Cannabidiol suppresses silica-induced pulmonary inflammation and fibrosis through regulating NLRP3/TGF-ß1/Smad2/3 pathway.

Silica-induced pulmonary fibrosis is an irreversible and progressive lung disease with limited treatments available. In this work, FDA-approved cannabidiol (CBD) was studied for its potential medical use in silicosis. In silicosis female C57BL/6 mice model, oral CBD or pirfenidone (PFD) on day 1 after intratracheal drip silica (150 mg/mL) and continued for 42 days. Lung inflammatory and fibrotic changes were studied using ELISA kits, H&E staining and Masson staining. Osteopontion (OPN) and α-smooth muscle actin (α-SMA) expression in lung tissues was determined using immunohistochemical staining. The results indicated that CBD attenuated silica-induced pulmonary inflammation and fibrosis. Human myeloid leukemia mononuclear cells (THP-1) were treated with silica (200 µg/mL) to induce cell damage, then CBD (10 µM, 20 µM) and PFD (100 µM) were incubated. In vitro experiments showed that CBD can effectively reduce the expression of NLRP3 inflammasome in THP-1 cells and subsequently block silica-stimulated transformation of fibromuscular-myofibroblast transition (FMT) by culturing human embryonic lung fibroblasts (MRC-5) in conditioned medium of THP-1 cells. Therefore, CBD exhibited the potential therapy for silicosis through inhibiting the silica-induced pulmonary inflammation and fibrosis via the NLRP3/TGF-ß1/Smad2/3 signaling pathway.

Cardamonin inhibits silicosis development through the PI3K-AKT signaling pathway.

Silicosis is one of the most severe occupational diseases characterized by inflammatory cell infiltration, fibroblasts activation, and fibrosis in the lung. However, specific drug treatments are lacking. Cardamonin (CDM) has been reported to possess antitumor, anti-inflammatory/fibrotic effects. While, the effect of CDM on the progression of silicosis remains unknown. In this study, we established a SiO2-M stimulated fibroblast cell model, and explored the antifibrotic effect of CDM and the related molecular mechanism using WB, RT-qPCR, and immunofluorescence. The results indicate that CDM inhibits SiO2-M-induced fibroblast activation, proliferation, and migration. Furthermore, a silicosis mouse model was established through injecting silica suspension intratracheally. The results revealed that CDM retards the progression of pulmonary fibrosis. The RNA sequencing results suggest that the antifibrotic effect of CDM may be mediated by the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway. In conclusion, the results of this study demonstrate that CDM inhibits the development of silicosis via the PI3K-AKT signaling pathway, which could provide guidance for the development of drugs for silicosis treatment.

GAMG ameliorates silica-induced pulmonary inflammation and fibrosis via the regulation of EMT and NLRP3/TGF-ß1/Smad signaling pathway.

Silicosis is an occupational disease caused by exposure to silica characterized by pulmonary inflammation and fibrosis, for which there is a lack of effective drugs. Glycyrrhetinic acid 3-O-ß-D-glucuronide (GAMG) can treat silicosis due to its anti-inflammatory and anti-fibrotic properties. Here, the effect of therapeutic interventions of GAMG was evaluated in early-stage and advanced silicosis mouse models. GAMG significantly improved fibrotic pathological changes and collagen deposition in the lungs, alleviated lung inflammation in the BALF, reduced the expression of TNF-α, IL-6, NLRP3, TGF-ß1, vimentin, Col-Ⅰ, N-cadherin, and inhibited epithelial-mesenchymal transition (EMT), thereby ameliorating pulmonary fibrosis. Moreover, the dose of 100 mg/kg GAMG can effectively prevent early-stage silicosis, while that of 200 mg/kg was recommended for advanced silicosis. In vitro and in vivo study verified that GAMG can suppress EMT through the NLRP3/TGF-ß1/Smad2/3 signaling pathway. Therefore, GAMG could be a promising preventive (early-stage silicosis) and therapeutic (advanced silicosis) strategy, which provides a new idea for formulating prevention and treatment strategies.

Exosomal miR-552-3p isolated from BALF of patients with silicosis induces fibroblast activation.

BACKGROUND: Silica particles can cause silicosis, a disease characterized by diffuse fibrosis of the lungs. Various signaling pathways composed of different types of cells and cytokines are involved in the development of silicosis. Exosomes have become a research hotspot recently. However, the role of exosomal microRNA (miRNA) in silicosis remains unclear. METHODS: In this study, we generated exosomal miRNA sequences from exosomes isolated from bronchoalveolar lavage fluid (BALF) of silicosis patients and the control group by high-throughput sequencing. Functional annotation and analysis of miRNA identified key target miRNAs. Levels of target miRNAs were analyzed in patient and animal samples and cells. Effects of increased miRNA were assessed through protein levels in target signaling pathways in cells treated with silica, miRNA mimics, and inhibitors. RESULTS: Our study identified 40 up-regulated and 70 down-regulated miRNAs, with miR-552-3p and its putative target gene Caveolin 1 (CAV1) as targets for further research. We found that the levels of exosomal miR-552-3p increased in silicosis patients' BALF samples, silicosis model mice, and A549 cells exposed to silica. Inhibition of miR-552-3p suppressed the expression of fibrosis markers. The increased miR-552-3p leads to the up-regulation of fibronectin and α-smooth muscle actin (α-SMA) and the suppression of caveolin 1 in fibroblast cells. Mitogen-activated protein kinase (MAPK) signaling pathways are activated in cells treated with silica and miR-552-3p mimics. CONCLUSIONS: These results help to understand exosomal miRNA-mediated intercellular communication and its key role in fibroblast activation and silicosis.

Inhibition of the ITGB1 gene attenuates crystalline silica-induced pulmonary fibrosis via epithelial-mesenchymal transformation.

Silicosis is a systemic disease caused by long-term exposure to high concentrations of free silica dust particles in the workplace. It is characterized by a persistent inflammatory response, fibroblast proliferation, and excessive collagen deposition, leading to pulmonary interstitial fibrosis. Epithelial interstitial transformation (EMT) can cause epithelial cells to lose their tight junctions, cell polarity, and epithelial properties, thereby enhancing the properties of interstitial cells, which can lead to the progression of fibrosis and the formation of scar tissue. Integrin 1 (ITGB1) is considered an important factor for promoting EMT and tumor invasion in a variety of tumors and also plays an important role in the progression of fibrotic diseases. Therefore, ITGB1 can be used as a potential target for the treatment of silicosis. In this study, we found that silica exposure induced epithelial-mesenchymal transformation in rats and that the expression of integrin ITGB1 was elevated along with the EMT. We used CRISPR/Cas9 technology to construct integrin ITGB1 knockdown cell lines for in vitro experiments. We compared the expression of the EMT key proteins E-cadherin and vimentin in the ITGB1 knockdown cells and wild-type cells simultaneously stimulated by silica and detected the aggregation point distribution of E-cadherin and vimentin in the cells using laser confocal microscopy. Our results showed that ITGB1 knockout inhibited the ITGB1/ILK/Snail signaling pathway and attenuated the EMT occurrence compared to control cells. These results suggested that ITGB1 is associated with silica-induced EMT and may be a potential target for the treatment of silicosis.

Glycolytic reprogramming governs crystalline silica-induced pyroptosis and inflammation through promoting lactylation modification.

Prolonged inhalation of environmental crystalline silica (CS) can cause silicosis, characterized by persistent pulmonary inflammation and irreversible fibrosis, but the mechanism has not been elucidated. To uncover the role and underlying mechanism of glycolytic reprogramming in CS-induced pulmonary inflammation, the mouse silicosis models and glycolysis inhibition models were established in vivo. And the CS-induced macrophage activation models were utilized to further explore the underlying mechanism in vitro. The results showed that CS induced lung inflammation accompanied by glycolytic reprogramming and pyroptosis. The application of glycolysis inhibitor (2-DG) suppressed CS-induced pyroptosis and alleviated lung inflammation. In vitro, 2-DG effectively impeded CS-induced macrophage pyroptosis and inflammatory response. Mechanistically, 2-DG suppressed pyroptosis by inhibiting NLRP3 inflammasome activation both in vivo and in vitro. Furtherly, metabolite lactate facilitated NLRP3-dependent pyroptosis synergistically with CS particles, while blocking the source of lactate largely alleviated NLRP3 inflammasome activation and subsequent pyroptosis triggered by CS. More profoundly, the increment of lactate induced by CS might drive NLRP3-dependent pyroptosis by increasing histone lactylation levels. In conclusion, our findings demonstrated inhibiting glycolytic reprogramming could alleviate CS-induced inflammatory response through suppressing NLRP3 -dependent pyroptosis. Increased glycolytic metabolite lactate and protein lactylation modifications might represent significant mechanisms during CS-induced NLRP3 activation and macrophage pyroptosis.

Exosome miRNA profile and mitigating effect of miR-23a-3p/Cul3 axis on apoptosis in the pathogenesis of SiO2 dust-induced lung fibrosis.

Silicosis is an irreversible interstitial lung fibrosis resulting from persistent inflammation induced by long-term inhalation of SiO2 dust. Treatment and early diagnosis are extremely challenging due to the lack of specific targets and biomarkers. MiRNAs play an important role in the early diagnosis and treatment of various diseases, due to their stability, small variations, and easy detection. Exosomes have become fashionable candidates to deliver miRNAs. However, the specific role of exosomes-loaded miRNAs in silicosis inflammation and fibrosis remains unclear. In the present study, the expression profile of serum exosomal miRNAs in the peripheral blood of silicosis patients was determined by transcritome sequencing. MiR-23a-3p was recognized as a protector against silicosis by bioinformatic analysis. The expression and regulatory axis of miR-23a-3p and its predicted target gene CUL3 were then confirmed. The therapeutic role of the miR-23a-3p/CUL3 axis and its alleviating effect on SiO2-induced apoptosis were verified in mice and in epithelial cells. Furthermore, the communication of exosomes carrying miR-23a-3p between macrophages and epithelial cells was demonstrated using a cell co-culture model. Our results suggest that exosomal miR-23a-3p could be prospective as a biomarker in early diagnose for SiO2-induced lung fibrosis, and provided new threads for the treatment of silicosis.

Integrated multi-omics analyses reveal the pro-inflammatory and pro-fibrotic pulmonary macrophage subcluster in silicosis.

BACKGROUND: Silicosis is a lethal occupational disease caused by long-term exposure to respirable silica dust. Pulmonary macrophages play a crucial role in mediating the initiation of silicosis. However, the phenotypic and functional heterogeneities of pulmonary macrophages in silicosis have not been well-studied. METHODS: The silicosis mouse model was established by intratracheal administration of silica suspension. Bronchoalveolar lavage fluids (BALFs) of mice were collected for the multiplex cytokine analysis. Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics were performed to reveal the heterogeneity and spatial localization of macrophages in the lung tissues. The formation of the fibrotic nodules was characterized by histology, hydroxyproline assay, and immunohistochemical staining, respectively. The expression of the pro-inflammatory or pro-fibrotic genes was investigated by quantitative polymerase chain reaction (qPCR). RESULTS: We found that the level of pro-inflammatory cytokines and chemokines is significantly increased in the BALFs of silicosis mice. Apparent collagen deposition can also be observed in the silicotic lung tissues. By scRNA-seq, we have identified a subpopulation of Mmp12hi macrophages significantly expanding in the lung tissues of mice with silicosis. Spatial transcriptomics analysis further confirmed that the Mmp12hi macrophages are mainly enriched in silicosis nodules. Pseudotime trajectory showed that these Mmp12hi macrophages, highly expressing both pro-inflammatory and pro-fibrotic genes, are derived from Ly6c+ monocytes. Additionally, 4-octyl itaconate (4-OI) treatment, which can alleviate pulmonary fibrosis in silicosis mice, also reduces the enrichment of the Mmp12hi macrophages. Moreover, we found a subset of macrophages in BALFs derived from patients with silicosis exhibited similar characteristics of Mmp12hi macrophages in silicosis mice models. CONCLUSIONS: Our study suggested that a group of Mmp12hi macrophages highly express both pro-inflammatory and pro-fibrotic factors in silicosis mice, and thus may contribute to the progression of fibrosis. The findings have proposed new insights for understanding the heterogeneity of lung macrophages in silicosis, suggesting that the subset of Mmp12hi macrophages may be a potential therapy target to further halt the progression of silicosis.

RELA-mediated upregulation of LINC03047 promotes ferroptosis in silica-induced pulmonary fibrosis via SLC39A14.

Long non-coding RNAs play a key role in silicosis, a fatal fibrotic lung disease, and there is an urgent need to develop new treatment targets. Long intergenic non-protein-coding RNA 3047 (LINC03047) is associated with cancer, but its role and mechanism in the progression of silicosis require further elucidation. This study investigated the function of LINC03047 in the epithelial-mesenchymal transition (EMT) during silicosis progression. LINC03047 expression was upregulated in SiO2-treated BEAS-2B and A549 cells, promoting SiO2-induced ferroptosis and subsequent EMT. Moreover, knockdown of LINC03047 significantly decreased the expression of solute carrier family 39 member 14 (SLC39A14), a ferrous iron transporter, and inhibition of SLC39A14 alleviated the ferroptosis and EMT caused by LINC03047 overexpression. We further investigated that NF-κB p65 (RELA) was critical for LINC03047 transcription in SiO2-treated BEAS-2B and A549 cells. In vivo experiments showed that SLC39A14 deficiency improved SiO2-induced lipid peroxidation and EMT. Collectively, our study reveals the function of the RELA/LINC03047/SLC39A14 axis in SiO2-induced ferroptosis and EMT, thereby contributing to the identification of novel drug targets for silicosis therapy.

Understanding the molecular basis of anti-fibrotic potential of intranasal curcumin and its association with mitochondrial homeostasis in silica-exposed mice.

Silicosis is an occupational disease of the lungs brought in by repeated silica dust exposures. Inhalation of crystalline silica leads to persistent lung inflammation characterized by lung lesions due to granuloma formation. The specific molecular mechanism has not yet been identified, though. The Present study investigated the impact of silica-exposed lung fibrosis and probable molecular mechanisms. Here, Curcumin, derived from Curcuma longa shown to be an effective anti-inflammatory and anti-fibrotic molecule has been taken to investigate its therapeutic efficacy in silica-induced lung fibrosis. An experimental model of silicosis was established in mice where curcumin was administered an hour before intranasal silica exposure every alternate day for 35 days. Intranasal Curcumin treatment reduced silica-induced oxidative stress, inflammation marked by inflammatory cell recruitment, and prominent granuloma nodules along with aberrant collagen repair. Its protective benefits were confirmed by reduced MMP9 activities along with EMT markers (Vimentin and α-SMA). It has restored autophagy and suppressed the deposition of damaged mitochondria after silica exposure. Intranasal Curcumin also inhibited oxidative stress by boosting antioxidant enzyme activities and enhanced Nrf2-Keap1 expressions. Higher levels of PINK1, PARKIN, Cyt-c, P62/SQSTM, and damaged mitochondria in the silicosis group were significantly lowered after curcumin and dexamethasone treatments. Curcumin-induced autophagy resulted in reduced silica-induced mitochondria-dependent apoptosis. We report that intranasal curcumin treatment showed protective properties on pathological features prompted by silica particles, suggesting that the compound may constitute a promising strategy for the treatment of silicosis in the near future.

Mefunidone alleviates silica-induced inflammation and fibrosis by inhibiting the TLR4-NF-κB/MAPK pathway and attenuating pyroptosis in murine macrophages.

AIMS: Silicosis is the most common and severe type of pneumoconiosis, imposing a substantial disease burden and economic loss on patients and society. The pathogenesis and key targets of silicosis are not yet clear, and there are currently no effective treatments available. Therefore, we conducted research on mefunidone (MFD), a novel antifibrotic drug, to explore its efficacy and mechanism of action in murine silicosis. METHODS: Acute 7-day and chronic 28-day silicosis models were constructed in C57BL/6J mice by the intratracheal instillation of silica and subsequently treated with MFD to assess its therapeutic potential. The effects of MFD on silica-induced inflammation, pyroptosis, and fibrosis were further investigated using immortalized mouse bone marrow-derived macrophages (iBMDMs). RESULTS: In the 7-day silica-exposed mouse models, MFD treatment significantly alleviated pulmonary inflammation and notably reduced macrophage infiltration into the lung tissue. RNA-sequencing analysis of silica-induced iBMDMs followed by gene set enrichment analysis revealed that MFD profoundly influenced cytokine-cytokine receptor interactions, chemokine signaling, and the toll-like receptor signaling pathways. MFD treatment also markedly reduced the secretion of inflammatory cytokines and chemokines from silica-exposed iBMDMs. Moreover, MFD effectively downregulated the activation of the TLR4-NF-κB/MAPK signaling pathway induced by silica and mitigated the upregulation of pyroptosis markers. Additionally, MFD treatment significantly suppressed the activation of fibroblasts and alveolar epithelial cells co-cultured with silica-exposed mouse macrophages. Ultimately, in the 28-day silica-exposed mouse models, MFD administration led to a substantial reduction in the severity of pulmonary fibrosis. CONCLUSION: MFD mitigates silica-induced pulmonary inflammation and fibrosis in mice by suppressing the TLR4-NF-κB/MAPK signaling pathway and reducing pyroptotic responses in macrophages. MFD could potentially emerge as a novel therapeutic agent for the treatment of silicosis.

Anti-pneumoconiosis effect of schisantherin A in PMA-induced A549 cells and SiO2/TiO2nanoparticles-induced acute pulmonary injury in mice.

There has been significant global interest in respiratory health driven by the coronavirus disease (COVID-19) and severe environmental pollution. This study explored the potential of schisantherin A (SchA), a compound derived from Schisandra chinensis, to protect against acute pneumoconiosis. We assessed the effects of SchA on phorbol 12-myristate 13-acetate (PMA)-stimulated A549 alveolar epithelial cells and SiO2/TiO2-induced pulmonary injury in mice. In A549 cells, SchA significantly decreased pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and interleukin (IL)-8 levels. SchA-mediated reduction in inflammatory mediators was associated with the downregulation of PMA-stimulated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling activation. In SiO2/TiO2-induced lung-injured mice, SchA administration significantly reduced MUC5AC production in lung tissue. SchA administration significantly downregulated the overexpression of NK-κB and the subsequent production of COX-2, iNOS, and NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasomes. It significantly suppressed expected increases in total cell numbers and pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and IL-1ß in the bronchoalveolar lavage fluid (BALF) in SiO2/TiO2-stimulated mice. In contrast, the SiO2/TiO2-mediated decrease in IL-10 levels was significantly improved by SchA treatment. These fundamental results can be used to develop potential treatments involving SchA for acute pneumoconiosis.

Crystalline silica-induced recruitment and immuno-imbalance of CD4+ tissue resident memory T cells promote silicosis progression.

Occupational crystalline silica (CS) particle exposure leads to silicosis. The burden of CS-associated disease remains high, and treatment options are limited due to vague mechanisms. Here we show that pulmonary CD4+ tissue-resident memory T cells (TRM) accumulate in response to CS particles, mediating the pathogenesis of silicosis. The TRM cells are derived from peripheral lymphocyte recruitment and in situ expansion. Specifically, CD69+CD103+ TRM-Tregs depend more on circulating T cell replenishment. CD69 and CD103 can divide the TRM cells into functionally distinct subsets, mirroring the immuno-balance within CD4+ TRM cells. However, targeting CD103+ TRM-Tregs do not mitigate disease phenotype since the TRM subsets exert immunosuppressive but not pro-fibrotic roles. After identifying pathogenic CD69+CD103- subsets, we highlight IL-7 for their maintenance and function, that present a promising avenue for mitigating silicosis. Together, our findings highlight the distinct role of CD4+ TRM cells in mediating CS-induced fibrosis and provide potential therapeutic strategies.

Bicyclol attenuates pulmonary fibrosis with silicosis via both canonical and non-canonical TGF-ß1 signaling pathways.

BACKGROUND: Silicosis is an irreversible fibrotic disease of the lung caused by chronic exposure to silica dust, which manifests as infiltration of inflammatory cells, excessive secretion of pro-inflammatory cytokines, and pulmonary diffuse fibrosis. As the disease progresses, lung function further deteriorates, leading to poorer quality of life of patients. Currently, few effective drugs are available for the treatment of silicosis. Bicyclol (BIC) is a compound widely employed to treat chronic viral hepatitis and drug-induced liver injury. While recent studies have demonstrated anti-fibrosis effects of BIC on multiple organs, including liver, lung, and kidney, its therapeutic benefit against silicosis remains unclear. In this study, we established a rat model of silicosis, with the aim of evaluating the potential therapeutic effects of BIC. METHODS: We constructed a silicotic rat model and administered BIC after injury. The FlexiVent instrument with a forced oscillation system was used to detect the pulmonary function of rats. HE and Masson staining were used to assess the effect of BIC on silica-induced rats. Macrophages-inflammatory model of RAW264.7 cells, fibroblast-myofibroblast transition (FMT) model of NIH-3T3 cells, and epithelial-mesenchymal transition (EMT) model of TC-1 cells were established in vitro. And the levels of inflammatory mediators and fibrosis-related proteins were evaluated in vivo and in vitro after BIC treatment by Western Blot analysis, RT-PCR, ELISA, and flow cytometry experiments. RESULTS: BIC significantly improved static compliance of lung and expiratory and inspiratory capacity of silica-induced rats. Moreover, BIC reduced number of inflammatory cells and cytokines as well as collagen deposition in lungs, leading to delayed fibrosis progression in the silicosis rat model. Further exploration of the underlying molecular mechanisms revealed that BIC suppressed the activation, polarization, and apoptosis of RAW264.7 macrophages induced by SiO2. Additionally, BIC inhibited SiO2-mediated secretion of the inflammatory cytokines IL-1ß, IL-6, TNF-α, and TGF-ß1 in macrophages. BIC inhibited FMT of NIH-3T3 as well as EMT of TC-1 in the in vitro silicosis model, resulting in reduced proliferation and migration capability of NIH-3T3 cells. Further investigation of the cytokines secreted by macrophages revealed suppression of both FMT and EMT by BIC through targeting of TGF-ß1. Notably, BIC blocked the activation of JAK2/STAT3 in NIH-3T3 cells required for FMT while preventing both phosphorylation and nuclear translocation of SMAD2/3 in TC-1 cells necessary for the EMT process. CONCLUSION: The collective data suggest that BIC prevents both FMT and EMT processes, in turn, reducing aberrant collagen deposition. Our findings demonstrate for the first time that BIC ameliorates inflammatory cytokine secretion, in particular, TGF-ß1, and consequently inhibits FMT and EMT via TGF-ß1 canonical and non-canonical pathways, ultimately resulting in reduction of aberrant collagen deposition and slower progression of silicosis, supporting its potential as a novel therapeutic agent.

SiO 2 Induces Iron Overload and Ferroptosis in Cardiomyocytes in a Silicosis Mouse Model.

Objective: The aim of this study was to explore the role and mechanism of ferroptosis in SiO 2-induced cardiac injury using a mouse model. Methods: Male C57BL/6 mice were intratracheally instilled with SiO 2 to create a silicosis model. Ferrostatin-1 (Fer-1) and deferoxamine (DFO) were used to suppress ferroptosis. Serum biomarkers, oxidative stress markers, histopathology, iron content, and the expression of ferroptosis-related proteins were assessed. Results: SiO 2 altered serum cardiac injury biomarkers, oxidative stress, iron accumulation, and ferroptosis markers in myocardial tissue. Fer-1 and DFO reduced lipid peroxidation and iron overload, and alleviated SiO 2-induced mitochondrial damage and myocardial injury. SiO 2 inhibited Nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream antioxidant genes, while Fer-1 more potently reactivated Nrf2 compared to DFO. Conclusion: Iron overload-induced ferroptosis contributes to SiO 2-induced cardiac injury. Targeting ferroptosis by reducing iron accumulation or inhibiting lipid peroxidation protects against SiO 2 cardiotoxicity, potentially via modulation of the Nrf2 pathway.

Pulmonary osteoclast-like cells in silica induced pulmonary fibrosis.

The pathophysiology of silicosis is poorly understood, limiting development of therapies for those who have been exposed to the respirable particle. We explored mechanisms of silica-induced pulmonary fibrosis in human lung samples collected from patients with occupational exposure to silica and in a longitudinal mouse model of silicosis using multiple modalities including whole-lung single-cell RNA sequencing and histological, biochemical, and physiologic assessments. In addition to pulmonary inflammation and fibrosis, intratracheal silica challenge induced osteoclast-like differentiation of alveolar macrophages and recruited monocytes, driven by induction of the osteoclastogenic cytokine, receptor activator of nuclear factor κΒ ligand (RANKL) in pulmonary lymphocytes, and alveolar type II cells. Anti-RANKL monoclonal antibody treatment suppressed silica-induced osteoclast-like differentiation in the lung and attenuated pulmonary fibrosis. We conclude that silica induces differentiation of pulmonary osteoclast-like cells leading to progressive lung injury, likely due to sustained elaboration of bone-resorbing proteases and hydrochloric acid. Interrupting osteoclast-like differentiation may therefore constitute a promising avenue for moderating lung damage in silicosis.

Jun and JunB members of the AP-1 complex are potential therapeutic targets for silicosis.

Silicosis is a systemic disease with predominantly diffuse fibrosis of the lungs due to prolonged inhalation of free SiO2 dust during the manufacturing process, for which there is no effective treatment. In this study, we used a combined epigenetic and transcriptomic approach to reveal the chromatin-opening features of silicosis and identify the key transcription factor activator protein 1 (AP-1) that responds to silicosis fibrosis. Therapeutic administration of an AP-1 inhibitor inhibits the PI3K/AKT signaling pathway, reduces fibrosis marker proteins, and significantly ameliorates lung fibrosis in a mouse model of silicosis. In addition, it was observed that the expression of Jun and JunB was significantly up-regulated in a TGF-ß1-induced in vitro transdifferentiation model of NIH/3T3 cells, and Co-IP confirmed that a protein complex could be formed between Jun and JunB. Mechanistically, silencing of Jun and JunB expression reversed the activation of the PI3K/AKT signaling pathway and the upregulation of fibrosis marker proteins in NIH/3 T3 cells after TGF-ß1 stimulation. Taken together, Jun/JunB is expected to be a potential therapeutic target for silicosis fibrosis.

The aryl hydrocarbon receptor pathway is a marker of lung cell activation but does not play a central pathologic role in engineered stone-associated silicosis.

Engineered stone-associated silicosis is characterised by a rapid progression of fibrosis linked to a shorter duration of exposure. To date, there is lack of information about molecular pathways that regulates disease development and the aggressiveness of this form of silicosis. Therefore, we compared transcriptome responses to different engineered stone samples and standard silica. We then identified and further tested a stone dust specific pathway (aryl hydrocarbon receptor [AhR]) in relation to mitigation of adverse lung cell responses. Cells (epithelial cells, A549; macrophages, THP-1) were exposed to two different benchtop stone samples, standard silica and vehicle control, followed by RNA sequencing analysis. Bioinformatics analyses were conducted, and the expression of dysregulated AhR pathway genes resulting from engineered stone exposure was then correlated with cytokine responses. Finally, we inhibited AhR pathway in cells pretreated with AhR antagonist and observed how this impacted cell cytotoxicity and inflammation. Through transcriptome analysis, we identified the AhR pathway genes (CYP1A1, CYP1B1 and TIPARP) that showed differential expression that was unique to engineered stones and common between both cell types. The expression of these genes was positively correlated with interleukin-8 production in A549 and THP-1 cells. However, we only observed a mild effect of AhR pathway inhibition on engineered stone dust induced cytokine responses. Given the dual roles of AhR pathway in physiological and pathological processes, our data showed that expression of AhR target genes could be markers for assessing toxicity of engineered stones; however, AhR pathway might not play a significant pathologic role in engineered stone-associated silicosis.

Trigonelline hydrochloride attenuates silica-induced pulmonary fibrosis by orchestrating fibroblast to myofibroblast differentiation.

BACKGROUND: Silicosis represents a paramount occupational health hazard globally, with its incidence, morbidity, and mortality on an upward trajectory, posing substantial clinical dilemmas due to limited effective treatment options available. Trigonelline (Trig), a plant alkaloid extracted mainly from coffee and fenugreek, have diverse biological properties such as protecting dermal fibroblasts against ultraviolet radiation and has the potential to inhibit collagen synthesis. However, it's unclear whether Trig inhibits fibroblast activation to attenuate silicosis-induced pulmonary fibrosis is unclear. METHODS: To evaluate the therapeutic efficacy of Trig in the context of silicosis-related pulmonary fibrosis, a mouse model of silicosis was utilized. The investigation seeks to elucidated Trig's impact on the progression of silica-induced pulmonary fibrosis by evaluating protein expression, mRNA levels and employing Hematoxylin and Eosin (H&E), Masson's trichrome, and Sirius Red staining. Subsequently, we explored the mechanism underlying of its functions. RESULTS: In vivo experiment, Trig has been demonstrated the significant efficacy in mitigating SiO2-induced silicosis and BLM-induced pulmonary fibrosis, as evidenced by improved histochemical staining and reduced fibrotic marker expressions. Additionally, we showed that the differentiation of fibroblast to myofibroblast was imped in Trig + SiO2 group. In terms of mechanism, we obtained in vitro evidence that Trig inhibited fibroblast-to-myofibroblast differentiation by repressing TGF-ß/Smad signaling according to the in vitro evidence. Notably, our finding indicated that Trig seemed to be safe in mice and fibroblasts. CONCLUSION: In summary, Trig attenuated the severity of silicosis-related pulmonary fibrosis by alleviating the differentiation of myofibroblasts, indicating the development of novel therapeutic approaches for silicosis fibrosis.

Role of TRIM59 in regulating PPM1A in the pathogenesis of silicosis and the intervention effect of tanshinone IIA.

This study examines the involvement of TRIM59 in silica-induced pulmonary fibrosis and explores the therapeutic efficacy of Tanshinone IIA (Tan IIA). In vivo experiments conducted on rats with silica-induced pulmonary fibrosis unveiled an increase in TRIM59 levels and a decrease in PPM1A levels. Subsequent investigations using in vitro silicosis cell models demonstrated that modulation of TRIM59 expression significantly impacts silicosis fibrosis, influencing the levels of PPM1A and activation of the Smad2/3 signaling pathway. Immunofluorescence and co-immunoprecipitation assays confirmed the interaction between TRIM59 and PPM1A in fibroblasts, wherein TRIM59 facilitated the degradation of PPM1A protein via proteasomal and ubiquitin-mediated pathways. Furthermore, employing a rat model of silica-induced pulmonary fibrosis, Tan IIA exhibited efficacy in mitigating lung tissue damage and fibrosis. Immunohistochemical analysis validated the upregulation of TRIM59 and downregulation of PPM1A in silica-induced pulmonary fibrosis, which Tan IIA alleviated. In vitro studies elucidated the mechanism by which Tan IIA regulates the Smad2/3 signaling pathway through TRIM59-mediated modulation of PPM1A. Treatment with Tan IIA in silica-induced fibrosis cell models resulted in concentration-dependent reductions in fibrotic markers and attenuation of relevant protein expressions. Tan IIA intervention in silica-induced fibrosis cell models mitigated the TRIM59-induced upregulation of fibrotic markers and enhanced PPM1A expression, thereby partially reversing Smad2/3 activation. Overall, the findings indicate that while overexpression of TRIM59 may activate the Smads pathway by suppressing PPM1A expression, treatment with Tan IIA holds promise in counteracting these effects by inhibiting TRIM59 expression.