Osteopontin (SPP1) plays a critical role in the initiation and progression of silicosis, but whether macrophage-derived SPP1 regulates lipid metabolism during silicosis remains unclear. In this study, RNA sequencing of lung tissues from silicotic mice and silica-stimulated alveolar macrophages identified Spp1 as a differentially expressed gene, and pathway enrichment analysis revealed significant enrichment of lipid metabolism-related signaling pathways. Using in vivo and in vitro models of silicosis, we assessed changes in SPP1 expression and key fatty acid synthesis markers. Silica stimulation increased SPP1 expression, induced lipid droplet formation, and upregulated fatty acid synthesis-related proteins. Subsequent in vitro experiments, including Spp1 knockdown, overexpression, and recombinant protein treatment, demonstrated that Spp1 knockdown effectively suppressed silica-induced lipid droplet formation and fatty acid synthesis. Mechanistically, SPP1 activated the fatty acid synthesis pathway by binding to its receptor CD44, thereby promoting SREBP-1 processing and nuclear translocation. Notably, Cd44 silencing abrogated SPP1-induced lipid droplet formation, the upregulation of fatty acid synthesis enzymes, and the production of pro-inflammatory cytokines (TNF-α and IL-6). Moreover, macrophage-specific knockout of Spp1 significantly alleviated pulmonary fibrosis, improved lung function, and inhibited the fatty acid synthesis pathway in vivo. Collectively, this study elucidates a novel mechanism by which SPP1 regulates macrophage fatty acid metabolism through the CD44/SREBP-1 axis, providing a mechanistic basis for targeting SPP1 in silicosis treatment.

Pulmonary fibrosis, particularly idiopathic pulmonary fibrosis (IPF), is a chronic and progressive interstitial lung disease characterized by alveolar epithelial injury, fibroblast activation, and excessive extracellular matrix deposition, which collectively lead to respiratory failure. Despite the availability of antifibrotic agents, disease-modifying therapies remain limited. Emerging evidence has identified dysregulated sphingolipid metabolism, especially ceramide accumulation, as a key driver of fibrotic pathogenesis. Ceramide is a central bioactive lipid in the sphingolipid pathway that regulates multiple cellular processes, including apoptosis, inflammation, endothelial barrier dysfunction, and fibroblast activation, all of which contribute to pulmonary fibrosis. This review is a narrative review that systematically summarizes the biosynthetic and metabolic pathways of ceramide, with an emphasis on chain length-specific functions and the ceramide to S1P rheostat. We further discuss the mechanistic roles of ceramide in alveolar epithelial cell apoptosis, inflammatory responses, and vascular barrier disruption in fibrotic lung disease. Finally, we highlight emerging therapeutic strategies that target ceramide metabolism, including inhibitors of acid sphingomyelinase (ASMase) and serine palmitoyltransferase (SPT), and propose future directions for clinical translation.

Silicosis is an irreversible occupational disease, but its pathogenesis remains incompletely understood. Although secreted phosphoprotein 1 (SPP1) and lipid dysregulation have been implicated in silicosis, their functional interplay is unclear. In this study, we demonstrated that macrophage-specific Spp1 knockout in mice attenuated silica-induced pulmonary fibrosis, improved lung function, and suppressed aberrant lipid metabolism both in vivo and in vitro. Specifically, silica-induced intracellular SPP1 (iSPP1) regulated the ceramide synthesis pathway by promoting the ubiquitination of fibronectin type III domain-containing 5 (FNDC5). This action attenuated FNDC5-mediated ubiquitination and subsequent downregulation of Δ4-dihydroceramide desaturase 1 (DEGS1), a key enzyme in de novo ceramide synthesis within the endoplasmic reticulum. Overall, we identified the interaction of SPP1, FNDC5, and DEGS1, providing a mechanistic basis and a potential therapeutic strategy for targeting lipid dysregulation in silicosis.

Silicosis, one of the most common and severe forms of pneumoconiosis, remains a major occupational health concern worldwide. Given the lack of effective therapies, understanding the underlying molecular mechanisms is urgently needed. Here, we report that ALKB homolog 1 (ALKBH1), an N6-methyladenosine (m6A) demethylase, is upregulated in silica-induced pulmonary fibrosis and plays a pro-fibrotic role. The antifibrotic peptide Ac-SDKP inhibited Alkbh1 expression and alleviated pulmonary fibrosis. Mechanistically, ALKBH1 suppressed the biosynthesis of miR-129-5p by removing m6A modification from pri-miR-129-5p, thereby reducing DGCR8-mediated processing and leading to decreased mature miR-129-5p levels. Ac-SDKP reversed this process, restoring miR-129-5p expression. Functionally, overexpression of miR-129-5p attenuated silica-induced pulmonary fibrosis by suppressing macrophage activation. Collectively, these findings identify the Ac-SDKP-ALKBH1-miR-129-5p axis as a critical regulatory mechanism, with ALKBH1-mediated m6A demethylation of pri-miR-129-5p representing a key node and a promising therapeutic target for silicosis.

Late-onset silicosis is characterized by the progression of pulmonary fibrosis long after cessation of silica exposure, yet its underlying mechanisms remain poorly understood. This study aimed to determine whether a brief silica exposure could initiate a sustaining fibrotic process and to characterize the associated pathological and molecular alterations. A rat model of silicosis was established by short-term silica inhalation for two weeks, followed by a recovery period of up to 46 weeks. Histopathological and micro-CT analyses demonstrated progressive fibrotic development even after exposure cessation. Notably, fibrotic pulmonary dust foci (fPDFs) emerged as a predominant lesion, characterized by alveolar remodeling and diffuse collagen deposition distinct from classical silicotic nodules. These lesions were associated with alveolar type II (AT2) cell dysfunction, evidenced by aberrant differentiation, loss of homeostatic markers such as ATP-binding cassette subfamily A member 3 (ABCA3), lysophosphatidylcholine acyltransferase 1 (LPCAT1), and fatty acid synthase (FAS), and gain of transitional markers keratin 8 (KRT8) and stratifin (SFN). Integrated proteomic and lipidomic analyses revealed profound metabolic reprogramming, with significant dysregulation of lipid metabolism pathways. Key enzymes involved in lipid synthesis and remodeling were identified in AT2 cells and downregulated in fPDF regions. Our findings establish that short-term silica exposure initiates a sustaining fibrotic cascade, highlight fPDFs as a critical pathological entity, and suggest that AT2 cell dysfunction and metabolic reprogramming are closely associated with the progression of silicosis, highlighting them as potential key correlates of the sustaining fibrotic cascade. These insights provide a novel framework for understanding disease progression and identifying therapeutic targets.

Silicosis remains a critical occupational health concern worldwide, lacking effective treatments due to unclear mechanisms. In this study, we investigated the citrullinated proteomic profile and its effects in mice exposed to silica. Our findings demonstrated elevated levels of citrullinated peptides and citrullinated vimentin (Cit-Vim) in silicotic mice and silica-treated macrophages, regulated by peptidylarginine deiminase (PADI2). Unlike vimentin, Cit-Vim amplified the production of tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6), and IL-1β in silica-treated macrophages through interaction with Toll-like receptor 4 (TLR4) signaling. RNA sequencing revealed that early growth response protein 1 (EGR1) is a target of PADI2, with Cit-Vim inducing lung inflammation via EGR1 signaling. Pharmacological inhibition or genetic knockout of Padi2 attenuated silica-induced lung inflammation and fibrosis. These findings suggest that targeting PADI2 may represent a novel therapeutic strategy of silicosis.

The role and mechanism of protein tyrosine phosphatase non-receptor type 2 (PTPN2) in silicosis remain unclear. In this study, transcriptome sequencing, microcomputed tomography, histological examination, and western blot analysis were used to evaluate the effects of PTPN2 on the retinol metabolism signaling pathway, the transforming growth factor-beta 1 (TGF-β1) signaling pathway, cellular senescence, and pulmonary fibrosis in silicosis. The results demonstrated that overexpression of PTPN2 significantly reduced the degree of pulmonary fibrosis in silicosis model mice, whereas alveolar endothelial cell-specific knockout of PTPN2 exacerbated fibrosis. PTPN2 overexpression ameliorated fibrosis by regulating key enzymes in retinol metabolism, downregulating the TGF-β1 signaling pathway, and decreasing the expression of senescence-related signals. Both in vitro and in vivo results showed that PTPN2 overexpression upregulated the expression of retinol metabolism signaling pathway-related molecules (ALDH1A2, RDH10, and DHRS3), reversed the expression of TGF-β1 signaling pathway-related components (TGF-β1, TGF-βR1, and TGF-βR2) and the phosphorylation of Smad2/3, and downregulated the expression of senescence-associated factors (β-galactosidase, p-p53, p21, and p16). These findings suggest that PTPN2 may alleviate silicotic pulmonary fibrosis by inhibiting the senescence of type II alveolar epithelial cells via influencing retinol metabolism regulated by ALDH1A2.

BACKGROUND:It is well recognized that developing new animal models, refining the existing mouse models, and thoroughly characterizing their features are essential for gaining a deeper understanding of rosacea pathogenesis and for advancing therapeutic strategies in this direction. Accordingly, we aimed to characterize the pathological features of a long-term LL-37-induced mouse model of rosacea and to compare the disease manifestations and pathophysiological characteristics between short-term and long-term LL-37-induced models. A key focus was to investigate differential gene expression and the underlying mechanisms of immune system dysregulation in these models. METHODS:We comparatively assessed skin lesion manifestations, the extent of inflammatory infiltration, sebaceous gland alterations, fibrosis, and angiogenesis in both models. Assessments were performed using photographic documentation, hematoxylin-eosin (HE) staining, Van Gieson's (VG) staining, immunohistochemistry, and Western blotting. Furthermore, we employed RNA sequencing to analyze differential gene expression in mouse skin. The RNA sequencing data were validated using immunofluorescence staining and Western blotting, with a specific focus on gene variations and mechanisms related to immune system dysregulation. RESULTS:Mice subjected to long-term LL-37 induction developed rosacea-like pathological features, including angiogenesis, thickened skin tissue, and sebaceous gland hypertrophy. In the short-term LL-37-induced model, immune dysregulation primarily involved the innate immune response. However, long-term LL-37 induction resulted in significant activation of both innate and adaptive immune responses. CONCLUSION:The long-term LL-37-induced mouse model offers a valuable animal model for the detailed investigation of the pathological mechanisms driving moderate-to-severe rosacea with prolonged disease duration. Importantly, this model provides a significant experimental foundation for exploring the potential role of immune system dysregulation in rosacea pathogenesis.

Targeting macrophage SCTR mitigates integrated profibrotic, inflammatory, ER stress, and senescent pathways, preserving lung function and revealing a novel therapeutic strategy for silicosis.

BackgroundThe senescence of alveolar type II epithelial cells is an important driving factor for the progression of silicotic fibrosis, and the regulatory effects of oxamate on the senescence of alveolar type II epithelial cells is still unclear.ObjectiveTo explore whether lactate dehydrogenase inhibitor oxamate can alleviate silicotic fibrosis in mice by inhibiting senescence of alveolar type II epithelial cellsMethodsThis study was divided into two parts: in vivo experiments and in vitro experiments. In the first part, forty SPF C57BL/6J male mice were randomly divided into four groups with 10 in each group: control group, silicosis model group, low-dose oxamate treatment group, and high-dose oxamate treatment group. The silicotic mouse model was established by intratracheal instillation of 50 μL SiO2 suspension (100 mg·mL−1). The treatment models were prepared by intraperitoneal injection of 100 μL oxamate (225 mmol·L−1 and 1125 mmol·L−1). In the second part, induction of MLE-12 mouse alveolar type II epithelial cells was conducted with SiO2. The in vitro experimental groups were ① SiO2 induction groups: control group, 50 μg·mL−1 SiO2 group, 100 μg·mL−1 SiO2 group, and 200 μg·mL−1 SiO2 group, and ② oxamate treatment groups: control group, SiO2 group (100 μg·mL−1), low-dose oxamate (25 mmol·L−1) treatment group, and high-dose oxamate (50 mmol·L−1) treatment group. Pathological morphology of lung tissues was evaluated after hematoxylin-eosin (HE) staining; deposition of collagen in lung tissues was evaluated after sirius red staining; positive co-expression of prosurfactant protein C (Pro-SPC) and β-galactosidase was detected by immunofluorescence staining; positive expression of β-galactosidase in MLE-12 cells was detected by immunofluorescence staining. The protein expression levels of collagen type I (CoL I), fibronectin1 (FN1), hexokinase 2 (HK2), pyruvate kinase isozyme type M2 (PKM2), lactate dehydrogenase A (LDHA), p-ataxia telangiectasia and Rad3-related kinase (ATR), and cyclin-dependent kinase inhibitors p21, and p16 were detected by Western blotting.ResultsCompared with the control group, the protein expression levels of HK2, PKM2, LDHA, p-ATR, p21, and p16 were significantly upregulated in the silicosis model group and the SiO2-induced MLE-12 cells (P＜0.05). The in vivo studies showed that, compared with the control group, the silicon nodule area, the collagen deposition area, the proportion of β-galactosidase positive cells, and the protein expression levels of CoL I, FN1, LDHA, p-ATR, p21, and p16 were significantly upregulated in the silicosis model group (P＜0.05). Compared with the silicosis model group, the oxamate treatment groups showed significant downregulation of the silicon nodule area, the collagen deposition area, the proportion of β-galactosidase positive cells, and the the CoL I, FN1, LDHA, p-ATR, p21, and p16 protein expression levels, and the high-dose oxamate treatment group showed a higher efficacy on these indicators than the low-dose oxamate treatment group (P＜0.05). The in vitro studies showed that, compared with the control group, the proportion of β-galactosidase positive cells and the protein expression levels of p-ATR, p21, and p16 were significantly upregulated in the SiO2-induced group (P＜0.05). Compared with the SiO2 group, the proportion of β-galactosidase positive cells and the LDHA, p-ATR, p21 and p16 protein expression levels were significantly downregulated in the oxamate treatment groups, and the high-dose oxamate treatment group showed a higher efficacy on these indicators than the low-dose oxamate treatment group (P＜0.05).ConclusionLactate dehydrogenase inhibitor oxamate can alleviate silicotic fibrosis in mice by inhibiting the senescence of alveolar type II epithelial cells.

Background:The aim was to explore the effect of macrophage polarization and macrophage-to-myofibroblast transition(MMT)in silicosis. Methods:Male Wistar rats were divided into a control group and a silicosis group developed using a HOPE MED 8050 dynamic automatic dusting system.Murine mac-rophage MH-S cells were randomly divided into a control group and an SiO 2 group.The pathological changes in lung tissue were observed using hematoxylin and eosin(HE)and Van Gieson(VG)staining.The distribution and location of macrophage marker(F4/80),M1 macrophage marker(iNOS),M2 macrophage marker(CD206),and myofibroblast marker(α-smooth muscle actin[α-SMA])were detected using immu-nohistochemical and immunofluorescent staining.The expression changes in iNOS,Arg,α-SMA,vimentin,and type I collagen(Col I)were measured using Western blot. Results:The results of HE and VG staining showed obvious silicon nodule formation and the distribution of thick collagen fibers in the lung tissue of the silicosis group.Macrophage marker F4/80 increased gradually from 8 to 32 weeks after exposure to silica.Immunohistochemical and immunofluorescent staining results revealed that there were more iNOS-positive cells and some CD206-positive cells in the lung tissue of the silicosis group at 8 weeks.More CD206-positive cells were found in the silicon nodules of the lung tissues in the silicosis group at 32 weeks.Western blot analysis showed that the expressions of Inducible nitric oxide synthase and Arg protein in the lung tissues of the silicosis group were upregulated compared with those of the con-trol group.The results of immunofluorescence staining showed the co-expression of F4/80,α-SMA,and Col I,and CD206 and α-SMA were co-expressed in the lung tissue of the silicosis group.The extracted rat alveolar lavage fluid revealed F4/80+α-SMA+,CD206+α-SMA+,and F4/80+α-SMA+Col I+cells using immunofluorescence staining.Similar results were also found in MH-S cells induced by SiO 2. Conclusions:The development of silicosis is accompanied by macrophage polarization and MMT.

BackgroundSilicosis presents a significant clinical challenges and economic burdens, with Traditional Chinese Medicine (TCM) emerging as a potential therapeutic avenue. However, the precise effects and mechanisms of TCM in treating silicosis remain uncertain and subject to debate.ObjectiveThe study aims to elucidate the therapeutic role and mechanisms of the Yang-Yin-Qing-Fei Decoction (YYQFD) and its key component, paeoniflorin, in silicosis using a murine model.MethodsSilicotic mice were treated with YYQFD, pirfenidone (PFD), or paeoniflorin. RAW264.7 cells and mouse lung fibroblasts (MLF) were stimulated with silica, matrix metalloproteinase-12 (MMP-12), or TGF-β1, followed by treatment with paeoniflorin, PFD, or relevant inhibitors. YYQFD constituents were characterized using High-Performance Liquid Chromatography (HPLC). Lung fibrosis severity was assessed via histopathological examination, micro-CT imaging, lung functions, and Western blot analysis. Transcriptome sequencing and bioinformatics analysis were employed to delineate the gene expression profile and target genes modulated by YYQFD in silicosis.ResultsTreatment with YYQFD ameliorated silica-induced lung fibrosis. Transcriptome sequencing identified MMP-12 as a potential common target of YYQFD and PFD. Additionally, a potential pro-inflammatory role of MMP-12, regulated by silica-induced TLR4 signaling pathways, was revealed. Paeoniflorin, one of the most distinctive compounds in YYQFD, attenuated silica-induced MMP-12 increase and its derived inflammatory factors in macrophages through a direct binding effect. Notably, paeoniflorin treatment exerted anti-fibrotic effects by inhibiting MMP-12-derived inflammatory factors and TGF-β1-induced myofibroblast differentiation in silica-exposed mice.ConclusionsThis study underscores paeoniflorin as one of the most principal bioactive compounds in YYQFD, highlighting its capacity to attenuate lung inflammation driven by macrophage-derived MMP-12 and reduce lung fibrosis both in vivo and in vitro.

Exposure to crystalline silica leads to health effects beyond occupational silicosis. Exercise training's potential benefits on pulmonary diseases yield inconsistent outcomes. In this study, we utilized experimental silicotic mice subjected to exercise training and pharmacological interventions, including interleukin-17A (IL-17A) neutralizing antibody or clodronate liposome for macrophage depletion. Findings reveal exercise training's ability to mitigate silicosis progression in mice by suppressing scavenger receptor B (SRB)/NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and Toll-like receptor 4 (TLR4) pathways. Macrophage-derived IL-17A emerges as primary source and trigger for silica-induced pulmonary inflammation and fibrosis. Exercise training effectively inhibits IL-17A-CXC motif chemokine ligand 5 (CXCL5)-Chemokine (C-X-C motif) Receptor 2 (CXCR2) axis in silicotic mice. Our study evidences exercise training's potential to reduce collagen deposition, preserve elastic fibers, slow pulmonary fibrosis advancement, and enhance pulmonary function post silica exposure by impeding macrophage-derived IL-17A-CXCL5-CXCR2 axis.

BackgroundPneumoconiosis is the most serious occupational disease in China, and silicosis accounts for about half of it. Any intervention effect of physical exercise as the key and core of lung rehabilitation training on silicosis is still unclear.ObjectiveTo explore potential intervention effect of physical exercise on silicotic mice.MethodsForty SPF C57BL/6 male mice were randomly divided into four groups, 10 in each group, including a control group, a physical exercise group, a silicosis model group, and a silicosis model + physical exercise intervention group. Silicotic mouse model was established by using 50 μL SiO2 suspension (200 mg·mL−1). A treadmill was used to prepare mice receiving physical exercise at 0° inclination, 12.3 m·min−1, 60 min·d−1, 5 d·week−1 for 4 weeks. Pathological morphology of lung tissues was evaluated after hematoxylin-eosin (HE) staining; deposition of collagen in lung tissues was evaluated after Van Gieson (VG) staining; expression of p-protein kinase R-like endoplasmic reticulum kinase (PERK) was detected by immunofluorescence staining; expressions of cyclin dependent kinase inhibitors (p21) and p-p38 mitogen activated protein kinase (p38) were detected by immunohistochemistry. The protein expressions of endoplasmic reticulum stress signal factors [p-inositol-requiring enzyme-1α (p-IRE-1α), p-PERK, and p-eukaryotic initiation factor-2α (p-eIF-2α)], senescence signal factors (p-p53, p21, and p16), mitogen-activated protein kinase (MAPK) signal factors [p-p38, p-extracellular regulated protein kinases (p-ERK), and p-stress-activated protein kinase (p-JNK)] were detected by Western blotting.ResultsAfter designed acute SiO2 exposure, the images of micro computed tomography (CT) showed high density shadows in lung tissues of the silicotic mice and less shadows in lung tissues of the physical exercise intervention mice. After HE staining, the proportions of silicotic nodule area in lung tissues was (18.67±3.89) % in the silicosis model group, and significantly decreased to (8.78±1.05) % in the silicosis model + physical exercise intervention group (P<0.05). After VG staining, the proportion of collagen fiber area of lung tissues was (10.37±2.18) % in the silicosis model group, and significantly decreased to (4.35±0.89) % in the silicosis model + physical exercise intervention group (P<0.05). The results of immunofluorescence staining showed that in the silicosis model group, the expression of p-PERK increased at the location of silicotic nodules, while in the silicotic model + physical exercise intervention group, the expression of p-PERK decreased. The immunohistochemical staining results showed that the expression of p21 and p-p38 increased in the lung tissues of the silicosis model group; the expression of p21 and p-p38 decreased in the lung tissues of the silicosis model + physical exercise intervention group. The results of Western blotting showed that compared with the control group, the expression levels of p-IRE-1α (0.11±0.03), p-PERK (0.95±0.40), p-eIF-2α (3.53±0.91), p-p53 (1.78±0.07), p21 (1.98±0.10), p16 (1.26±0.17), p-p38 (0.41±0.09), p-ERK (0.42±0.05), and p-JNK (3.20±1.23) of the silicosis model group were all upregulated (P<0.05). Compared with the silicosis model group, the expression levels of p-IRE-1α (0.03±0.01), p-PERK (0.31±0.12), p-eIF-2α (0.30±0.06), p-p53 (0.76±0.08), p21 (0.18±0.11), p16 (0.70±0.24), p-p38 (0.03±0.00), p-ERK (0.19±0.03), and p-JNK (0.46±0.21) of the silicosis model + physical exercise intervention group were downregulated (P<0.05).ConclusionPhysical exercise may alleviate pulmonary fibrosis in silicotic mice, and inhibit abnormal expressions of endoplasmic reticulum stress signal, MAPK signal, and senescent signal.

The role and mechanisms of integrated stress response inhibitor (ISRIB) on silicosis are still not well defined. In the present study, the effects of ISRIB on cellular senescence and pulmonary fibrosis in silicosis were evaluated by RNA sequencing, micro-computed tomography, pulmonary function assessment, histological examination, and Western blot analysis. The results showed that ISRIB significantly reduced the degree of pulmonary fibrosis in mice with silicosis and reduced the expression of type I collagen, fibronectin, α-smooth muscle actin, and transforming growth factor-β1. Both in vivo and in vitro results showed that ISRIB reversed the expression of senescence-related factors β-galactosidase, phosphor-ataxia telangiectasia mutated, phosphor-ataxia telangiectasia and Rad3-related protein, p-p53, p21, p16, and plasminogen activator inhibitor type 1. The aforementioned results were consistent with the sequencing results. These findings implied that ISRIB might reduce the degree of pulmonary fibrosis in mice with silicosis by inhibiting the cellular senescence of alveolar epithelial cell type II.

Silicosis is a pulmonary disease caused by the inhalation of silica. There is a lack of early and effective prevention, diagnosis, and treatment methods, and addressing silicotic fibrosis is crucial. Quercetin, a flavonoid with anti-carcinogenic, anti-inflammatory, and antiviral properties, is known to have a suppressive effect on fibrosis. The present study aimed to determine the therapeutic effect of quercetin on silicotic mice and macrophage polarity. We found that quercetin suppressed silicosis in mice. It was observed that SiO2 activated macrophage polarity and the macrophage-to-myofibroblast transition (MMT) by transforming the growth factor-β (TGF-β)-Smad2/3 signaling pathway in silicotic mice and MH-S cells. Quercetin also attenuated the MMT and the TGF-β-Smad2/3 signaling pathway in vivo and in vitro. The present study demonstrated that quercetin is a potential therapeutic agent for silicosis, which acts by regulating macrophage polarity and the MMT through the TGF-β-Smad2/3 signaling pathway.

Kinesin family member 3 A (KIF3A) decrease have been reported in silicotic patients and rats. However, the detailed mechanisms of KIF3A in silicosis remain unknown. In this study, we demonstrated that KIF3A effectively blocked the expression of beta-catenin and downstream myocardin-related transcription factor (MRTF)-A/serum response factor (SRF) signaling, thus inhibiting silica-induced epithelial myofibroblast transition (EMyT). Moreover, KIF3A was identified as a downstream mediator of an antifibrotic tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP). Knockdown of KIF3A expression reactivated beta-catenin/myocardin-related transcription factor (MRTF)-A/serum response factor (SRF) signaling that was attenuated by Ac-SDKP in vitro. Collectively, our findings suggest that Ac-SDKP plays its anti-fibrosis role via KIF3A-mediated beta-catenin suppression, at least in part, in both in vivo model of silicosis and in vitro model of EMyT.

[Background]Silicosis is one of the most serious occupational diseases in China,requiring new treatment targets and therapies.The effects and mechanisms of integrated stress response in-hibitors(ISRIB)on silicosis are still unknown. [Objective]To observe the effects of ISRIB on silicosis fibrosis and its possible mechanisms. [Methods]The study was divided into two parts:in vivo and in vitro experiments.For the in vivo part,40 SPF grade male C57BL/6J mice were randomly divided into four groups:control group,ISRIB group,silicotic model group,and ISRIB treatment group,with 10 mice in each group.A silicotic mouse model was established by using a single tracheal infusion of 50 μL 200 mg·mL-1 SiO2 suspension.After one week of perfusion with SiO2(the control group and the ISRIB group were perfused with an equal amount of sodium chloride solution),the ISRIB group and the ISRIB treatment group were intraperitoneally injected with 200 μL 2.5 mg·kg-1 ISRIB for four weeks,and mice of other groups were injected with equal amounts of sodium chloride solution.A micro-CT instrument was used to observe the lung field clarity and lung texture of each group;hematoxylin eosin(HE)staining was used to observe the histopathology morphology of the lung and the formation of silicon nodules;Van Gieson(VG)staining was used to observe the deposition of collagen in silicotic nodules;im-munofluorescence assay was used to detect the expression and localization of p-protein kinase RNA-like ER kinase(PERK)in lung tissue;immunoblotting was used to detect the expression of collagen I(Col I)and endoplasmic reticulum stress signal related proteins p-PERK,p-inositol-requiring enzyme-1α(p-IRE-1α),p-eukaryotic initiation factor 2α(p-eIF-2α),activating transcription factor 4(ATF4),and NOD-like receptor thermal protein domain associated protein 3(NLRP3).For the in vitro part,mouse alveolar macrophages MH-S cells were cultured in vitro and divided into a control group,an ISRIB(1 μg·mL-1)group,a SiO2 induction group(100 μg·mL-1),and an ISRIB treatment group(1 μg·mL-1 ISRIB treatment for 1 h,followed by 100 μg·mL-1 SiO2 induction).Immunofluorescence assay was used to detect the expression of p-PERK in MH-S cells;immunoblotting was used to determine expressions of endoplasmic reticulum stress signal related proteins p-PERK,p-IRE-1α,p-eIF-2α,and ATF4. [Results]The CT images showed that the lung markings of the silicotic model group mice were thickened,and several high-density shadows of varying sizes were observed in the lung field,mainly distributed around the bronchi.Compared with the silicotic model group,the ISRIB treatment group showed a decrease in the number and volume of high-density shadows.The HE staining results showed that lung tissues in the silicotic model group lost their normal structure,with the formation of silicon nodules,around which were thickened alveoli around the silicon nodules,and infiltration of inflammatory cells;the area and number of silicon nodules in the ISRIB treatment group were significantly reduced,and the range of silicon nodules was limited.The results of VG staining showed that the proportion of collagen fiber area in the lung tissue of the silicotic model group was 21.47%±2.59%,and it decreased to 9.34%±1.06%in the ISRIB treatment group,with a statistically significant difference(P<0.05).The immunofluorescence results showed that p-PERK was strongly expressed in the silicotic nodules and localized in macrophages;the expression of p-PERK was also significantly increased in the SiO2 induced MH-S cells,while the intensity of p-PERK was weakened in the ISRIB treatment groups both in vitro and in vivo.The results of immunoblotting showed that compared with the control group,the expressions of endoplasmic reticulum stress signal related proteins p-PERK,p-IRE-1α,p-eIF-2α,and ATF4 were upregulated after SiO2 stimulation in vivo and in vitro;compared with the SiO2 induction group,the expressions of p-PERK,p-IRE-1α,p-eIF-2α,and ATF4 were significantly downregulated in the ISRIB treatment group in vivo and in vitro,and the differ-ences were statistically significant(P<0.05). [Conclusion]ISRIB antagonizes silicosis fibrosis by inhibiting the activation of macrophage endoplasmic reticulum stress signals.

Silicosis is the most prevalent occupational disease in China. It is a form of pulmonary fibrosis caused by the inhalation of silicon particles. As there is no cure for the potentially lethal and progressive condition, the treatment of silicotic fibrosis is an important and difficult problem to address. Thalidomide, a drug with anti-inflammatory and immunoregulatory properties, has been reported to have lung-protective effects. The purpose of this study was to observe the therapeutic effect of thalidomide on silicotic mice and to determine the protective mechanism. By using silicotic mice models and MH-S cells, we found the expression of endoplasmic reticulum stress (ER stress) and Toll-like receptor 4 (TLR4)-nuclear factor kappa-B (NF-κB) pathway as well as inflammation-related factors were upregulated in the macrophages of silicotic mice. The same indexes were detected in silica-stimulated MH-S cells, and the results were consistent with those in vivo. That is, silica activated ER stress and the TLR4-NF-κB pathway as well as the inflammatory response in vitro. Treating both silicotic mice and silica-stimulated MH-S cells with thalidomide inhibited ER stress and the TLR4-NF-κB pathway as well as the inflammatory response. The present study demonstrates thalidomide as a potential therapeutic agent against silicosis.