Potential Involvement of the IL-6/STAT3/MMP12 Signaling Axis in DMSO-Mediated Anti-Fibrotic Effects in Experimental Silicosis

Summary

This study suggests that dimethyl sulfoxide (DMSO) may alleviate silica-induced pulmonary inflammation and fibrosis in mice. Transcriptomic and experimental analyses suggest that its protective effects may involve suppression of the IL-6/STAT3 signaling pathway and downregulation of MMP12 expression, providing potential therapeutic insights for silicosis treatment.

Abstract

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.

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 1 month. Lung tissues were collected from all mice; a subset was subjected to transcriptomic analysis, and differentially expressed genes were identified using the limma package. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were conducted using ClusterProfiler to investigate gene functions and associated pathways. The remaining samples were subjected to histopathological assessment by hematoxylin and eosin staining (HE) and Masson’s trichrome staining, while Western blot analysis was performed to validate transcriptomic results.

This study suggests that DMSO may alleviate the fibrotic process in silicosis by modulating the IL-6/STAT3-MMP12 signaling 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, including MMP12, which 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.

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

Introduction

Silicosis is one of the major forms of occupational pneumoconiosis1, characterized by persistent pulmonary inflammation and irreversible pulmonary fibrosis resulting from prolonged exposure to silica dust^2,3. The pathogenesis of this silicosis is highly complex. Although previous studies have demonstrated that aberrant macrophage 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 remodeling 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 suggest 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 signaling 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 of MMP expression. Therefore, this study represents the investigation of the anti-inflammatory and anti-fibrotic effects of DMSO in a silicosis mouse model, aiming to reveal the key regulatory gene network and molecular mechanism of DMSO intervention and provide theoretical support for innovative MMP-based treatment strategies.

Protocol

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. Figure 1 shows the study design.

Preparation of solutions​

Preparation of silica suspension:

Crystalline silica (SiO₂, 80%, particle size 1–5 µm) was sterilized by autoclaving. The sterile silica powder was then dispersed in sterile saline to obtain a final concentration of 10 mg/mL, and the suspension was thoroughly homogenized using an ultrasonic homogenizer immediately before administration to ensure a uniform particle suspension.

Preparation of DMSO solution:

Under aseptic conditions, DMSO (≥99.9%) was diluted with sterile physiological saline to prepare an approximately 10% (v/v) DMSO solution, which was homogenized by ultrasonic agitation. Each mouse was injected with the DMSO solution at a dose of 0.9 g/kg, as previously described^14,15.

Experimental animals

Thirty-two healthy male C57BL/6J mice aged 8–12 weeks were used in this study and acclimatized for 1 week before experimentation. Mice were randomly assigned to four experimental groups (n = 8 per group) using a computer-generated randomization method: control, DMSO, silica, and silica+DMSO groups. The sample size was determined based on previous studies using the same murine silicosis model and our preliminary experimental experience, which indicated that 8 animals per group were sufficient to detect histopathological and molecular differences between groups. Animals were housed under standard laboratory conditions with controlled temperature, a 12 h light/dark cycle, and free access to food and water. Humane endpoints were established before the study, and no animals met the predefined criteria for early euthanasia.

Silicosis model establishment and drug intervention

Silicosis was induced by a single intranasal instillation of 60 µL of sterile crystalline silica suspension (10 mg/mL). Following induction of anesthesia, mice were placed in the supine position. Once deep and slow breathing was observed, 60 µL of the silica suspension was carefully instilled dropwise into the nostrils, allowing the suspension to be inhaled spontaneously into the lungs¹⁶. Mice in the DMSO administration group received intraperitoneal injections of DMSO solution at a fixed dose twice weekly, with a 3–4 day interval between injections, for one consecutive month. Mice in the remaining groups were treated identically with an equal volume of sterile saline instead. Daily body weight monitoring was performed throughout the experiment. On the 30th day after modeling, all mice were euthanized, and lung tissues were collected under sterile conditions. Euthanasia was performed by cervical dislocation following intraperitoneal administration of tribromoethanol (0.2 mL/10 g) to induce anesthesia. Partial lung tissues were preserved in liquid nitrogen for transcriptome sequencing. A portion of tissues was fixed in 4% paraformaldehyde for 48 h prior to pathological staining, and the remaining tissues were stored at -80 °C for subsequent molecular experiments. All animal operations complied with institutional animal ethics guidelines.

Histopathological evaluation, including the Ashcroft fibrosis score, was independently performed by two investigators blinded to the experimental group allocation.

Pulmonary pathological examination

Fixed lung tissues were dehydrated, transparentized, and embedded in paraffin blocks, then sliced into 5 µm-thick sections.

hematoxylin and eosin (HE) staining: Sections were stained with hematoxylin for 3–5 min, differentiated in acidic solution, blued under running water, and counterstained with eosin for 5 min. After dehydration through graded ethanol, sections were cleared in xylene and sealed with neutral resin¹⁷.

Masson’s trichrome staining: Sections were stained sequentially with hematoxylin, Lichun Red–acid fuchsin, phosphomolybdic acid, and Bright Green. After acidic differentiation, sections were dehydrated, cleared in xylene, and mounted with neutral resin^18,19.

Transcriptome sequencing

Mouse lung tissues from the four experimental groups were collected immediately after sacrifice and snap-frozen in liquid nitrogen. Total RNA was extracted using TRIzol reagent according to the manufacturer's instructions. RNA concentration and purity were assessed, and RNA integrity was evaluated using a Bioanalyzer with built-in software. Samples with RIN values greater than 8.0 were considered acceptable. Library preparation included poly(A) mRNA enrichment using Oligo(dT) beads, RNA fragmentation, cDNA synthesis, adapter ligation, and PCR amplification. Sequencing was performed on an Illumina platform, generating approximately 6.2–6.4 Gb of clean data per sample. All raw sequencing data generated in this study have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE311671.

Clean reads were aligned to the Mus musculus reference genome (mm8) using the STAR aligner. Gene expression levels were quantified as raw read counts. Differential expression analysis was performed using the limma package in R. Prior to linear modeling, raw count data were transformed using the voom function to estimate the mean-variance relationship and generate precision weights. Raw P values were adjusted for multiple testing using the Benjamini-Hochberg (BH) method, and genes with |log₂FoldChange| > 1.5 and false discovery rate (FDR) < 0.05 were considered differentially expressed. Functional enrichment analyses were performed using the clusterProfiler package in R. Gene ontology (GO) enrichment analysis was conducted to identify significantly enriched biological process (BP), cellular component (CC), and molecular function (MF) categories, whereas Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis was performed to identify significantly enriched signaling pathways. Enrichment significance was assessed using a hypergeometric test, and P values were adjusted for multiple comparisons using the Benjamini-Hochberg method. GO terms and KEGG pathways with an adjusted P value (padj) < 0.05 were considered significantly enriched^20,21,22,23.

Western blot detection

Fresh mouse lung tissue was collected and homogenized with an appropriate volume of radioimmunoprecipitation assay (RIPA) lysis buffer. After centrifugation, the supernatant was collected, and protein concentration was determined using the BCA protein assay kit. The samples were then mixed with loading buffer and heated at 100 °C for 10 min. Protein separation was carried out on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels at a current of 200 V for 30 min. Subsequently, proteins were electrotransferred to polyvinylidene fluoride (PVDF) membranes with 300 mA constant current for 30 min. The membrane was blocked with 5% skim milk, then incubated overnight at 4 °C with primary antibodies against β-Actin (1:1500), IL-6 (1:1000), STAT3 (1:800), p-STAT3 (1:1500) and MMP12 (1:800). The following day, the membrane was washed with TBST and incubated with secondary antibody at room temperature for 1 h, followed by additional washes with TBST. Protein bands were visualized using a chemiluminescent substrate, and protein quantification was performed using ImageJ software.

Western blot analysis was performed using three independent biological replicates from each experimental group. Protein samples were prepared from individual mouse lung tissues, and each biological replicate represented one independent animal.

Statistical analysis

All experimental data were analyzed using GraphPad Prism 9.5. Normality and homogeneity of variance tests were conducted beforehand. Student’s t-test was used for two-group comparison, and one-way ANOVA combined with Tukey’s post-hoc test was adopted for multi-group comparison. P ≤ 0.05 was regarded as statistically significant.

Results

DMSO alleviates silica-induced pulmonary fibrosis in mice (Figure 2)

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 1-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 HE staining (Figure 2B) and Masson’s trichrome staining (Figure 2C). Pulmonary fibrosis was scored on HE -stained sections using the Ashcroft method²⁴, revealing a lower fibrosis score in the Silica+DMSO group compared to the Silica group (Figure 2D). Masson’s staining further indicated that collagen fiber deposition in the Silica group was extensive and dense, occupying approximately 40% of the lung tissue area, which was 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.

Transcriptome analysis identifies differentially expressed genes associated with DMSO treatment in silica-exposed mice

To investigate the anti-inflammatory and anti-fibrotic effects of dimethyl sulfoxide (DMSO) in a silicosis model, transcriptomic sequencing was performed on lung tissue samples collected from experimental mice (Figure 3). DEGs were identified using the limma algorithm, with selection criteria defined as |log2(fold change)| > 1.5 and a corrected P-value (false discovery rate [FDR]) < 0.05.

Transcriptome analysis demonstrated that, compared with the control group, the Silica group exhibited 132 significantly differentially expressed genes (Figure 3A), including 117 that were upregulated, and 15 that 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.

The IL-6/STAT3/MMP12 signaling axis may be involved in the protective effects of DMSO in silica-induced pulmonary fibrosis

To investigate the relationships among differentially expressed genes (DEGs), a gene interaction network was constructed based on the transcriptomic data (Figure 4A). MMP12 appeared to occupy a central position within the network. To further examine the potential protein-level associations, a predicted protein–protein interaction (PPI) network was generated using the STRING database (Figure 4B). The STRING analysis suggested potential functional associations between MMP12 and several key DEGs, including IL-6, STAT3, SPP1, and MMP19. Because STRING integrates evidence from different sources, these associations are predicted functional relationships.

Western blot analysis was performed to quantify the protein expression levels of IL-6, STAT3, p-STAT3, and MMP12 (Figure 4C–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, and MMP12 protein expression also showed a trend toward increased expression. 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.

DATA AVAILABILITY:

All raw sequencing data generated in this study have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE311671. The raw images of the study are available in a supplementary folder (Supplementary File 1).

Figure 1: Schematic of the study design. Please click here to view a larger version of this figure.

Figure 2: DMSO alleviates pulmonary fibrosis in silicosis mice. (A) Schematic diagram of the experimental design. Arrows of different colors represent different administration regimens. (B) Hematoxylin 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 20 µm. (C) Masson staining of lung tissue (blue: collagen fibers; red: muscle fibers). At a magnification of 200×, the scale bar is 50 µm; at a magnification of 400×, the scale bar is 20 µm. (D). Fibrosis scores of different groups based on HE staining results. n = 8, P ≤ 0.0001. (E) Relative collagen area of Masson staining in the lung tissue of different groups. n = 8, P ≤ 0.0001. (F) Body weight changes of mice within 30 days. Data were 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. n = 8, P(time) ≤ 0.0001, P(group) ≤ 0.0001. N: Control group; S: Silica group; SD: Silica + DMSO group; D: DMSO group. Please click here to view a larger version of this figure.

Figure 3: Transcriptome sequencing reveals the gene regulation of DMSO in alleviating silicosis. (A,B) Volcano plot of differentially expressed genes between the Silica (S) group and the Control (N) group, the Silica + DMSO (SD) group, and the Silica (S) group. (C,D) Heatmap of the Silica (S) group compared with the Control (N) group, and the Silica + DMSO (SD) group compared with the Silica (S) group. Please click here to view a larger version of this figure.

Figure 4: 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 (*P = 0.0257, *P = 0.0195), MMP12 (**P = 0.0013, *P = 0.0318), STAT3 (P = 0.3205, P = 0.2347), and p-STAT3 (*P = 0.0123, **P = 0.0096) proteins via Western blotting. Immunoblotting analysis was performed in triplicate (n = 3). N: Control group; S: Silica group; SD: Silica + DMSO group; D: DMSO group. Please click here to view a larger version of this figure.

Supplementary File 1: Raw data of this study.Please click here to download this file.

Discussion

Silicosis remains one of the most severe occupational lung diseases worldwide, characterized by persistent inflammation and progressive pulmonary fibrosis, which substantially impair lung function and patient quality of life²⁵. In this study, using a silica-induced murine model, we systematically examine the potential protective effects of dimethyl sulfoxide (DMSO) during the early stage. The findings here suggest that DMSO treatment attenuates inflammatory cell infiltration and mitigates fibrotic pathological changes, suggesting DMSO may exert a modulatory role in the early phases of silicosis. Previous experimental studies have demonstrated that DMSO exerts protective effects against pulmonary fibrosis in both rats and mice. Haschek et al. reported that DMSO attenuated pulmonary fibrotic changes in experimental animal models, although the underlying molecular mechanisms remained unclear²⁶. Consistent with these findings, our transcriptomic and protein expression analyses further suggest that DMSO may alleviate silica-induced pulmonary fibrosis through modulation of inflammatory signaling pathways, thereby providing additional mechanistic insight into its potential anti-fibrotic activity.

DMSO, a polar aprotic solvent with high tissue permeability²⁷, 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^28,29,30. In addition, previous studies have demonstrated that DMSO attenuates pulmonary fibrosis and lung injury in experimental animal models, suggesting that DMSO possesses protective effects against fibrotic lung diseases, possibly through its anti-inflammatory and free radical-scavenging properties²⁶. At the molecular level, DMSO has been reported to suppress NF-κB and MAPK signaling pathways, leading to reduced IL-6 expression in LPS-stimulated RAW264.7 macrophages³¹. Notably, high doses of DMSO may cause systemic toxicity, with multi-organ damage reported at doses exceeding 8 g/kg¹⁴; 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 indicated a reduction in MMP12 expression following DMSO treatment. MMP12, predominantly secreted by macrophages³², 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 CYR61^33,34,35. In the present study, the observed downregulation of MMP12 suggests its potential involvement in the anti-inflammatory and anti-fibrotic effects associated with DMSO treatment. Importantly, MMP12 facilitates degradation of excessive extracellular matrix deposits during fibrosis resolution, thereby promoting tissue remodeling and scar clearance³⁶.

Macrophages are key drivers of pro-inflammatory cytokine production in silica-induced silicosis³⁷. Activated macrophages secrete IL-6 and TNF-α, which contribute to inflammation and fibrosis³⁸. 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^39,40,41,42. 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^43,44. STAT3 has been proposed as an important signaling hub linking inflammatory responses to fibrotic remodeling, and its activation may regulate MMP12, thereby enhancing its transcriptional activity⁴⁵. In addition, STAT3 signaling is involved in macrophage polarization, driving the transition of macrophages from the pro-inflammatory M1 phenotype to the pro-fibrotic M2 phenotype^46,47, while M2 macrophages represent one of the primary cellular sources of MMP12⁴⁸. Therefore, the concomitant downregulation of STAT3 and MMP12 observed in the present study suggests that DMSO may reduce MMP12 expression, at least in part, through modulation of STAT3 signaling. However, it should be noted that the present study was primarily designed to identify potential molecular mechanisms based on transcriptomic profiling combined with in vivo validation. Although the coordinated changes in IL-6, STAT3, and MMP12 support the involvement of the IL-6/STAT3/MMP12 signaling axis in the anti-fibrotic effects of DMSO, the current evidence does not establish a direct causal relationship between STAT3 activation and MMP12 regulation. Additional mechanistic studies are therefore required to further clarify the direct regulatory interactions within this signaling pathway.

This study has several limitations. First, the conclusions of this study are based on transcriptomic profiling combined with histopathological and protein expression analyses in a murine silicosis model. Although these findings support the involvement of the IL-6/STAT3/MMP12 signaling axis, they do not establish a direct causal relationship between STAT3 activation and MMP12 regulation. Second, mechanistic validation, including macrophage-based in vitro studies and rescue experiments using genetic or pharmacological manipulation of the IL-6/STAT3 pathway and MMP12, was not performed. In addition, no positive anti-fibrotic control was included, limiting direct comparison of DMSO with established therapies. Future studies incorporating mechanistic validation and clinically relevant anti-fibrotic drugs, such as pirfenidone or nintedanib, will further clarify the molecular mechanism and therapeutic potential of DMSO in silicosis.

In conclusion, the findings suggest that DMSO attenuates macrophage-mediated inflammation and fibrosis in silica-induced silicosis, possibly by modulating the IL-6/STAT3/MMP12 signaling axis. These findings provide preliminary evidence supporting the potential involvement of the IL-6/STAT3/MMP12 signaling axis in the anti-fibrotic effects of DMSO and highlight MMP12 as a potential molecular target for future investigation.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10% SDS-PAGE	Servicebio	G2177
BCA Protein Assay Kit	Servicebio	G2026
C57BL/6J mice	Changzhou Cavens Laboratory Animal Co., Ltd.	No: SCXY(Su)2011-0003
Crystalline silica	Sigma Aldrich	No.S5631
DMSO	Servicebio	GC203006
IL-6	Affinity Biosciences	DF6087
loading buffer	Bioteke	P0015L
MMP12	Servicebio	GB115475
p-STAT3	Servicebio	GB150001
RIPA lysis buffer	Servicebio	G2002
Secondary antibody	Servicebio	GB23303
skim milk	Servicebio	GC310001
STAT3	Servicebio	GB11176
β-Actin	Servicebio	GB11001