The Molecular Mechanism of DMSO in Anti-Silicosis Fibrosis Mediated by the IL-6/STAT3-MMP12 Axis Based on Transcriptome Sequencing Analysis

DOI:https://doi.org/10.65281/709729

Yukang Zhu1, 2*,Guoling Yao1, 2*, Lei Xu1, 2, Wei You3, Wenhao Liu1, 2, Zihan Liu4, Deyong Ge2#

* Co-first authors: Yukang Zhu, Guoling Yao

1 Department of Nuclear Medicine, The First Hospital of Anhui University of Science and Technology, Huainan City, Anhui Province, Postcode: 232000, People’s Republic of China.

2 Anhui University of Science and Technology, Huainan City, Anhui Province, Postcode: 232001, People’s Republic of China.

3 Ningbo University, Ningbo City, Zhejiang Province, Postcode: 315211, People’s Republic of China.

4 Anhui University of Science and Technology School of Medicine, Huainan City, Anhui Province, Postcode: 232001, People’s Republic of China.

#Correspondence: Deyong Ge, Anhui University of Science and Technology, No. 168, Taifeng Street, Huainan City, Anhui Province, Postcode: 232001, People’s Republic of China, Email： dyge@aust.edu.cn.

Abstract

 Aims: This study aims to investigate the anti-inflammatory and anti-fibrotic effects of dimethyl sulfoxide (DMSO) in a mouse model of silicosis, thereby exploring its potential therapeutic value.

Methods: A mouse model of silicosis was established by intranasal instillation, and DMSO treatment was administered via intraperitoneal injection. The experiment was conducted over a period of one month.Lung tissues were collected from all mice; a subset was subjected to transcriptomic analysis, and were differentially expressed genes were identified using the limma package. GO and KEGG enrichment analyses were conducted using ClusterProfiler to investigate gene functions and associated pathways. The remaining samples were subjected to

histopathological assessment by Haematoxylin and eosin staining and Masson ’ s trichrome

staining, while Western blot analysis was performed to validate transcriptomic results. Results: This study demonstrates that DMSO may alleviate the fibrotic process in silicosis by modulating the IL-6/STAT3-MMP12 signalling axis. In the silica-induced silicosis mouse model, DMSO attenuated disease-associated weight loss and reduced collagen deposition. Transcriptomic analysis indicated that DMSO suppressed the activity of multiple fibrosis- related pathways and identified 51 key genes, among which MMP12 was significantly downregulated. Western blot analysis further confirmed reduced MMP12 expression, accompanied by markedly decreased levels of IL-6 and p-STAT3, suggesting the IL-6/STAT3 pathway may play a crucial role in regulating MMP12 expression.

Conclusion: DMSO may attenuate inflammatory responses and pulmonary fibrosis in silicosis by inhibiting activation of the IL-6/STAT3 signalling pathway, thereby reducing MMP12 expression.

Keywords: Silicosis;DMSO; MMP12; IL-6/STAT3 pathway; Transcriptomic analysis

Introduction

 Silicosis is one of the major forms of occupational pneumoconiosis1, characterised by persistent pulmonary inflammation and irreversible pulmonary fibrosis resulting from Prolonged exposure to silica dust2,3. The pathogenesis of this silicosis is highly complex. Although previous studies have demonstrated that aberrant macrophages activation4, excessive proliferation of fibroblasts, epithelial-mesenchymal transition, and abnormal deposition of extracellular matrix (ECM) components are critical contributors to fibrotic development 5,6, the core molecular mechanisms underlying the inflammatory-fibrotic vicious

cycle remain incompletely understood. Notably, the matrix metalloproteinase (MMP) family plays a crucial role in ECM remodelling and the progression of pathological fibrosis7. Among

these, matrix metalloproteinase 12（MMP12）which is specifically secreted by macrophages,

which degrades key ECM components such as elastin, fibronectin, and type IV collagen, and plays an important role in the pathogenesis of fibrotic diseases including chronic obstructive pulmonary disease8. Recent studies further suggests that MMP12 derived from macrophages may further promote fibrosis by damaging endothelial cells during the process of pulmonary fibrosis9.

Dimethyl sulfoxide (DMSO), a low-molecular-weight compound with diverse biological activities10, has demonstrated anti-inflammatory effects in several inflammatory diseases including cystitis and has been approved by the U.S. Food and Drug Administration for the treatment of interstitial cystitis11. Recent studies suggest that DMSO may slow the progression of fibrosis to some extent by inhibiting the release of inflammatory factors and reducing oxidative stress responses12. Research conducted by Ingrid Elisia et al. reported that DMSO specifically inhibits the activation of ERK1/2, p38 MAPK, JNK, and PI3K/Akt signalling pathways in human monocytes in whole blood models, thereby exerting its anti- inflammatory effects13.

Although the MMP family has been shown to play a critical role in the fibrosis process of silicosis, no studies have conclusively demonstrated whether DMSO can affect the pathological progression of silicosis through regulation MMP expression. Therefore, this study represents the first investigation of the anti-inflammatory and anti-fibrotic effects of DMSO in a silicosis mouse model. Through transcriptome sequencing, differential gene expression analysis, and enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, this study aims to identify the key regulatory gene networks and molecular mechanisms underlying DMSO intervention, providing theoretical support for MMP-based innovative therapeutic strategies.

Materials and methods

 

 

Preparation of Solutions

Preparation of Silica Suspension: Crystalline silica (SiO2, 80%, particle size 1-5 μm) was purchased from Sigma Aldrich (Cat.No.S5631). A 20 mg sample was sterilised under high pressure and then dissolved in 1 ml of sterile saline under sterile conditions, resulting in a silica suspension with a final concentration of 20 mg/ml. The solution was thoroughly dispersed using an ultrasonic homogeniser.

Preparation of DMSO Solution: Under aseptic conditions, DMSO( ≥ 99.9%, GC203006,

Servicebio, Wuhan, China) was diluted with sterile physiological saline to prepare an approximately 10% (v/v) DMSO solution, which was homogenised by ultrasonic agitation. Each mouse was injected with the DMSO solution at a dose of 0.9 g/kg, as previously described14,15.

Experimental Animals

A total of 32 male C57BL/6J mice (8–12 weeks old; average weight 24.23 ± 1.66 g at study initiation) were used. Mice were obtained from Changzhou Cavens Laboratory Animal Co., Ltd. (License No: SCXY(Su)2011-0003) and allowed to acclimatise for one week. Animals were randomly allocated to four experimental groups (n=8/group) using a block randomisation method: Control, DMSO, Silica, and Silica + DMSO. Mice were housed in a specific facility under controlled environmental conditions (20–25°C, 12 h:12 h light‑dark cycle) with ad libitum access to standard chow and water. Euthanasia was performed by cervical dislocation following intraperitoneal administration of tribromoethanol (200 mg/kg)

to induce anaesthesia. Humane endpoints were predefined as ≥ 20% loss of body‑weight,

persistent loss of appetite or thirst, or inability to stand independently; animal meeting any of these criteria would have been euthanised immediately. The maximum body weight loss observed before the scheduled endpoint was 18.38%, and no animals reached a humane endpoint.

Silicosis Mouse Model

The silicosis model was established by intranasal instillation, as previously described 16. Mice in the Silica and Silica + DMSO groups were anaesthetised with isoflurane and then received a single intranasal instillation of 80 µl silica suspension. Mice in the DMSO and Silica + DMSO groups received intraperitoneal injections of 200 µl DMSO solution twice weekly for one month, all remaining groups received an equivalent volume of sterile physiological saline on the same schedule. Body weight was monitored daily throughout the experimental period. On the 30th day after the model establishedment, mice were anaesthetised and euthansied by cervical dislocation. Under sterile surgical technique, the mice were dissected, and their lung tissues were collected. The lung tissues of four mice per group were used for transcriptome sequencing     analysis.   For       the                     remaining mics, the   left              lungs   were fixed in         4% paraformaldehyde at room temperature for 48 hours, while the right lungs were stored at

−80 °C for subsequent experiments. All animal experiments were performed in accordance with the relevant guidelines of the Animal Ethics Committee.

Lung Tissue Histopathological Staining Analysis

Fixed mouse lung tissues were dehydrated through xylene and graded ethanol, followed by paraffin infiltration and embedded to form paraffin blocks. Thin sections of 5 μm were cut from the paraffin blocks and subjected to histopathological analysis. Haematoxylin and eosin

（ H&E ）  staining and Masson’s trichrome staining were performed to prepare the

pathological slides, which were subsequently examined under an Olympus FV3000 microscope to assess tissue morphology and the degree of fibrosis.

Haematoxylin and eosin staining

The tissue sections were first stained in haematoxylin solution for 3-5 minutes, followed by differentiation in an acidic solution for 2-5 seconds. The sections were then treated with a bluing solution for an additional 2-5 seconds and rinsed under running water. Subsequently, the sections were counterstained in eosin solution for 5 minutes, dehydrated through graded ethanol (85%, 95%, and 100%), for 5 minutes each step. Finally, the sections were cleared in xylene for two consecutive 5-minute intervals and mounted using neutral resin for sealing.

Masson trichrome staining

The sections were initially stained with haematoxylin for 5 minutes, followed by differentiation in acidic solution for 30 seconds and bluing for 10 minutes under running water. The sections were then stained with Lichun Red–acid fuchsin solution for 10 minutes, differentiated in phosphomolybdic acid for 2 minutes, and sussequently stained with Bright Green for 1 minute. After another 1-minute treatment with an acidic differentiating solution, the sections were dehydrated through graded ethanol and cleared in xylene. Finally, they were mounted using neutral resin for sealing.

Transcriptome sequencing workflow

Mouse lung tissue samples from four experimental groups were collected and immediately frozen in liquid nitrogen. Sequencing was performed by Novier Biotechnology Company. Total RNA was extracted from mouse lung tissues, and RNA purity and concentration were assessed. RNA integrity was evaluated using the RNA Integrity Number (RIN), a standardised metric ranging from 0 to 10, with higher values indicating better RNA quality and integrity17. RIN values were automatically calculated from the RNA electropherograms using the Agilent ProSize data analysis system in conjunction with the Agilent 5300 instrument. The resulting RIN scores for each group were as follows: 9.45 for the N group, 8.85 for the ND group, 9.06 for the S group, and 9.15 for the SD group. During library preparation, one sample from the silicosis group failed to meet the RNA concentration quality control criteria, therefore, three

samples from the silica group were included in the subsequent transcriptomic sequencing analysis. The eukaryotic reference transcriptome sequencing depth reached approximately

6.2–6.4 Gb per sample. Subsequently, mRNA was enriched using Oligo dT, fragmented, reverse transcribed to cDNA, and ligated with adapters. Finally, sequencing was performed on the Illumina platform. All raw sequencing data generated in this study have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE311671, and are available upon reasonable request.

Differentially expressed gene screening and functional enrichment analysis.

The core analysis of transcriptomics involves assessing the significance of differential gene expression. This process includes six main steps: quality control, alignment, quantification, differential analysis, and functional enrichment. Differentially expressed genes were analysed using the limma package (data normalisation via the Median Ratio Method of DESeq2, FDR correction via the Benjamini–Hochberg strategy, using the Mus musculus mm8 genome  as the reference), with a fold change threshold of |log₂FoldChange| > 1.5 and a P-value < 0.05 set as the selection criteria. Subsequently, the clusterProfiler software was used for GO functional enrichment analysis and KEGG pathway enrichment analysis, with a threshold adjusted P value (padj) < 0.05 considered significantly enriched.

Western Blot Analysis

Fresh mouse lung tissue was collected and homogenised with an appropriate volume of RIPA lysis buffer (G2002, Servicebio, Wuhan). After centrifugation, the supernatant was collected collected and protein concentration was determined using the BCA Protein Assay Kit (G2026, Servicebio, Wuhan). The samples were then mixed with loading buffer (P0015L, Bioteke, Beijing) and heated at 100°C for 10 minutes. Protein separation was performed using a 10% SDS-PAGE gel (G2177, Servicebio, Wuhan), followed by electrotransfer to a PVDF membrane. The membrane was blocked with 5% skim milk (GC310001, Servicebio, Wuhan), then incubated overnight at 4°C with primary antibodies against β-Actin (GB11001, Servicebio, Wuhan), IL-6 (DF6087, Affinity Biosciences, Jiangsu), STAT3 (GB11176, Servicebio, Wuhan), p-STAT3 (GB150001, Servicebio, Wuhan) and MMP12 (GB115475, Servicebio, Wuhan). The following day, the membrane was washed with TBST and incubated with secondary antibody (GB23303, Servicebio, Wuhan) at room temperature for 1 hour, followed by additional washes with TBST. Protein bands were visualised using a chemiluminescent substrate (G2014, Servicebio, Wuhan), and protein quantification was performed using ImageJ software.

Statistical Analysis

All data were analysed using GraphPad Prism 9.5 software (GraphPad Software, USA). Data normality was assessed using the Shapiro–Wilk test, and homogeneity of variance was evaluated using the Brown–Forsythe test. Comparisons between two groups were performed using a t-test, whereas comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA), and followed by Tukey’s post hoc test. Statistical significance was defined as a P-value ≤ 0.05. *P<0.05, **P<0.01, ***P<0.001,  ****P<0.0001.

Results

 

 

Pulmonary Inflammation and Fibrosis in Silicosis Mice

As shown in Figure 1A, experimental mouse models were established for the control

and Silicosis groups. Mice in the silicosis group were intranasally instilled with a silicon dioxide suspension, and body weight was monitored on daily basis throughout the experimental period. Compared with the control group, mice in the silicosis group exhibited a

significant reduction in body weight（Figure1B）.

Histopathological evaluation of lung tissue, conducted using H&E and Masson’s trichrome staining (Figures 1C and 1D), revealed pronounced pathological alterations in the silicosis group. These changes included marked thickening of the alveolar walls, areas of pulmonary consolidation, and extensive infiltration of inflammatory cells. The severity of pulmonary fibrosis was further quantified using the Ashcroft scoring system18, as shown in Figure 1E. Additionally, Masson’s trichrome staining demonstrated substantial deposition of collagen fibres within the lung interstitium, as shown in Figure 1F. Collectively, these pathological changes confirm the successful establishment of the silicosis mouse model.

Figure 1. Characteristics of fibrosis in the mouse silicosis model. (A) Schematic diagram of the mouse silicosis experiment. Arrows of different colours represent different drug

administration schemes. (B) Haematoxylin and eosin staining of lung tissue. Scale bar: 50 μ

m(C) Masson staining of lung tissue (blue: collagen fibres; red: muscle fibres). Scale bar: 25μ m (D) Changes in body weight of mice in the Control group and the Silica group. (E, F) The

comparison of the inflammation score in HE staining results and the relative collagen area between the Control group and the Silica group.

DMSO Alleviates Silica-Induced Pulmonary Fibrosis in Mice

To evaluate the effects of DMSO on pathological processes and lung tissue alterations in a silicosis model, four experimental groups were established (Figure 2A). Body weight changes were monitored over a one month period.

The results demonstrated that mice in the Silicosis group exhibited a continuous decline in body weight, whereas the Silica+DMSO group showed an initial weight loss followed by a

recovery after DMSO intervention (Figure 2F). After 30 days of silica exposure, lung tissues were collected and assessed for inflammation and fibrosis changes using H&E staining (Figure 2B) and Masson’s trichrome staining (Figure 2C). Pulmonary fibrosis was scored on H&E -stained sections using the Ashcroft method, revealing a significantly lower fibrosis score in the Silica+DMSO group compared to the Silica group (Figure 2D). Masson’s staining further indicated that collagen fibre deposition in the Silica group was extencive and dense, occupying approximately 40% of the lung tissue area, which was markedly higher than the 10% observed in the control group (Figure 2E), suggesting that silica exposure induced severe pulmonary fibrosis. In contrast, collagen deposition in the Silica+DMSO group was reduced to 20%, indicating that DMSO may alleviate silica-induced pulmonary fibrosis by inhibiting excessive collagen accumulation.

Figure 2. DMSO alleviates pulmonary fibrosis in silicosis mice. (A) Schematic diagram of the experimental design. Arrows of different colours represent different administration regimens. (B) Haematoxylin and eosin staining of lung tissue. At a magnification of 200×, the scale bar is 50 μm; at a magnification of 400×, the scale bar is 25 μm. (C) Masson staining of lung tissue (blue: collagen fibres; red: muscle fibres). At a magnification of 200×, the scale bar is 50 μm; at a magnification of 400×, the scale bar is 25 μm. (D). Relative collagen area of Masson staining in the lung tissue of different groups. (E) Inflammation scores of different groups based on HE staining results. (F) Body weight changes of mice within 30 days. Data

was presented as mean ± SEM. The comparison of multiple sets of data was conducted using

one-way analysis of variance (ANOVA), and post hoc tests were carried out using the Tukey method.

Transcriptome Sequencing Reveals the Regulation of Differentially Expressed Genes in Silicosis Mice by DMSO Intervention

To investigate the anti-inflammatory and anti-fibrotic effect of dimethyl sulfoxide (DMSO) in a silicosis model, transcriptome sequencing was performed on lung tissue samples collected from the experimental mice. DEGs were identified using the limma algorithm, with selection criteria defined as |log2(fold change)| > 1.5 and a corrected P-value (FDR) < 0.05.

Transcriptome Analysis demonstrated that, Compare with the control group, the Silica group exhibited 132 significantly differentially expressed genes (Figure 3A), including 117 were upregulated, and 15 were downregulated. Notably, genes such as Saa3 and MMP12 were markedly upregulated. This expression pattern was further supported by hierarchical clustering heatmap analysis (Figure 3C), which demonstrated consistent gene expression profiles across individual samples.

In the comparison between the Silica+DMSO and Silica groups, a total of 3,054 significantly differentially expressed genes were identified (Figure 3B), comprising 1,399 upregulated and 1,655 downregulated genes. The corresponding heatmap (Figure 3D) revealed substantial transcriptomic alterations following DMSO treatment, with prominent downregulation of genes including Lcn2 and MMP12. Specifically, key genes that were upregulated in the Silica group such as MMP12, Saa3, and Spp1, showed pronounced downregulation in the Silica+DMSO group. These findings suggest that DMSO may mitigate silicosis-related pathology by suppressing the expression of pro-inflammatory and pro-fibrotic mediators.

Figure 3. Transcriptome sequencing reveals the gene regulation of DMSO in alleviating silicosis. (A, B) Volcano plot of differentially expressed genes between the Silica group and the Control group, the Silica + DMSO group and the Silica group. (C, D) Heatmap of the Silica group compared with the Control group, and the Silica + DMSO group compared with the Silica group.

DMSO Inhibits the Expression of MMP12 in Silicosis Mice

To elucidate the potential molecular targets of dimethyl sulfoxide (DMSO) in silicosis-related inflammation and fibrosis, an intersection analysis of differentially expressed genes (DEGs) was conducted between the Silica vs. Control and Silica+DMSO vs. Silica comparison groups. This analysis identified 51 common DEGs (Figure 4A), that were significantly upregulated in the silicosis mouse model but exhibited marked downregulation following DMSO treatment. Among these genes, MMP12 showed the most substantial changes in expression (Figure 4B). Further characterisation revealed that these 51 genes included several well-established fibrotic markers, such as MMP12, MMP9, SPP1, CCL3, and CCL8, suggesting their potential involvement in the anti-inflammatory and anti-fibrotic mechanisms of DMSO.

To further investigate the regulatory effect of DMSO on MMP12 and its role in the fibrotic progression, MMP12 expression levels were assessed in lung tissues from four experimental

groups (Control, DMSO, Silica, and Silica+DMSO) using transcriptome sequencing and Western blot analysis (Figure 4C). The results demonstrated that MMP12 expression was significantly elevated in the Silica group compared to the Control group, whereas this upregulation was markedly suppressed in the Silica+DMSO group.

Figure 4. Transcriptome sequencing indicated that DMSO reduces MMP12 expression. (A) Venn diagrams of differentially expressed genes (DEGs) between the Silica group and the Control group, and between the Silica+DMSO group and the Silica group. (B) Heatmap of significantly differentially expressed genes. (C) Expression levels of MMP12 in each group.

GO/KEGG Analysis Elucidates the Mechanism by Which DMSO Regulates Inflammation and Fibrosis in Silicosis

 

To elucidate the molecular regulatory mechanisms underlying the effects of different treatments, GO functional enrichment analysis and KEGG pathway enrichment analysis were performed on DEGs identified from transcriptome sequencing.

In the comparison between the Silica vs. Control group, GO functional enrichment results demonstrated that DEGs were primarily enriched in biological processes and cellular components, including “collagen-containing extracellular matrix” and “regulation of inflammatory response” (Figure 5A). These findings indicate that silica exposure significantly activates inflammatory chemokine signalling and ECM remodelling processes. KEGG pathway enrichment analysis further revealed that DEGs were significantly enriched in the NF-κB signalling pathway, TNF signalling pathway, and Toll-like receptor signalling pathway (Figure 5B). Collectively, these results suggest that dysregulated inflammatory signalling cascades (NF-κB/TNF/Toll-like receptor) and ECM structural abnormalities (e.g., collagen deposition) constitute the core molecular features of silicosis-induced pulmonary fibrosis.

In the comparison between the DMSO+Silica vs. Silica group, GO enrichment analysis showed that DEGs were mainly involved in “chemotaxis” and “protein tyrosine kinase activity” (Figure 5C). Meanwhile, KEGG enrichment results focused on the PI3K-Akt signalling pathway, Rap1 signalling pathway, and p53 signalling pathway (Figure 5D). These results suggest that DMSO intervention may inhibit inflammatory cell infiltration and reverse excessive collagen deposition by regulating cell migration and the PI3K-Akt/Rap1/p53 signalling axes, thereby exerting anti-fibrotic effects in silicosis.

Figure 5. GO/KEGG enrichment analysis revealing the ameliorative effect of DMSO on silicosis via transcriptome sequencing. (A) GO functional enrichment analysis and (B) KEGG pathway enrichment analysis for the comparison between the Silica group and the Control group. (C) GO functional enrichment analysis and (D) KEGG pathway enrichment analysis for the comparison between the Silica+DMSO group and the Silica group.

DMSO May Inhibit MMP12 Gene Expression in Silicosis Mice via the IL-6/STAT3 Signaling Pathway

To investigate potential regulatory relationships among differentially expressed genes (DEGs), a gene interaction network was constructed (Figure 6A) . The analysis revealed that most DEGs exhibited direct or indirect interactions with MMP12. Further detailed analysis identified interactions between MMP12 and several significantly differentially expressed genes, including key regulators such as IL-6, STAT3, SPP1, and MMP19 (Figure 6B).

Western blot analysis was performed to quantify the protein expression levels of IL-6, STAT3, p-STAT3 and MMP12 (Figure 6C-F). The results showed that compared with the Control group, the protein expression levels of IL-6 and p-STAT3 were significantly elevated in the Silica group, while MMP12 protein expression also exhibited an increasing trend. Following

DMSO treatment, the expression levels of these three proteins were significantly reduced. Notably, total STAT3 protein expression remained relatively consistent across all experimental groups.

 
   

Figure 6. DMSO reduces IL-6 and STAT3 protein expression. (A) Network diagram of significantly differentially expressed genes. (B) STRING network interaction diagram of MMP12 and related genes. (C-F) Western blot analysis of β-Actin, IL-6, MMP12, STAT3, and p-STAT3 protein expression. Gray value analysis of β-Actin, IL-6, MMP12, STAT3, and p- STAT3 proteins via Western blotting. Immunoblotting analysis was performed in triplicate (n

= 3).

Discussion

Silicosis remains one of the most severe occupational lung diseases worldwide, characterised by persistent inflammation and progressive pulmonary fibrosis, which substantially impair lung function and patient quality of life 19. In this study, using a silica-induced murine model, we systematically examine the potential protective effects of dimethyl sulfoxide (DMSO) during early stage. Our findings demonstrate that DMSO treatment attenuates inflammatory cell infiltration and mitigates fibrotic pathological changes, suggesting DMSO may exert amodulatory role in the early phases of silicosis.

DMSO, a polar aprotic solvent with high tissue permeability 20, and has well-documented anti-inflammatory and analgesic properties, that has been applied in various inflammation- related disorders, including interstitial cystitis, rheumatoid arthritis, and osteoarthritis 21–23. At the molecular level, DMSO has been reported to suppress NF-κB and MAPK signalling pathways, leading to reduced IL-6 expression in LPS-stimulated RAW264.7 macrophages 24. Notably, high doses of DMSO may cause systemic toxicity, with multi-organ damage reported at doses exceeding 8 g/kg 14; therefore, a low-dose, repeated administration protocol was adopted therapeutic effects and toxicity.

By Integrating transcriptomic and histopathological analyses, we observed that DMSO treatment attenuated pulmonary inflammation and fibrosis in silica-exposed mice, accompanied by downregulation of IL-6 expression. Transcriptomic profiling further revealed a pronounced suppression of MMP12 expression following DMSO treatment. MMP12, predominantly secreted by macrophages 25, has been implicated in the fibrotic progression of silicosis, COPD, and bleomycin-induced lung injury, through regulation of pro-fibrotic pathways including TGF-β1, EGR1, and CYR61 26–29. In the present study, the observed downregulation of MMP12 suggests its involvement in the anti-inflammatory and anti-fibrotic effects mediated by DMSO. Importantly, MMP12 facilitates degradation of excessive extracellular matrix deposits during fibrosis resolution, thereby promoting tissue remodelling and scar clearance 30.

Macrophages are key drivers of pro-inflammatory cytokine production in silica-induced silicosis31. Activated macrophages secrete IL-6 and TNF-α, which contribute to inflammation and fibrosis32. IL-6 engages the gp130 receptor complex to activate the JAK–STAT pathway, particularly STAT3, thereby influencing the macrophage phenotype and promoting pro- fibrotic processes 33–36.In this study, DMSO treatment significantly reduced expression levels of IL-6, STAT3, and MMP12, indicating that DMSO may modulate the IL-6/STAT3 axis and indirectly downregulate MMP12. Previous studies have demonstrated that the IL-6–STAT3 axis plays a pivotal regulatory role in various models of pulmonary fibrosis, and its sustained activation is closely associated with fibrosis severity and disease progression 37,38. STAT3 is

considered a critical hub linking inflammatory responses to fibrotic remodelling, and its activation can regulate MMP12, thereby enhancing its transcriptional activity 39. In addition, STAT3 signalling is involved in macrophage polarisation, driving the transition of macrophages from the pro-inflammatory M1 phenotype to the pro-fibrotic M2 phenotype 40,41, while M2 macrophages represent one of the primary cellular sources of MMP12 42. Therefore, the concomitant downregulation of STAT3 and MMP12 observed in the present study suggests that DMSO may reduce MMP12 expression by interfering with STAT3 signalling. This mechanism may, at least in part, explain the inhibitory effects of DMSO on inflammatory responses and fibrotic progression, and further supports the regulatory role of the IL-6/STAT3/MMP12 axis in the pathogenesis of silicosis.

In conclusion, our findings suggest that DMSO attenuates macrophage-mediated inflammation and fibrosis in silica-induced silicosis by modulating the IL-6/STAT3/MMP12 signalling axis. These results provide novel mechanistic insights into the inflammatory– fibrotic progression of silicosis and identify MMP12 as a potential molecular target for DMSO-based interventions, offering a theoretical basis for future translational research and the development of new therapeutic strategies.

Conclusions

This study, through transcriptome sequencing analysis, revealed that DMSO treatment significantly alters the expression of various inflammatory factors in silicosis, with the most prominent change observed in MMP12. Further experimental evidence suggest that DMSO may mediate the changes in MMP12 through the IL-6/STAT3 signaling pathway. These findings indicate thatMMP12 could be one of the key targets for the treatment of silicosis, providing a new direction for future research in the disease’s therapy.

Ethical approval and consent participate.

All animal experiments conducted in this study were approved by the Science and Technology Ethics Committee of Anhui University of Science and Technology (Approval No. GZ2025- 048) and strictly adhered to the national standards for laboratory animal welfare (GB/T 35892-2018) of China.

Funding

The Open Research Fund of Key Laboratory of Industrial Dust Deep Reduction and Occupational Health and Safety of Anhui Higher Education Institutes (No. AYZJSGXLK202202004). Huainan Science and Technology Plan Project (No.2024N013). Anhui Provincial Quality Engineering Project in 2024 (No.2024jyxm0699). Anhui University

of Science and Technology (Class of 2024) Undergraduate Innovation and Entrepreneurship Training Program Funding (Project No.: 202410361124).

Author contributions

YZ, GY, DG conceived and designed the studies; LX, WY, WL carried out the high- throughput sequencing experiments and performed the bioinformatics analysis; YZ, LX, WY, Z L performed experiments and analysed the data; YZ, GY, WL performed the histopathology

examination of the lung; YZ、GY wrote the paper, DG amended the manuscript. All authors

read and approved the final manuscript.

Competing interests

Authors state no competing interests.

Availability of data and materials

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Acknowledgments

Sincere gratitude to my mentor for their dedicated guidance, the Shendong Laboratory team for collaborative support, my family for their selfless encouragement, and the grant project for funding. This work reflects collective contributions from all parties, acknowledged with deep appreciation.

References

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