in the lung tissues of mice treated with silica at 1 week and 4 weeks.Immunohistochemical and Western blotting results showed that the level of Pgrn protein was increased in the lung tissues of mice exposed to silica at 1 week,4 weeks,and 24 weeks.The results of single-cell sequencing and immunofluorescence indicated that macrophages were the main cells expressing Pgrn in lung tissue.Immunohistochemical results further suggested that Pgrn was highly expressed in alveolar macrophages.ELISA,Western blotting and q PCR results showed that the Pgrn gene and protein levels were increased in murine alveolar macrophage line MH-S cells treated with 125μg/m L and 250μg/m L silica for 24 h and 48 h.（2）Compared with wild-type mice,the m RNA and protein expression levels of Pgrn in Pgrn-/-mice were close to 0.The inflammation score in the lung tissue of Pgrn-/-mice treated with silica at 1 and 4 weeks was decreased with the comparation to Pgrnwt/wtmice treated with silica,and the inflammation was most significantly reduced at the time point of 4 weeks.In addition,Pgrn deficiency decreased the number of F4/80+macrophages,lymphocyte antigen 6 complex,locus G,lymphocyte antigen 6 complex,locus G（Ly6g）+neutrophils,and CD4+lymphocytes in mice treated with silica.The results of q PCR and inflammatory cytokine microarray further demonstrate that Pgrn deficiency reduced the expression of many inflammatory cytokines,including Il-1βand tumor necrosis factor（Tnf）-α,in mice after silica instillation for 1 week and 4 weeks.Masson staining results showed that compared with Pgrnwt/wtmice exposed to silica for 4 weeks,the collagen fibers in lung tissue of Pgrn-/-mice treated with silica for 4 weeks were reduced.（3）In this study,transcriptome sequencing revealed that 1175 genes were down-regulated and 570 genes were-up regulated after Pgrn deficiency.GO-CC results showed that these differentially expressed genes were mainly located in extracellular regions;GO-MF results showed that the molecular functions of the differential genes were mainly involved in the formation of extracellular matrix,protein binding,and regulation of cytokine and chemokine activities;GO-BP results revealed that Pgrn deficiency affected the immune response and inflammatory response process of silicosis,and KEGG analysis further demonstrated that Pgrn mainly regulated the cytokine cytokine receptor signal pathway.Through in-depth analysis of GO-BP results,the production process of cytokine Il-6 was abnormal after Pgrn deficiency,suggesting that Pgrn might promote silicosis inflammation by regulating the expression of Il-6.The results of q PCR and ELISA showed that the Il-6 gene and protein decreased after Pgrn deficiency.H&E staining showed that silicosis inflammation was alleviated after Il-6 knockout.Compared with the Il-6wt/wtmice treated with silica,the m RNA levels of Il-1βand Tnf-αin the lung tissue of Il-6-/-mice with the silica stillation were decreased.The transcriptome sequencing results suggested that a total of 30 genes might be involved in the Pgrn regulation of the expression of Il-6,the most significant was C-type lectin domain family 7,member a（Clec7a,log2 Fold Change=-3.43）,KEGG analysis further showed that differentially expressed genes involved in Pgrn regulation of Il-6 production were mainly enriched in toll-like receptor signaling pathway.（4）100 ng/m L Pgrn promoted the m RNA expression of Tnf-αand Il-6 in MH-S cells,but had no effect on the expression of Il-1β.The expression levels of Tnf-α,Il-6,and Il-1βwere all increased in MH-S treated with 500 ng/m L for 48 h.Considering that Pgrn had the most obvious promoting effect on Il-6 expression,Western blotting was used to detect the Il-6 protein level.The results showed that100 ng/m L and 500 ng/m L Pgrn increased Il-6 protein levels in MH-S cells.Knockdown of Pgrn inhibited the Il-6 increase in MH-S cells induced by silica.The Il-6 promoter contains a variety of cis-elements and can be activated by homologous transcription factors including c AMP response element-binding protein 1（Creb1）.The results showed that the phosphorylation level of Creb1 was increased after treatment of MH-S cells with 100 ng/m L and 500 ng/m L Pgrn factor for 48 h.Knockdown of Pgrn inhibited Creb1 phosphorylation induced by 250μg/m L silica.Inhibition of Creb1 phosphorylation by 0.5μM and 1.0μM 666-15（Creb1phosphorylation inhibitors）reduced the Il-6 elevation caused by Pgrn.Transcriptome sequencing results showed that 151 genes were up-regulated and 181genes were down-regulated in MH-S cells treated with 500 ng/m L Pgrn for 48 hours.GO analysis showed that Pgrn mainly regulated cell migration and calcium ion transport.A total of 83 potential Pgrn binding proteins were identified by co-immunoprecipitation-mass spectrometry.Based on the results of transcriptomics and mass spectrometry,the interaction between Pgrn effectant molecules was analyzed using String database,and the molecular interaction network of Pgrn in MH-S cells was constructed.A total of 10 regulatory networks were screened.The results of literature research showed that these molecular interaction networks mainly regulated cell proliferation,apoptosis,migration,and glucolipid metabolism.Conclusions（1）Pgrn was increased in silica-induced lung inflammation,and alveolar macrophages were the main cell type with the high expression of Pgrn,suggesting that Pgrn might be involved in silica-induced lung inflammation.（2）Pgrn deficiency reduced lung tissue inflammation and fibrosis in mice exposed to silica,and the decrease was more significant at 4 weeks,indicating that alteration of Pgrn expression in the early stage of silica exposure can effectively inhibit the progression of lung tissue inflammation and fibrosis induced by silica.（3）Pgrn deficiency could reduce lung tissue injury,inflammatory cell infiltration and inflammatory factor expression in mice treated with silica by decreasing Il-6 production.（4）Pgrn up-regulated Il-6 expression in alveolar macrophages by increasing Creb1 phosphorylation level.Omics studies have revealed that Pgrn might regulate calcium ion transport,cell migration,and proliferation in alveolar macrophages. |
