Targeting acid sphingomyelinase/ceramide/S1P ameliorates silica-induced pulmonary fibrosis through Hippo/YAP signaling pathway in mice

Targeting acid sphingomyelinase/ceramide/S1P ameliorates silica-induced pulmonary fibrosis through Hippo/YAP signaling pathway in mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Targeting acid sphingomyelinase/ceramide/S1P ameliorates silica-induced pulmonary fibrosis through Hippo/YAP signaling pathway in mice Ruimin Ma, Di Sun, Yuanying Wang, Yawen Song, Qiao Ye This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7052670/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Silicosis is irreversible lung disease caused by exposure to dust-containing silica particles. Our study aims to explore the potential effects of acid sphingomyelinase (aSMase), ceramide (Cer) and sphingosine-1-phosphate (S1P) on silica-induced pulmonary fibrosis. We also explored the expression of the downstream signaling pathway of Hippo/YAP. The high expression of SMPD1 which encodes aSMase were given. The level of aSMase in silicosis was lower than those in controls. Additionally, the level of aSMase in lung fibroblasts, following stimulation with TGF-β1, was observed to decrease. The phagocytosis of silica particles by macrophages promoted lung fibroblast transdifferentiation and activated the aSMase/Cer/S1P signaling pathway. The activated Hippo/YAP signaling pathway is involved in silica-induced pulmonary fibrosis. Moreover, the collagen content within silicotic nodules and the number of large nodules were reduced by the intervention with the high expression of SMPD1 in vivo. Furthermore, the levels of fibrotic genes in TGF-β1-induced lung fibroblasts activation were diminished with the overexpression of SMPD1. ASMase was downregulated in silica-induced lung fibrosis, however the administration of exogenous SMPD1 overexpression has the potential to alleviate this condition. This process may be related to the downstream Hippo/YAP signaling pathway. Biological sciences/Cell biology Health sciences/Diseases Biological sciences/Molecular biology Health sciences/Pathogenesis silica sphingolipid metabolism acid sphingomyelinase Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Fibrotic diseases encompass a range of conditions characterized by the formation of scar tissue, affecting various organs. These diseases pose a significant burden, impacting roughly a quarter of the global population due to their severity 1 . According to the Global Burden of Disease (GBD) study, the incidence of pneumoconiosis, including silicosis, increased by 66.0% between 1990 and 2017 2 . Silicosis, a form of pulmonary fibrosis caused by silica exposure, manifests through the injury to alveolar epithelial cells, fibroblast differentiation, and extracellular matrix deposition 3 . During tissue repair, fibroblasts become activated and transition into myofibroblasts, which contribute to extracellular matrix production. The inflammatory and fibrotic factors within the tissue microenvironment stimulate epithelial cells to undergo epithelial-mesenchymal transition, ultimately leading to pulmonary fibrosis 4 . Sphingolipids have recently been found to play a critical role in regulating cell proliferation and differentiation during pulmonary fibrosis 5 . Sphingomyelin, a specific sphingolipid, serves as a major component of mammalian cell membranes 6 . It is hydrolyzed by three principal enzymes known as sphingomyelinases (SMases): acid, basic, and neutral sphingomyelinase. Among these, acid sphingomyelinase (aSMase) is considered the most active 6 . ASMase is a Zn2+-stimulated SMase that is encoded by the Sphingomyelin Phosphodiesterase 1 (SMPD1) gene 7 . This hydrolytic process generates ceramide, a molecule essential for various cellular functions 8 . Ceramide can be further metabolized by ceramidase to produce sphingosine, which can be phosphorylated by either sphingosine kinase 1 (Sphk1) or sphingosine kinase 2 (Sphk2) to generate sphingosine-1-phosphate (S1P) 9 . The aSMase/Cer/S1P signaling pathway has been implicated in the pathogenesis of various diseases. Activation of aSMase in emphysema leads to increased ceramide production, which contributes to the loss of alveolar septa in the lungs and excessive apoptosis of endothelial cells 10 . In aSMase knockout mice, elevated levels of fibrotic markers, including α-SMA and COL1A1, were observed in the liver, while the presence of foam cells was noted in lung tissue 11 . Additionally, Sphk1 expression was found to be increased in the lung tissue of bleomycin (BLM)-induced pulmonary fibrosis. Inhibiting Sphk1 expression enhanced resistance to BLM-induced pulmonary fibrosis in mice 12 . Previous research has demonstrated that the Sphk1/S1P signaling pathway promotes fibrosis by activating the Hippo/YAP axis in lung fibroblasts 13 . Our previous studies have highlighted abnormalities in sphingolipid metabolism, as well as arginine and proline metabolism, in both dust-exposed workers and silicosis patients 14 . Additionally, we observed a decrease in sphingomyelinase and ceramide levels in a mouse model of silicosis, along with an increase in downstream levels of Sphk1 and S1P 15 . These findings strongly suggest the involvement of the aSMase/Cer/S1P signaling pathway in silica-induced pulmonary fibrosis. However, the precise mechanisms underlying its role remain unclear. In this study, we aim to elucidate the metabolites and key enzymes associated with the aSMase/Cer/S1P pathway in silica-induced pulmonary fibrosis and the downstream signaling pathway. Material and Methods Materials Silica (consisting of 99% silicon dioxide, with a diameter ranging from 1 to 5 µm, purchased from Sigma-Aldrich) was free of endotoxin. Prior to use in animal and cell experiments, it was suspended in sterile phosphate-buffered saline (PBS). For the induction of NIH-3T3 cells, recombinant transforming growth factor (TGF)-β1 (obtained from Pepro Tech, USA) was dissolved in ddH2O. Mice model construction and treatments Male C57BL/6J mice, 8 weeks old, were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). The mice were housed in a specific pathogen-free environment. Each group consists of 8 mice. Animal procedures were performed in accordance with the standard guidelines and were approved by the institutional animal care and treatment committee of the Capital Medical University. The animal experimental procedures complied with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Pulmonary fibrosis was induced by intratracheal instillation of silica (0.4g/kg, 0.2mL/kg), while the control group received PBS. Lung lobes were collected at 7, 14, 28, and 42 days for analysis. To generate Smpd1-overexpressing mice, adeno-associated virus (AAV) was administered for a period of 3 weeks. Silica was then administered to induce the silicosis mouse model. The mice were divided into four groups: AAV-Control group, AAV-Control-Silicosis group, AAV-SMPD1 group and AAV-SMPD1-Silicosis group. Single-cell RNA sequencing Samples from the control and the silica-induced pulmonary fibrosis groups were integrated and clustered using the Seurat R package (v4.0.5). Single cells that passed quality control were annotated into 8 distinct clusters based on the gene expression patterns. Read alignment and quality control were performed using Cell Ranger software (v3.1.0) from 10x Genomics. Cell culture and treatments NIH-3T3 cells (Wuhan Procell Life Science & Technology Co.) were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin, and incubated at 37°C with 5% CO2. A 0.4 µm porous membrane transwell device (Corning, New York, USA) was placed in a 6-well culture dish. RAW264.7 cells (1×10 5 ) were cultured in the upper chamber, while NIH-3T3 cells (2×10 5 ) were cultured in the lower chamber of the six-well plate. After a 6-hour stimulation of the macrophages with 100µg/mL silica, NIH-3T3 cells were collected for further analysis. Lentivirus transfection The Smpd1-overexpression lentivirus (HBLV-m-Smpd1-3xflag-ZsGreen-PURO) and the control lentivirus (HBLV-ZsGreen-PURO) were procured from Hanbio Biotechnology Co. Ltd (Shanghai). A population of 4×10 3 NIH-3T3 cells was transfected with these lentiviruses in 96-well plates, followed by selection with puromycin. The transfected cell lines were subsequently treated with TGF-β1 (2.5 ng/mL) for further experimentation. Histology of the lungs Lung tissue samples from mice were fixed with 4% paraformaldehyde overnight. Following this, the samples were dehydrated and embedded in paraffin. Sections of 4µm thickness were prepared for Hematoxylin and Eosin (HE) and Masson staining. The Szapiel method was used to evaluate pulmonary inflammation by HE staining 16 and the King method to score pulmonary fibrosis by Masson staining 17 . For further details, please refer to previous studies 15 . Paraffin-embedded tissue sections with 4µm were prepared for immunohistochemical staining. The sections were incubated overnight at 4°C with anti-collagen I antibody (1:200 dilution) and SMPD1 antibody (1:200 dilution), followed by washing and incubation with the secondary antibody for 1 hour at room temperature. The positively stained areas were then quantified under a microscope. Quantitative real-time PCR Lung tissues from mice or cultured cells were placed in an Eppendorf tube with 1 mL of TRIzol reagent for RNA extraction. The concentration and purity of the isolated RNA were measured using a NanoDrop instrument. Complementary DNA (cDNA) was synthesized using a reverse transcription kit (KR103, Tiangen Biotechnology, Beijing, China) according to the procedures. mRNA levels were quantitated using the SYBR Green PreMix kit (FP205, Tiangen Biotechnology, Beijing, China) on an ABI7500 Fast Real-Time System. Data analysis was performed using the ΔΔCT method. Primer sequence details are provided in Table S1 . Western blot analysis Proteins from fresh lung tissues and cells were extracted utilizing RIPA lysis buffer (P0013b, Beyotime, Shanghai, China), supplemented with a protease and phosphatase inhibitor cocktail. Protein concentration was measured with the BCA Protein Assay Kit (P0010, Beyotime, Shanghai, China). Proteins were then separated on 10% SDS–polyacrylamide gels and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk at room temperature for 1 hour, followed by overnight incubation with primary antibodies at 4°C. Afterward, the membranes were incubated with secondary antibodies at room temperature for 1 hour and visualized using the LICOR Odyssey fluorescent imaging system (LICOR Biotechnology). The primary antibodies for Western blot were listed in Table S2. Wound healing assay NIH-3T3 cells were grown in a six-well plate until reaching 90% confluency, then incubated in serum-free DMEM medium overnight. A vertical scratch was made using a 200µL micropipette tip. The Cells were subsequently cultured in DMEM supplemented with 1% fetal bovine serum and treated with ceramide, with or without TGF-β1 (2.5 ng/mL), for 0, 6, 12, 24 and 30 hours. Scratch images were captured under a microscope, and the scratch width was analyzed using ImageJ software. Cell counting kit (CCK)-8 assays Cell proliferation was assessed using the CCK-8 assay. NIH-3T3 cells were cultured in 96-well plates at a density of 5000 cells/well overnight. The next day, cells were treated with ceramide and TGF-β1 for 0, 6, 12 and 24 hours. Subsequently, the medium was replaced with serum-free medium, and 10µL of CCK-8 solution was added to each well. After incubating the plate for 90 minutes, optical densities (OD) were measured at 450nm. Enzyme-linked immunosorbent assay (ELISA) ELISA was used to determine the protein levels of the inflammatory cytokine TNF-α and the fibrotic cytokine TGF-β1 in the cell supernatant, following the manufacturer's instructions. Acid Sphingomyelinase Activity Assay ASMase activity in tissues and cells was detected using the ASMase assay kit (ab190554, Abcam). Tissues and cells were lysed using Mammalian Cell Lysis Buffer (ab179835, Abcam), and the samples were then reacted with the ASMase assay reagent according to the manufacturer's protocol. The fluorescence of each sample was measured at Ex/Em = 540/590 nm using a microplate reader. Samples were also assayed for protein content and further tested for enzyme activity. Graph visualization Heatmaps and bubble maps were also generated using Bioladder platform ( https://www.bioladder.cn/web/#/pro/index ). Statistical analysis Statistical analyses were performed using GraphPad Prism 8.0 software. Data are presented as mean ± SEM. A Student's t -test was employed for comparisons between two groups. Comparisons among multiple groups were performed using one-way ANOVA followed by an SNK test. P < 0.05 was considered statistically significant. Results The metabolites and key enzymes of the aSMase/CerS1P signaling pathway are associated with silica-induced pulmonary fibrosis Establishing the silica-induced pulmonary fibrosis mice model (0.4g/kg), observing the formation of silicotic nodules by day 7 via HE and Masson staining ( Supplementary Fig. 1a ). As the duration of silica exposure increased, typical silicotic nodules with collagen fibers arranged in concentric circles were formed ( Supplementary Fig. 1b ). To verify the successful construction of the model, we examined the expression of fibrosis-related genes and found that these proteins were also upregulated ( Supplementary Fig. 1c ). These data indicate that exposure to silica (0.4 g/kg) results in pulmonary fibrosis. Sphingolipids, an essential class of lipids, play a pivotal role in regulating cellular functions. Within the aSMase/Cer/S1P signaling pathway, aSMase, an enzyme located in lysosomes, converts sphingomyelin into ceramide. Subsequently, ceramidase breaks down ceramide to generate sphingosine, which is further metabolized by Sphk1 into S1P 10 . We conducted scRNA-seq to identify the cell populations of lung tissues from a silica-induced pulmonary fibrosis model. scRNA-seq of silicosis model and control lung tissue revealed eight distinct cell populations (Fig. 1 a). Among these cells, fibroblasts expressed the highest levels of SMPD1 (Fig. 1 b). Further analysis showed that SMPD1 expression was lower in the silicosis group (Fig. 1 c). Our previously published data demonstrated that in a high-dose silica (0.4 g/kg) induced pulmonary fibrosis mouse model, metabolomic analysis was performed to assess the trends of key metabolites in this pathway 15 . The analysis revealed that the relative levels of SM, Cer, and sphingosine were lower in the silicosis group compared to controls. In contrast, S1P levels were higher and correlated with the severity of silicosis (Figs. 1 d-f). To gain deeper insights into changes in metabolic enzyme expression, qRT-PCR and Western blotting were employed to evaluate mRNA and protein levels of aSMase and Sphk1, the key enzymes in this metabolic pathway. The data indicated a decrease in aSMase expression and an increase in Sphk1 expression in the experimental group compared to the control group (Figs. 1 g-j, Supplementary Fig. 2 ). A consistent decrease in aSMase enzyme activity was also observed (Fig. 1 k). These findings suggest the potential involvement of aSMase and Sphk1 in silica-induced lung fibrosis, underscoring their importance in the dysregulation of the aSMase/Cer/S1P signaling pathway. The activated Hippo/YAP signaling pathway is involved in silica-induced pulmonary fibrosis The Hippo/YAP signaling pathway is intricately linked to tissue homeostasis, cell proliferation, metabolism, and the formation of fibrosis 18 . Therefore, we examined the levels of YAP and TAZ in the Hippo/YAP signaling pathway in a silica-induced pulmonary fibrosis model. Compared to controls, a significant decrease was observed in the mRNA expression levels of YAP and TAZ in lung tissues from mice exposed to 0.4g/kg of silica for 42 days (Figs. 2 a, b). Similarly, the protein levels of YAP, TAZ, and p-YAP showed a declining trend (Figs. 2 c-h). To further support these findings, we analyzed relevant data from a public database. In this bioinformatic analysis, lung tissues from mice exposed to 12mg of silica were collected at various time points (14d, 28d, 42d, 77d) 19 . The proteomic analysis revealed a downregulated trend in the protein levels of YAP and TAZ within the Hippo/YAP signaling pathway (Figs. 2 i-j) 19 . Upon phagocytosis of silica by lung macrophages, specific signaling factors are activated, leading to a reduced expression of both total and phosphorylated YAP in the cell cytoplasm. This reduction facilitates the nuclear translocation of unphosphorylated YAP, which activates TAZ and ultimately contributes to fibrosis formation. Therefore, we propose that the Hippo/YAP signaling pathway plays a pivotal role in the progression of silicosis. The metabolites and key enzymes are involved in the aSMase/Cer/S1P signaling pathway in vitro Upon stimulating NIH-3T3 mouse lung fibroblasts with TGF-β1, a noteworthy upregulation of COL1A1 and α-SMA expression was observed specifically with 2.5 ng/mL of TGF-β1 for 24 hours (Figs. 3 a-e). In evaluating the expression of aSMase and Sphk1 within the metabolic pathway, aSMase showed a decreasing trend at the transcriptional, translational, and activity levels following TGF-β1 stimulation in NIH-3T3 cells (Fig. 3 f-i). Meanwhile, the mRNA level of Sphk1 exhibited an increasing trend ( Supplementary Fig. 3 ). Additionally, S1P expression, a downstream component in the signaling pathway, was increased (Fig. 3 j). These findings suggest the involvement of the aSMase/Cer/S1P signaling pathway in the process of fibrosis. Phagocytosis of silica particles by macrophages promoted pulmonary fibroblast transdifferentiation and activated the aSMase/Cer/S1P signaling pathway To investigate whether macrophages can induce fibroblast transdifferentiation following the phagocytosis of silica particles, an in vitro co-culture model was established. Considering TNF-α and TGF-β1 as pro-inflammatory and pro-fibrotic factors, respectively, their levels in the supernatant of RAW264.7 cells exposed to silica (100 µg/mL at 12h, 24h, 48h, and 72h) were measured using ELISA. The data showed an increase in the levels of these cytokines (Figs. 4 a, b). Additionally, a significant rise in the expression of pro-fibrotic genes, specifically COL1A1, ACTA2, and fibronectin, was observed following silica phagocytosis by RAW264.7 for 6h in NIH-3T3(Fig. 4 c-e). An examination of enzymes and metabolites in the aSMase/Cer/S1P signaling pathway within the co-culture model revealed a decreasing trend in both the mRNA expression and activity of aSMase (Figs. 4 f-g). In contrast, an upward trend was observed in the levels of Sphk1 and S1P ( Supplementary Fig. 4 , Fig. 4 h). These findings suggest that silica phagocytosis by RAW264.7 cells may promote the transdifferentiation of NIH-3T3 cells and enhance the production of fibrotic factors. However, data from the Hippo/YAP signaling pathway in the co-culture model showed no significant changes in YAP and TAZ expression (Figs. 4 i, j). Alleviation of silica-induced lung fibrosis by intratracheal administration of AAV-SMPD1 Mice were intratracheally administered adeno-associated virus (AAV)-Control or AAV-SMPD1. One week later, they received intratracheal silica (0.4g/kg) and were subsequently sacrificed for lung tissue collection (Fig. 5 a). After confirming successful viral transfection into lung tissue with high SMPD1 expression, we assessed the degree of fibrosis (Fig. 5 b, c). Administration of AAV-SMPD1 significantly attenuated silica-induced pulmonary fibrosis in mice, as evidenced by a reduction in collagen content within silicotic nodules (Fig. 5 d). Notably, the analysis of SMPD1 expression in lung tissue revealed a relatively lower increase in the AAV-SMPD1-Silicosis group compared to the AAV-SMPD1 group. This observation aligned with our earlier findings, which indicated a decrease in SMPD1 expression in silica-induced pulmonary fibrosis (Fig. 5 e). Upon SMPD1 overexpression, a noticeable increase in the expression levels of YAP and TAZ was observed to a certain extent (Figs. 5 f, g). These results support the hypothesis that AAV-SMPD1 delivery inhibits lung fibrosis. Alleviation of lung fibrosis by overexpression of SMPD1 in NIH-3T3 cells To further elucidate the role of SMPD1, we investigated the impact of SMPD1 overexpression on the proliferation, migration, and differentiation of NIH-3T3 cells in vitro. The successful overexpression of SMPD1 in NIH-3T3 cells was first confirmed (Figs. 6 a, b). Following SMPD1 overexpression, a reduction was observed in the expression of pro-fibrotic genes, as well as in cell proliferation, migration and differentiation of fibroblasts into myofibroblasts when treated with 2.5ng/mL TGF-β1 for 24 hours (Figs. 6 c, d, e, g, h, i, j, k). The expression of SMPD1 was decreased in fibroblasts treated with TGF-β1, consistent with our previous observations (Fig. 6 f). Together, these findings demonstrate that restoring SMPD1 expression can effectively mitigate lung fibrosis. Discussion The present study demonstrates that the aSMase/Cer/S1P signaling pathway plays a critical role in the pathogenesis of silica-induced pulmonary fibrosis in mice. When macrophages phagocytose exogenous silica, they release significant amounts of pro-inflammatory and pro-fibrotic factors, which further stimulate the activation and transdifferentiation of fibroblasts into myofibroblasts in vivo. Evidence of the aSMase/Cer/S1P signaling pathway's involvement is seen in the reduced levels and activity of acid sphingomyelinase, alongside increased S1P levels. These metabolites regulate the downstream Hippo/YAP axis through specific activation pathways, influencing the migration, proliferation and differentiation of fibroblasts. This disruption of the lung tissue microenvironment ultimately leads to the formation of lung fibrosis. Notably, overexpression of SMPD1 may modulate the downstream Hippo/YAP signaling pathways, thereby mitigating the extent of silica-induced fibrosis. ASMase exhibits high biological activity among sphingomyelinases. It catalyzes the breakdown of sphingomyelin to generate Cer, which is deacylated by ceramide synthase to produce sphingosine. Sphingosine is rapidly phosphorylated by Sphk to produce S1P, which binds to its downstream G protein-coupled receptors (S1PR1-S1PR5), triggering the downstream inflammatory response 20 . ASMase is a Zn2+-stimulated SMase encoded by the SMPD1 gene 21 . In the late 1960s, researchers reported that a deficiency in aSMase leads to a rare autosomal recessive disorder known as Niemann-Pick disease (NPD), a lysosomal storage disease 22 . Deficiency of the SMPD1 gene results in the accumulation of cholesterol and sphingomyelin in the liver and brain, accompanied by atrophy of the cerebellum and deficiency of Purkinje cells 22 . A previous study revealed decrease in sphingolipid metabolism in lungs of idiopathic pulmonary fibrosis (IPF) patients, evidenced by decreased levels of sphingomyelin and sphingosine, along with reduced expression of SMPD1 and SMPD4 23 . In our study, we observed lower levels and activity of aSMase in lung tissue and foam cell deposition in the alveolar spaces of the silica-induced mouse model. Overexpression of the SMPD1 gene alleviated lung fibrosis, inhibited fibroblast proliferation and migration, and enhanced apoptosis. Similarly, we observed that SMPD1 exhibited a consistent increase at both the RNA and protein levels over time. We hypothesize that this may be associated with early-stage inflammatory responses and late-stage fibrotic reactions. Further investigation is required to determine whether SMPD1 levels eventually normalize as fibrosis progresses and whether this influences downstream metabolites. If so, early intervention may have greater therapeutic significance. Ceramide, a type of amide compound, is fundamental to sphingolipid metabolism. De novo ceramide biosynthesis involves the pathway of serine palmitoyltransferase complex and ceramide synthase, and a breakdown synthesis pathway via sphingomyelinase that catabolizes membrane sphingolipids 24 . Cer plays a crucial role in inhibiting cell proliferation and promoting apoptosis and also acts as an intracellular lipid second messenger, regulating cell proliferation, differentiation, senescence, and migration 25 . Beyond its role in apoptosis, ceramide has shown to be associated with cell cycle arrest, inducing cell in the G0/G1 phase 26 . In relation to tumor diseases, ceramide exerts anti-proliferative effects by inhibiting the growth and migration of cancer cells and inducing autophagy and apoptosis 27 . Its expression varies among tumor subtypes, with higher levels observed in ovarian cancer compared to normal tissues 28 , whereas ceramide levels are significantly reduced in patients with colon cancer 29 . Previous studies have shown that feeding mice with sphingomyelin or ceramide analogues reduces the incidence of colon cancer 30 . Therefore, the explore of ceramide may become our research in the future. Sphk1 and Sphk2 are two isozymes identified in mammals and both are commonly expressed in most tissues. However, Sphk1 is more abundantly expressed in the lungs and heart, while Sphk2 is primarily expressed in the liver and spleen 31 . S1P is a simple sphingolipid metabolite present in blood, plasma, bronchoalveolar lavage fluid and various organs. S1P is involved in numerous cellular responses, including proliferation, differentiation, adhesion, motility and apoptosis, by acting on specific G protein-coupled receptors (S1P1-5) 32 . A study of patients with IPF found elevated S1P levels in serum and BALF, as detected by ELISA, which negatively correlated with pulmonary function. Macrophages isolated from BALF exhibited increased Sphk1 expression at the transcriptional level, and lung tissue from IPF patients showed higher Sphk1 protein expression compared to controls. Immunohistochemistry revealed that Sphk1 was highly expressed, particularly in proliferating alveolar epithelial cells and fibroblasts. S1P was also found to promote the differentiation of alveolar epithelial cells into mesenchymal cells and fibroblasts into myofibroblasts 33 . In our silicosis model and in vitro studies, we similarly found increased levels of Sphk1 expression. These findings suggest that Sphk1 is a novel prognostic and therapeutic target for pulmonary fibrosis. Targeting Sphk1 to inhibit S1P production may provide a new therapeutic strategy for fibrotic lung diseases. The relationships among SM, ceramide, sphingosine, and S1P require further investigation. The Hippo/Yes-associated protein (YAP)1 signaling pathway is known to regulate cellular proliferation, differentiation, and tissue homeostasis 34 . YAP and TAZ are transcriptional co-activator proteins that shuttle between the nucleus and cytoplasm and regulate the expression of downstream factors by binding to the intracellular transcription factor TEAD 35 . Previous research has shown that the Sphk1/S1P signaling pathway promotes fibrosis by activating the Hippo/YAP axis in lung fibroblasts. Conditional knockdown of Sphk1 in mice lung fibroblasts led to reduced lung tissue fibrosis, accompanied by decreased YAP1 localization in fibrotic regions. Additionally, in primary lung fibroblasts stimulated with TGF-β, YAP1 nuclear translocation was significantly increased, while Sphk1 knockdown reduced nuclear YAP1 localization. The expression of fibrosis-related factors was significantly reduced when verteporfin, an inhibitor of YAP1, was added. All these data suggest that the Sphk1/S1P metabolic pathway and the Hippo/YAP axis may have a synergistic role in the development of fibrosis 13 . In our model, we found that YAP/TAZ showed a trend of low expression in silica-induced mice models, consistent with previous proteomics sequencing results in silicosis models 19 . However, in our in vitro co-culture model, we did not observe a significant trend in YAP and TAZ expression at the transcriptional level. These data suggest that targeting the YAP/TAZ pathway may be a promising therapeutic option for fibrosis patients, though the exact mechanism need further investigation. In conclusion, this study demonstrated that aSMase/Cer/S1P signaling plays a significant role in silica-induced pulmonary fibrosis, likely by impacting the downstream Hippo/YAP signaling axis. Overexpression of SMPD1 can mitigate silica-induced pulmonary fibrosis. Therefore, targeting these metabolites presents a promising therapeutic strategy for silicosis. Declarations Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethical Approval All animal protocols were approved by the institutional animal care and treatment committee of the Capital Medical University (AEEI-2023-077). Fundings National Natural Science Foundation of China (81970061). High Level Public Health Technology Talent Construction Project (DL-02-21). Reform and Development Program of Beijing Institute of Respiratory Medicine (Ggyfz202512). Author Contribution Ruimin Ma: Data analysis and wrote the manuscript. Di Sun: Carried out the experiments. Yuanying Wang and Yawen Song: Collected and analyzed the data. Qiao Ye: Conceived, designed the experiments and wrote the manuscript. Data Availability Data is provided within the manuscript or supplementary information files. References Zhao, X., Kwan, J., Yip, K., Liu, P. P. & Liu, F. F. Targeting Metabolic Dysregulation for Fibrosis Therapy. Nat. Rev. Drug Discov . 19 , 57–75 (2020). Shi, P. et al. Trends in Global, Regional and National Incidence of Pneumoconiosis Caused by Different Aetiologies: An Analysis From the Global Burden of Disease Study 2017. Occup. Environ. Med. 77 , 407–414 (2020). Adamcakova, J. & Mokra, D. New Insights Into Pathomechanisms and Treatment Possibilities for Lung Silicosis. Int J. Mol. Sci 22 , (2021). DeLeon-Pennell, K. Y., Barker, T. H. & Lindsey, M. L. Fibroblasts: The Arbiters of Extracellular Matrix Remodeling. 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Metabolic Heterogeneity of Idiopathic Pulmonary Fibrosis: A Metabolomic Study. BMJ Open. Respir Res. 4 , e000183 (2017). Xie, T. et al. Ceramide Sensing by Human SPT-ORMDL Complex for Establishing Sphingolipid Homeostasis. Nat. Commun. 14 , 3475 (2023). Markowski, A. R. et al. The Effect of Silencing the Genes Responsible for the Level of Sphingosine-1-Phosphate On the Apoptosis of Colon Cancer Cells. Int J. Mol. Sci 24 , (2023). Kinoshita, M. & Matsumori, N. Inimitable Impacts of Ceramides On Lipid Rafts Formed in Artificial and Natural Cell Membranes. Membranes 12 , (2022). Li, R. Z. et al. The Key Role of Sphingolipid Metabolism in Cancer: New Therapeutic Targets, Diagnostic and Prognostic Values, and Anti-Tumor Immunotherapy Resistance. Front. Oncol. 12 , 941643 (2022). Knapp, P., Bodnar, L., Blachnio-Zabielska, A., Swiderska, M. & Chabowski, A. Plasma and Ovarian Tissue Sphingolipids Profiling in Patients with Advanced Ovarian Cancer. Gynecol. Oncol. 147 , 139–144 (2017). Guo, W. et al. M6a Methylation of DEGS2, a Key Ceramide-Synthesizing Enzyme, is Involved in Colorectal Cancer Progression through Ceramide Synthesis. Oncogene 40 , 5913–5924 (2021). Marzo, F. et al. Effect of a Diet Supplemented with Sphingomyelin and Probiotics On Colon Cancer Development in Mice. Probiotics Antimicrob. Proteins . 14 , 407–414 (2022). Melendez, A. J., Carlos-Dias, E., Gosink, M., Allen, J. M. & Takacs, L. Human Sphingosine Kinase: Molecular Cloning, Functional Characterization and Tissue Distribution. Gene 251 , 19–26 (2000). Hu, Y. & Dai, K. Sphingosine 1-Phosphate Metabolism and Signaling. Adv. Exp. Med. Biol. 1372 , 67–76 (2022). Milara, J. et al. Sphingosine-1-Phosphate is Increased in Patients with Idiopathic Pulmonary Fibrosis and Mediates Epithelial to Mesenchymal Transition. Thorax 67 , 147–156 (2012). Warren, R., Lyu, H., Klinkhammer, K. & De Langhe, S. P. Hippo Signaling Impairs Alveolar Epithelial Regeneration in Pulmonary Fibrosis. Elife 12 , (2023). Heng, B. C. et al. An Overview of Signaling Pathways Regulating YAP/TAZ Activity. Cell. Mol. Life Sci. 78 , 497–512 (2021). Additional Declarations No competing interests reported. Supplementary Files SupplementaryInfoFile.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7052670","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":492544235,"identity":"af4ee618-f387-415a-a245-772b8fff2635","order_by":0,"name":"Ruimin Ma","email":"","orcid":"","institution":"Beijing Chao-Yang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ruimin","middleName":"","lastName":"Ma","suffix":""},{"id":492544237,"identity":"e8d26bbd-1a87-4440-bf8c-a9930159c68f","order_by":1,"name":"Di Sun","email":"","orcid":"","institution":"Beijing Chao-Yang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Di","middleName":"","lastName":"Sun","suffix":""},{"id":492544240,"identity":"3f4a4dcd-5147-487e-ac2d-6ef247f7e045","order_by":2,"name":"Yuanying Wang","email":"","orcid":"","institution":"Beijing Chao-Yang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuanying","middleName":"","lastName":"Wang","suffix":""},{"id":492544242,"identity":"8653dc3e-d391-4569-a78d-27a27c342ebc","order_by":3,"name":"Yawen Song","email":"","orcid":"","institution":"Beijing Chao-Yang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yawen","middleName":"","lastName":"Song","suffix":""},{"id":492544243,"identity":"d9e8c54a-6029-4f28-a7fe-a7eeddba5fa0","order_by":4,"name":"Qiao Ye","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCUlEQVRIiWNgGAWjYJCCAyDCAMqRY2AmVYsxUVoYkLUkNhBUebx544EfFQzy5uyHj0n83FGbPr+d9+AHhhqbaJxazhwrONhzhsFwZ09ammTvmeO5Gw7zJUswHEvLxWWdwY0cgwO8bQwJBgdyzCR4247lbmDmMZBgbDiMW8v9NwYH/4K0nH9jJvm37Vi6fDOP8Q+8Wm7wGBwG23Ijx0yat60mgeEwjxleWyTPpBUclgH6ZcONZ8nWsm0HDDcAtVgk4PEL3/HDmz++AYaYwfnkgzffttXJy/efMb7xocYGpxaFA+AY+Q9is0gwMByGCCfgUA4C8g3wSGRg/sDAUIdH7SgYBaNgFIxUAACjpl/EghoY2AAAAABJRU5ErkJggg==","orcid":"","institution":"Beijing Chao-Yang Hospital","correspondingAuthor":true,"prefix":"","firstName":"Qiao","middleName":"","lastName":"Ye","suffix":""}],"badges":[],"createdAt":"2025-07-05 11:08:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7052670/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7052670/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87952041,"identity":"2c924d5b-2380-48fd-a470-e9cf96111832","added_by":"auto","created_at":"2025-07-30 17:50:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":151862,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of silica exposure on the related metabolites and key enzymes in the aSMase/Cer/S1P signaling pathway. a, Cell clusters in lung tissue of silicosis mice; b, mRNA expression levels of SMPD1 in different cell types; c, mRNA expression levels of SMPD1 in lung tissue of control group and silicosis mice; d, e, Heatmaps show changes in SM and Cer. The color bars on the right of heatmap show the gene level (blue, downregulation, and red, upregulation); f, Relative levels of sphingosine and S1P; g, Images of immunohistochemical staining of SMPD1 in lung tissues on day 0 and day 42; h, mRNA expression of SMPD1 in lung tissues; i, Western blots of aSMase in lung tissues; j, Western blot analysis; k, Enzyme activity of aSMase in lung tissues. Magnification, ×200. Scale bar indicates 50 µm. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.0001\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7052670/v1/b359df0d83201827ab527732.png"},{"id":87953075,"identity":"1b8200ad-3437-40fd-9085-990538f2c2d5","added_by":"auto","created_at":"2025-07-30 18:06:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":73365,"visible":true,"origin":"","legend":"\u003cp\u003eSilica-induced pulmonary fibrosis activates Hippo/YAP signaling pathway. a, b, mRNA expression of YAP and TAZ in lung tissues; c, d, Western blots of YAP, TAZ and p-YAP in lung tissues; e, f, g, h, Western blot analysis; i, j, Relative protein levels of YAP and TAZ. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7052670/v1/28592f04748fbf48fbd97d0a.png"},{"id":87952043,"identity":"a9d63ecf-cfa6-4ea4-b5f8-5c687cb65c33","added_by":"auto","created_at":"2025-07-30 17:50:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":77467,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of silica exposure on metabolites and key enzymes in the aSMase/Cer/S1P signaling pathway in vitro model. a, b, mRNA expression of COL1A1 and ACTA2 in NIH-3T3 cells treated with TGF-β1 (2.5ng/mL, 6h, 12h, 24h, 48h); d, Western blots of COL1A1 and α-SMA in NIH-3T3 cells treated with TGF-β1 (2.5ng/mL, 24h); c, e, Western blot analysis; f, mRNA expression of aSMase in NIH-3T3 cells treated with TGF-β1 (2.5ng/mL, 24h); g, Western blots of aSMase in NIH-3T3 cells treated with TGF-β1 (2.5ng/mL, 24h); h, Western blot analysis; i, Enzyme activity of aSMase in NIH-3T3 cells treated with TGF-β1 (2.5ng/mL, 24h); j, Relative levels of S1P in NIH-3T3 cells treated with TGF-β1 (2.5ng/mL, 24h). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7052670/v1/bc5d3640e6b40b2eb7a70988.png"},{"id":87952742,"identity":"c07145f9-4200-451d-b7c9-5dc4743d4680","added_by":"auto","created_at":"2025-07-30 17:58:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":37271,"visible":true,"origin":"","legend":"\u003cp\u003eExpression analysis of metabolites and key enzymes in the aSMase/Cer/S1P signaling pathway in the co-culture model. a, b, TNF-α and TGF-β1 was determined by ELISA in the supernate of RAW264.7 cells treated with silica (100μg/mL, 12h, 24h, 48h, 72h); c, d, e, mRNA expression of α-SMA, COL1A1 and Fibronectin in the co-culture model (100μg/mL, 6h); f, mRNA expression of aSMase in the co-culture model (100μg/mL, 6h); g, Enzyme activity of aSMase in the co-culture model (100μg/mL, 6h); h, Relative levels of S1P in the co-culture model (100μg/mL, 6h) ; i, j, mRNA expression of YAP and TAZ in the co-culture model (100 μg/mL, 6h). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7052670/v1/d16c5fe2921589339240a668.png"},{"id":87952744,"identity":"716a41f3-d2fa-43e8-b58f-ff89b38a72a3","added_by":"auto","created_at":"2025-07-30 17:58:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":131442,"visible":true,"origin":"","legend":"\u003cp\u003eAlleviation of silica-induced mouse pulmonary fibrosis by intratracheal administration of AAV-SMPD1. a, Schematic of the AAV-SMPD1 therapy in the silica-induced mouse pulmonary fibrosis model; b, Frozen tissue sections of the lung were used to determine the transfection efficiency of AAV, the green signal confirms successful AAV transduction; c, mRNA expression of SMPD1 in the groups of AAV-Control and AAV-SMPD1; d, HE staining and Masson staining in lung tissues of AAV treatment; e, f, g mRNA expression of SMPD1, YAP and TAZ in lung tissues of AAV treatment. Magnification, ×50. Scale bar indicates 200 µm. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7052670/v1/b08da9f31fa13d792afdd337.png"},{"id":87952050,"identity":"62aad921-6812-4cdd-91f4-efc72917d121","added_by":"auto","created_at":"2025-07-30 17:50:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":154666,"visible":true,"origin":"","legend":"\u003cp\u003eSMPD1 can alleviate TGF-β1-induced pulmonary fibrosis and alleviate the proliferation and migration. a, NIH-3T3 cells were infected with a control lentivirus encoding SMPD1 or a lentivirus encoding SMPD1 for 48h or 72h, respectively, and transfected with 6μg/mL polybrene. b, mRNA expression of SMPD1 in the groups of Control and SMPD1; c, Western blots of COL1A1 and α-SMA in NIH-3T3 cells infected with a control lentivirus encoding SMPD1 or a lentivirus encoding SMPD1 and treated with 2.5ng/mL TGF-β1 for 24h; d, e, Western blot analysis; f, g, h, mRNA expression of SMPD1, COL1A1 and α-SMA in NIH-3T3 cells infected with a control lentivirus encoding SMPD1 or a lentivirus encoding SMPD1 and treated with 2.5ng/mL TGF-β1 for 24h; i, Cell viability in NIH-3T3 cells infected with a control lentivirus encoding SMPD1 or a lentivirus encoding SMPD1 and treated with 2.5ng/mL TGF-β1 for 24h; j, Wound healing assays in NIH-3T3 cells infected with a control lentivirus encoding SMPD1 or a lentivirus encoding SMPD1 and treated with 2.5ng/mL TGF-β1 for 24h; k, The scratch area analysis. Magnification, ×40. Scale bar indicates 1000 µm. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7052670/v1/3534e42bc3df4f07662f73ce.png"},{"id":90116516,"identity":"28b92ad1-a577-407c-960b-77705fe1d3d0","added_by":"auto","created_at":"2025-08-28 16:23:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1826572,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7052670/v1/05e52606-8aea-4d0a-808c-d95f263f9922.pdf"},{"id":87952049,"identity":"af57bb6c-f69a-4ae7-a0a0-fa4f2e15d3ee","added_by":"auto","created_at":"2025-07-30 17:50:15","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1841937,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInfoFile.docx","url":"https://assets-eu.researchsquare.com/files/rs-7052670/v1/5924fbd35d4d663ea650aeea.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Targeting acid sphingomyelinase/ceramide/S1P ameliorates silica-induced pulmonary fibrosis through Hippo/YAP signaling pathway in mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFibrotic diseases encompass a range of conditions characterized by the formation of scar tissue, affecting various organs. These diseases pose a significant burden, impacting roughly a quarter of the global population due to their severity \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. According to the Global Burden of Disease (GBD) study, the incidence of pneumoconiosis, including silicosis, increased by 66.0% between 1990 and 2017 \u003csup\u003e2\u003c/sup\u003e. Silicosis, a form of pulmonary fibrosis caused by silica exposure, manifests through the injury to alveolar epithelial cells, fibroblast differentiation, and extracellular matrix deposition \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. During tissue repair, fibroblasts become activated and transition into myofibroblasts, which contribute to extracellular matrix production. The inflammatory and fibrotic factors within the tissue microenvironment stimulate epithelial cells to undergo epithelial-mesenchymal transition, ultimately leading to pulmonary fibrosis \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSphingolipids have recently been found to play a critical role in regulating cell proliferation and differentiation during pulmonary fibrosis \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Sphingomyelin, a specific sphingolipid, serves as a major component of mammalian cell membranes \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. It is hydrolyzed by three principal enzymes known as sphingomyelinases (SMases): acid, basic, and neutral sphingomyelinase. Among these, acid sphingomyelinase (aSMase) is considered the most active \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. ASMase is a Zn2+-stimulated SMase that is encoded by the Sphingomyelin Phosphodiesterase 1 (SMPD1) gene \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. This hydrolytic process generates ceramide, a molecule essential for various cellular functions \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Ceramide can be further metabolized by ceramidase to produce sphingosine, which can be phosphorylated by either sphingosine kinase 1 (Sphk1) or sphingosine kinase 2 (Sphk2) to generate sphingosine-1-phosphate (S1P) \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe aSMase/Cer/S1P signaling pathway has been implicated in the pathogenesis of various diseases. Activation of aSMase in emphysema leads to increased ceramide production, which contributes to the loss of alveolar septa in the lungs and excessive apoptosis of endothelial cells \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In aSMase knockout mice, elevated levels of fibrotic markers, including α-SMA and COL1A1, were observed in the liver, while the presence of foam cells was noted in lung tissue \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Additionally, Sphk1 expression was found to be increased in the lung tissue of bleomycin (BLM)-induced pulmonary fibrosis. Inhibiting Sphk1 expression enhanced resistance to BLM-induced pulmonary fibrosis in mice \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Previous research has demonstrated that the Sphk1/S1P signaling pathway promotes fibrosis by activating the Hippo/YAP axis in lung fibroblasts \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOur previous studies have highlighted abnormalities in sphingolipid metabolism, as well as arginine and proline metabolism, in both dust-exposed workers and silicosis patients \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Additionally, we observed a decrease in sphingomyelinase and ceramide levels in a mouse model of silicosis, along with an increase in downstream levels of Sphk1 and S1P \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. These findings strongly suggest the involvement of the aSMase/Cer/S1P signaling pathway in silica-induced pulmonary fibrosis. However, the precise mechanisms underlying its role remain unclear. In this study, we aim to elucidate the metabolites and key enzymes associated with the aSMase/Cer/S1P pathway in silica-induced pulmonary fibrosis and the downstream signaling pathway.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cb\u003eMaterials\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSilica (consisting of 99% silicon dioxide, with a diameter ranging from 1 to 5 \u0026micro;m, purchased from Sigma-Aldrich) was free of endotoxin. Prior to use in animal and cell experiments, it was suspended in sterile phosphate-buffered saline (PBS). For the induction of NIH-3T3 cells, recombinant transforming growth factor (TGF)-β1 (obtained from Pepro Tech, USA) was dissolved in ddH2O.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMice model construction and treatments\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMale C57BL/6J mice, 8 weeks old, were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). The mice were housed in a specific pathogen-free environment. Each group consists of 8 mice. Animal procedures were performed in accordance with the standard guidelines and were approved by the institutional animal care and treatment committee of the Capital Medical University. The animal experimental procedures complied with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Pulmonary fibrosis was induced by intratracheal instillation of silica (0.4g/kg, 0.2mL/kg), while the control group received PBS. Lung lobes were collected at 7, 14, 28, and 42 days for analysis.\u003c/p\u003e\u003cp\u003eTo generate Smpd1-overexpressing mice, adeno-associated virus (AAV) was administered for a period of 3 weeks. Silica was then administered to induce the silicosis mouse model. The mice were divided into four groups: AAV-Control group, AAV-Control-Silicosis group, AAV-SMPD1 group and AAV-SMPD1-Silicosis group.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSingle-cell RNA sequencing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSamples from the control and the silica-induced pulmonary fibrosis groups were integrated and clustered using the Seurat R package (v4.0.5). Single cells that passed quality control were annotated into 8 distinct clusters based on the gene expression patterns. Read alignment and quality control were performed using Cell Ranger software (v3.1.0) from 10x Genomics.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell culture and treatments\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNIH-3T3 cells (Wuhan Procell Life Science \u0026amp; Technology Co.) were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin\u0026ndash;streptomycin, and incubated at 37\u0026deg;C with 5% CO2.\u003c/p\u003e\u003cp\u003eA 0.4 \u0026micro;m porous membrane transwell device (Corning, New York, USA) was placed in a 6-well culture dish. RAW264.7 cells (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e) were cultured in the upper chamber, while NIH-3T3 cells (2\u0026times;10\u003csup\u003e5\u003c/sup\u003e) were cultured in the lower chamber of the six-well plate. After a 6-hour stimulation of the macrophages with 100\u0026micro;g/mL silica, NIH-3T3 cells were collected for further analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLentivirus transfection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe Smpd1-overexpression lentivirus (HBLV-m-Smpd1-3xflag-ZsGreen-PURO) and the control lentivirus (HBLV-ZsGreen-PURO) were procured from Hanbio Biotechnology Co. Ltd (Shanghai). A population of 4\u0026times;10\u003csup\u003e3\u003c/sup\u003e NIH-3T3 cells was transfected with these lentiviruses in 96-well plates, followed by selection with puromycin. The transfected cell lines were subsequently treated with TGF-β1 (2.5 ng/mL) for further experimentation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHistology of the lungs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLung tissue samples from mice were fixed with 4% paraformaldehyde overnight. Following this, the samples were dehydrated and embedded in paraffin. Sections of 4\u0026micro;m thickness were prepared for Hematoxylin and Eosin (HE) and Masson staining. The Szapiel method was used to evaluate pulmonary inflammation by HE staining \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and the King method to score pulmonary fibrosis by Masson staining \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. For further details, please refer to previous studies \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eParaffin-embedded tissue sections with 4\u0026micro;m were prepared for immunohistochemical staining. The sections were incubated overnight at 4\u0026deg;C with anti-collagen I antibody (1:200 dilution) and SMPD1 antibody (1:200 dilution), followed by washing and incubation with the secondary antibody for 1 hour at room temperature. The positively stained areas were then quantified under a microscope.\u003c/p\u003e\u003cp\u003e\u003cb\u003eQuantitative real-time PCR\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLung tissues from mice or cultured cells were placed in an Eppendorf tube with 1 mL of TRIzol reagent for RNA extraction. The concentration and purity of the isolated RNA were measured using a NanoDrop instrument. Complementary DNA (cDNA) was synthesized using a reverse transcription kit (KR103, Tiangen Biotechnology, Beijing, China) according to the procedures. mRNA levels were quantitated using the SYBR Green PreMix kit (FP205, Tiangen Biotechnology, Beijing, China) on an ABI7500 Fast Real-Time System. Data analysis was performed using the ΔΔCT method. Primer sequence details are provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern blot analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eProteins from fresh lung tissues and cells were extracted utilizing RIPA lysis buffer (P0013b, Beyotime, Shanghai, China), supplemented with a protease and phosphatase inhibitor cocktail. Protein concentration was measured with the BCA Protein Assay Kit (P0010, Beyotime, Shanghai, China). Proteins were then separated on 10% SDS\u0026ndash;polyacrylamide gels and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk at room temperature for 1 hour, followed by overnight incubation with primary antibodies at 4\u0026deg;C. Afterward, the membranes were incubated with secondary antibodies at room temperature for 1 hour and visualized using the LICOR Odyssey fluorescent imaging system (LICOR Biotechnology). The primary antibodies for Western blot were listed in Table S2.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWound healing assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNIH-3T3 cells were grown in a six-well plate until reaching 90% confluency, then incubated in serum-free DMEM medium overnight. A vertical scratch was made using a 200\u0026micro;L micropipette tip. The Cells were subsequently cultured in DMEM supplemented with 1% fetal bovine serum and treated with ceramide, with or without TGF-β1 (2.5 ng/mL), for 0, 6, 12, 24 and 30 hours. Scratch images were captured under a microscope, and the scratch width was analyzed using ImageJ software.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell counting kit (CCK)-8 assays\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCell proliferation was assessed using the CCK-8 assay. NIH-3T3 cells were cultured in 96-well plates at a density of 5000 cells/well overnight. The next day, cells were treated with ceramide and TGF-β1 for 0, 6, 12 and 24 hours. Subsequently, the medium was replaced with serum-free medium, and 10\u0026micro;L of CCK-8 solution was added to each well. After incubating the plate for 90 minutes, optical densities (OD) were measured at 450nm.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eELISA was used to determine the protein levels of the inflammatory cytokine TNF-α and the fibrotic cytokine TGF-β1 in the cell supernatant, following the manufacturer's instructions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAcid Sphingomyelinase Activity Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eASMase activity in tissues and cells was detected using the ASMase assay kit (ab190554, Abcam). Tissues and cells were lysed using Mammalian Cell Lysis Buffer (ab179835, Abcam), and the samples were then reacted with the ASMase assay reagent according to the manufacturer's protocol. The fluorescence of each sample was measured at Ex/Em\u0026thinsp;=\u0026thinsp;540/590 nm using a microplate reader. Samples were also assayed for protein content and further tested for enzyme activity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGraph visualization\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHeatmaps and bubble maps were also generated using Bioladder platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioladder.cn/web/#/pro/index\u003c/span\u003e\u003cspan address=\"https://www.bioladder.cn/web/#/pro/index\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using GraphPad Prism 8.0 software. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. A Student's \u003cem\u003et\u003c/em\u003e-test was employed for comparisons between two groups. Comparisons among multiple groups were performed using one-way ANOVA followed by an SNK test. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eThe metabolites and key enzymes of the aSMase/CerS1P signaling pathway are associated with silica-induced pulmonary fibrosis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEstablishing the silica-induced pulmonary fibrosis mice model (0.4g/kg), observing the formation of silicotic nodules by day 7 via HE and Masson staining (\u003cb\u003eSupplementary Fig.\u0026nbsp;1a\u003c/b\u003e). As the duration of silica exposure increased, typical silicotic nodules with collagen fibers arranged in concentric circles were formed (\u003cb\u003eSupplementary Fig.\u0026nbsp;1b\u003c/b\u003e). To verify the successful construction of the model, we examined the expression of fibrosis-related genes and found that these proteins were also upregulated (\u003cb\u003eSupplementary Fig.\u0026nbsp;1c\u003c/b\u003e). These data indicate that exposure to silica (0.4 g/kg) results in pulmonary fibrosis.\u003c/p\u003e\u003cp\u003eSphingolipids, an essential class of lipids, play a pivotal role in regulating cellular functions. Within the aSMase/Cer/S1P signaling pathway, aSMase, an enzyme located in lysosomes, converts sphingomyelin into ceramide. Subsequently, ceramidase breaks down ceramide to generate sphingosine, which is further metabolized by Sphk1 into S1P \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. We conducted scRNA-seq to identify the cell populations of lung tissues from a silica-induced pulmonary fibrosis model. scRNA-seq of silicosis model and control lung tissue revealed eight distinct cell populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Among these cells, fibroblasts expressed the highest levels of SMPD1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Further analysis showed that SMPD1 expression was lower in the silicosis group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Our previously published data demonstrated that in a high-dose silica (0.4 g/kg) induced pulmonary fibrosis mouse model, metabolomic analysis was performed to assess the trends of key metabolites in this pathway \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The analysis revealed that the relative levels of SM, Cer, and sphingosine were lower in the silicosis group compared to controls. In contrast, S1P levels were higher and correlated with the severity of silicosis (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-f).\u003c/p\u003e\u003cp\u003eTo gain deeper insights into changes in metabolic enzyme expression, qRT-PCR and Western blotting were employed to evaluate mRNA and protein levels of aSMase and Sphk1, the key enzymes in this metabolic pathway. The data indicated a decrease in aSMase expression and an increase in Sphk1 expression in the experimental group compared to the control group (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg-j, \u003cb\u003eSupplementary Fig.\u0026nbsp;2\u003c/b\u003e). A consistent decrease in aSMase enzyme activity was also observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek). These findings suggest the potential involvement of aSMase and Sphk1 in silica-induced lung fibrosis, underscoring their importance in the dysregulation of the aSMase/Cer/S1P signaling pathway.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe activated Hippo/YAP signaling pathway is involved in silica-induced pulmonary fibrosis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe Hippo/YAP signaling pathway is intricately linked to tissue homeostasis, cell proliferation, metabolism, and the formation of fibrosis \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Therefore, we examined the levels of YAP and TAZ in the Hippo/YAP signaling pathway in a silica-induced pulmonary fibrosis model.\u003c/p\u003e\u003cp\u003eCompared to controls, a significant decrease was observed in the mRNA expression levels of YAP and TAZ in lung tissues from mice exposed to 0.4g/kg of silica for 42 days (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). Similarly, the protein levels of YAP, TAZ, and p-YAP showed a declining trend (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-h). To further support these findings, we analyzed relevant data from a public database. In this bioinformatic analysis, lung tissues from mice exposed to 12mg of silica were collected at various time points (14d, 28d, 42d, 77d) \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The proteomic analysis revealed a downregulated trend in the protein levels of YAP and TAZ within the Hippo/YAP signaling pathway (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei-j) \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eUpon phagocytosis of silica by lung macrophages, specific signaling factors are activated, leading to a reduced expression of both total and phosphorylated YAP in the cell cytoplasm. This reduction facilitates the nuclear translocation of unphosphorylated YAP, which activates TAZ and ultimately contributes to fibrosis formation. Therefore, we propose that the Hippo/YAP signaling pathway plays a pivotal role in the progression of silicosis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe metabolites and key enzymes are involved in the aSMase/Cer/S1P signaling pathway in vitro\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUpon stimulating NIH-3T3 mouse lung fibroblasts with TGF-β1, a noteworthy upregulation of COL1A1 and α-SMA expression was observed specifically with 2.5 ng/mL of TGF-β1 for 24 hours (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-e). In evaluating the expression of aSMase and Sphk1 within the metabolic pathway, aSMase showed a decreasing trend at the transcriptional, translational, and activity levels following TGF-β1 stimulation in NIH-3T3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef-i). Meanwhile, the mRNA level of Sphk1 exhibited an increasing trend (\u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e). Additionally, S1P expression, a downstream component in the signaling pathway, was increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej). These findings suggest the involvement of the aSMase/Cer/S1P signaling pathway in the process of fibrosis.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhagocytosis of silica particles by macrophages promoted pulmonary fibroblast transdifferentiation and activated the aSMase/Cer/S1P signaling pathway\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate whether macrophages can induce fibroblast transdifferentiation following the phagocytosis of silica particles, an in vitro co-culture model was established. Considering TNF-α and TGF-β1 as pro-inflammatory and pro-fibrotic factors, respectively, their levels in the supernatant of RAW264.7 cells exposed to silica (100 \u0026micro;g/mL at 12h, 24h, 48h, and 72h) were measured using ELISA. The data showed an increase in the levels of these cytokines (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). Additionally, a significant rise in the expression of pro-fibrotic genes, specifically COL1A1, ACTA2, and fibronectin, was observed following silica phagocytosis by RAW264.7 for 6h in NIH-3T3(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003ec-e).\u003c/p\u003e\u003cp\u003eAn examination of enzymes and metabolites in the aSMase/Cer/S1P signaling pathway within the co-culture model revealed a decreasing trend in both the mRNA expression and activity of aSMase (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003ef-g). In contrast, an upward trend was observed in the levels of Sphk1 and S1P (\u003cb\u003eSupplementary Fig.\u0026nbsp;4\u003c/b\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). These findings suggest that silica phagocytosis by RAW264.7 cells may promote the transdifferentiation of NIH-3T3 cells and enhance the production of fibrotic factors. However, data from the Hippo/YAP signaling pathway in the co-culture model showed no significant changes in YAP and TAZ expression (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003ei, j).\u003c/p\u003e\u003cp\u003e\u003cb\u003eAlleviation of silica-induced lung fibrosis by intratracheal administration of AAV-SMPD1\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMice were intratracheally administered adeno-associated virus (AAV)-Control or AAV-SMPD1. One week later, they received intratracheal silica (0.4g/kg) and were subsequently sacrificed for lung tissue collection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). After confirming successful viral transfection into lung tissue with high SMPD1 expression, we assessed the degree of fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, c). Administration of AAV-SMPD1 significantly attenuated silica-induced pulmonary fibrosis in mice, as evidenced by a reduction in collagen content within silicotic nodules (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003eNotably, the analysis of SMPD1 expression in lung tissue revealed a relatively lower increase in the AAV-SMPD1-Silicosis group compared to the AAV-SMPD1 group. This observation aligned with our earlier findings, which indicated a decrease in SMPD1 expression in silica-induced pulmonary fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Upon SMPD1 overexpression, a noticeable increase in the expression levels of YAP and TAZ was observed to a certain extent (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef, g). These results support the hypothesis that AAV-SMPD1 delivery inhibits lung fibrosis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAlleviation of lung fibrosis by overexpression of SMPD1 in NIH-3T3 cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further elucidate the role of SMPD1, we investigated the impact of SMPD1 overexpression on the proliferation, migration, and differentiation of NIH-3T3 cells in vitro. The successful overexpression of SMPD1 in NIH-3T3 cells was first confirmed (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). Following SMPD1 overexpression, a reduction was observed in the expression of pro-fibrotic genes, as well as in cell proliferation, migration and differentiation of fibroblasts into myofibroblasts when treated with 2.5ng/mL TGF-β1 for 24 hours (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, d, e, g, h, i, j, k).\u003c/p\u003e\u003cp\u003eThe expression of SMPD1 was decreased in fibroblasts treated with TGF-β1, consistent with our previous observations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). Together, these findings demonstrate that restoring SMPD1 expression can effectively mitigate lung fibrosis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study demonstrates that the aSMase/Cer/S1P signaling pathway plays a critical role in the pathogenesis of silica-induced pulmonary fibrosis in mice. When macrophages phagocytose exogenous silica, they release significant amounts of pro-inflammatory and pro-fibrotic factors, which further stimulate the activation and transdifferentiation of fibroblasts into myofibroblasts in vivo. Evidence of the aSMase/Cer/S1P signaling pathway's involvement is seen in the reduced levels and activity of acid sphingomyelinase, alongside increased S1P levels. These metabolites regulate the downstream Hippo/YAP axis through specific activation pathways, influencing the migration, proliferation and differentiation of fibroblasts. This disruption of the lung tissue microenvironment ultimately leads to the formation of lung fibrosis. Notably, overexpression of SMPD1 may modulate the downstream Hippo/YAP signaling pathways, thereby mitigating the extent of silica-induced fibrosis.\u003c/p\u003e\u003cp\u003eASMase exhibits high biological activity among sphingomyelinases. It catalyzes the breakdown of sphingomyelin to generate Cer, which is deacylated by ceramide synthase to produce sphingosine. Sphingosine is rapidly phosphorylated by Sphk to produce S1P, which binds to its downstream G protein-coupled receptors (S1PR1-S1PR5), triggering the downstream inflammatory response \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. ASMase is a Zn2+-stimulated SMase encoded by the SMPD1 gene \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In the late 1960s, researchers reported that a deficiency in aSMase leads to a rare autosomal recessive disorder known as Niemann-Pick disease (NPD), a lysosomal storage disease \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Deficiency of the SMPD1 gene results in the accumulation of cholesterol and sphingomyelin in the liver and brain, accompanied by atrophy of the cerebellum and deficiency of Purkinje cells \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. A previous study revealed decrease in sphingolipid metabolism in lungs of idiopathic pulmonary fibrosis (IPF) patients, evidenced by decreased levels of sphingomyelin and sphingosine, along with reduced expression of SMPD1 and SMPD4 \u003csup\u003e23\u003c/sup\u003e. In our study, we observed lower levels and activity of aSMase in lung tissue and foam cell deposition in the alveolar spaces of the silica-induced mouse model. Overexpression of the SMPD1 gene alleviated lung fibrosis, inhibited fibroblast proliferation and migration, and enhanced apoptosis. Similarly, we observed that SMPD1 exhibited a consistent increase at both the RNA and protein levels over time. We hypothesize that this may be associated with early-stage inflammatory responses and late-stage fibrotic reactions. Further investigation is required to determine whether SMPD1 levels eventually normalize as fibrosis progresses and whether this influences downstream metabolites. If so, early intervention may have greater therapeutic significance.\u003c/p\u003e\u003cp\u003eCeramide, a type of amide compound, is fundamental to sphingolipid metabolism. De novo ceramide biosynthesis involves the pathway of serine palmitoyltransferase complex and ceramide synthase, and a breakdown synthesis pathway via sphingomyelinase that catabolizes membrane sphingolipids \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Cer plays a crucial role in inhibiting cell proliferation and promoting apoptosis and also acts as an intracellular lipid second messenger, regulating cell proliferation, differentiation, senescence, and migration \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Beyond its role in apoptosis, ceramide has shown to be associated with cell cycle arrest, inducing cell in the G0/G1 phase \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In relation to tumor diseases, ceramide exerts anti-proliferative effects by inhibiting the growth and migration of cancer cells and inducing autophagy and apoptosis \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Its expression varies among tumor subtypes, with higher levels observed in ovarian cancer compared to normal tissues \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, whereas ceramide levels are significantly reduced in patients with colon cancer \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that feeding mice with sphingomyelin or ceramide analogues reduces the incidence of colon cancer \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Therefore, the explore of ceramide may become our research in the future.\u003c/p\u003e\u003cp\u003eSphk1 and Sphk2 are two isozymes identified in mammals and both are commonly expressed in most tissues. However, Sphk1 is more abundantly expressed in the lungs and heart, while Sphk2 is primarily expressed in the liver and spleen \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. S1P is a simple sphingolipid metabolite present in blood, plasma, bronchoalveolar lavage fluid and various organs. S1P is involved in numerous cellular responses, including proliferation, differentiation, adhesion, motility and apoptosis, by acting on specific G protein-coupled receptors (S1P1-5) \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. A study of patients with IPF found elevated S1P levels in serum and BALF, as detected by ELISA, which negatively correlated with pulmonary function. Macrophages isolated from BALF exhibited increased Sphk1 expression at the transcriptional level, and lung tissue from IPF patients showed higher Sphk1 protein expression compared to controls. Immunohistochemistry revealed that Sphk1 was highly expressed, particularly in proliferating alveolar epithelial cells and fibroblasts. S1P was also found to promote the differentiation of alveolar epithelial cells into mesenchymal cells and fibroblasts into myofibroblasts \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In our silicosis model and in vitro studies, we similarly found increased levels of Sphk1 expression. These findings suggest that Sphk1 is a novel prognostic and therapeutic target for pulmonary fibrosis. Targeting Sphk1 to inhibit S1P production may provide a new therapeutic strategy for fibrotic lung diseases. The relationships among SM, ceramide, sphingosine, and S1P require further investigation.\u003c/p\u003e\u003cp\u003eThe Hippo/Yes-associated protein (YAP)1 signaling pathway is known to regulate cellular proliferation, differentiation, and tissue homeostasis \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. YAP and TAZ are transcriptional co-activator proteins that shuttle between the nucleus and cytoplasm and regulate the expression of downstream factors by binding to the intracellular transcription factor TEAD \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Previous research has shown that the Sphk1/S1P signaling pathway promotes fibrosis by activating the Hippo/YAP axis in lung fibroblasts. Conditional knockdown of Sphk1 in mice lung fibroblasts led to reduced lung tissue fibrosis, accompanied by decreased YAP1 localization in fibrotic regions. Additionally, in primary lung fibroblasts stimulated with TGF-β, YAP1 nuclear translocation was significantly increased, while Sphk1 knockdown reduced nuclear YAP1 localization. The expression of fibrosis-related factors was significantly reduced when verteporfin, an inhibitor of YAP1, was added. All these data suggest that the Sphk1/S1P metabolic pathway and the Hippo/YAP axis may have a synergistic role in the development of fibrosis \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In our model, we found that YAP/TAZ showed a trend of low expression in silica-induced mice models, consistent with previous proteomics sequencing results in silicosis models \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, in our in vitro co-culture model, we did not observe a significant trend in YAP and TAZ expression at the transcriptional level. These data suggest that targeting the YAP/TAZ pathway may be a promising therapeutic option for fibrosis patients, though the exact mechanism need further investigation.\u003c/p\u003e\u003cp\u003eIn conclusion, this study demonstrated that aSMase/Cer/S1P signaling plays a significant role in silica-induced pulmonary fibrosis, likely by impacting the downstream Hippo/YAP signaling axis. Overexpression of SMPD1 can mitigate silica-induced pulmonary fibrosis. Therefore, targeting these metabolites presents a promising therapeutic strategy for silicosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003ch2\u003eEthical Approval\u003c/h2\u003e\n\u003cp\u003eAll animal protocols were approved by the institutional animal care and treatment committee of the Capital Medical University (AEEI-2023-077).\u003c/p\u003e\n\u003ch2\u003eFundings\u003c/h2\u003e\n\u003cp\u003eNational Natural Science Foundation of China (81970061). High Level Public Health Technology Talent Construction Project (DL-02-21). Reform and Development Program of Beijing Institute of Respiratory Medicine (Ggyfz202512).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eRuimin Ma: Data analysis and wrote the manuscript. Di Sun: Carried out the experiments. Yuanying Wang and Yawen Song: Collected and analyzed the data. Qiao Ye: Conceived, designed the experiments and wrote the manuscript.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhao, X., Kwan, J., Yip, K., Liu, P. P. \u0026amp; Liu, F. F. Targeting Metabolic Dysregulation for Fibrosis Therapy. \u003cem\u003eNat. Rev. Drug Discov\u003c/em\u003e. \u003cb\u003e19\u003c/b\u003e, 57\u0026ndash;75 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShi, P. et al. 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Sphingosine-1-Phosphate is Increased in Patients with Idiopathic Pulmonary Fibrosis and Mediates Epithelial to Mesenchymal Transition. \u003cem\u003eThorax\u003c/em\u003e \u003cb\u003e67\u003c/b\u003e, 147\u0026ndash;156 (2012).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWarren, R., Lyu, H., Klinkhammer, K. \u0026amp; De Langhe, S. P. Hippo Signaling Impairs Alveolar Epithelial Regeneration in Pulmonary Fibrosis. \u003cem\u003eElife\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHeng, B. C. et al. An Overview of Signaling Pathways Regulating YAP/TAZ Activity. \u003cem\u003eCell. Mol. Life Sci.\u003c/em\u003e \u003cb\u003e78\u003c/b\u003e, 497\u0026ndash;512 (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"silica, sphingolipid metabolism, acid sphingomyelinase","lastPublishedDoi":"10.21203/rs.3.rs-7052670/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7052670/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSilicosis is irreversible lung disease caused by exposure to dust-containing silica particles. Our study aims to explore the potential effects of acid sphingomyelinase (aSMase), ceramide (Cer) and sphingosine-1-phosphate (S1P) on silica-induced pulmonary fibrosis. We also explored the expression of the downstream signaling pathway of Hippo/YAP. The high expression of SMPD1 which encodes aSMase were given. The level of aSMase in silicosis was lower than those in controls. Additionally, the level of aSMase in lung fibroblasts, following stimulation with TGF-β1, was observed to decrease. The phagocytosis of silica particles by macrophages promoted lung fibroblast transdifferentiation and activated the aSMase/Cer/S1P signaling pathway. The activated Hippo/YAP signaling pathway is involved in silica-induced pulmonary fibrosis. Moreover, the collagen content within silicotic nodules and the number of large nodules were reduced by the intervention with the high expression of SMPD1 in vivo. Furthermore, the levels of fibrotic genes in TGF-β1-induced lung fibroblasts activation were diminished with the overexpression of SMPD1. ASMase was downregulated in silica-induced lung fibrosis, however the administration of exogenous SMPD1 overexpression has the potential to alleviate this condition. This process may be related to the downstream Hippo/YAP signaling pathway.\u003c/p\u003e","manuscriptTitle":"Targeting acid sphingomyelinase/ceramide/S1P ameliorates silica-induced pulmonary fibrosis through Hippo/YAP signaling pathway in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-30 17:50:10","doi":"10.21203/rs.3.rs-7052670/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"86cc3380-a789-43c6-a106-8db8d7f75dd6","owner":[],"postedDate":"July 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":52298226,"name":"Biological sciences/Cell biology"},{"id":52298227,"name":"Health sciences/Diseases"},{"id":52298228,"name":"Biological sciences/Molecular biology"},{"id":52298229,"name":"Health sciences/Pathogenesis"}],"tags":[],"updatedAt":"2025-08-28T16:23:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-30 17:50:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7052670","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7052670","identity":"rs-7052670","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}