Surfactant protein A deficiency aggravates silica-induced pulmonary fibrosis by promoting intrinsic apoptosis of alveolar type II epithelial cells

Abstract

Background

Silicosis is an incurable occupational lung disease characterized by progressive fibrosis and respiratory failure, imposing a significant global health burden. Surfactant protein A (SP-A) plays a critical role in maintaining pulmonary homeostasis, yet its mechanistic role in silicosis remains unclear.

Methods

SP-A expression was assessed in lung tissues from patients with silicosis and in silica-exposed mice. Sftpa1 gene knockout (Sftpa1^-/-) mice were generated to evaluate the functional role of SP-A in vivo, including lung pathology, collagen deposition, and pulmonary function. RNA sequencing was performed to uncover underlying molecular mechanisms. A549 cells with SP-A silenced by siRNA were employed for in vitro experiments.

Results

SP-A levels were notably reduced in the lung tissue of silicosis patients and in experimental silicosis mice, correlating inversely with disease severity. Sftpa1^⁻/⁻ mice showed markedly exacerbated silica-induced pulmonary fibrosis, extracellular matrix deposition, and functional decline. RNA-seq analysis highlighted activation of intrinsic apoptosis pathways related to pulmonary fibrosis. Mechanistically, SP-A deficiency disrupted the balance of Bcl-2 and Bax, activated Caspase-3, and promoted epithelial apoptosis. Inhibition of the intrinsic apoptosis pathway mitigated the pro-apoptotic effects of SP-A silencing.

Conclusions

These findings demonstrate that SP-A deficiency exacerbates silica-induced pulmonary fibrosis by promoting epithelial apoptosis involving the Bcl-2/Bax/Caspase pathway, highlighting the role of SP-A in fibrogenesis progression and providing a basis for its potential therapeutic target for silicosis.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12931-025-03478-1.

Background

Silicosis is a common and ultimately fatal occupational disease caused by prolonged inhalation of high concentrations of free crystalline silica generated in industries including mining, quarrying, sandblasting, and construction [1, 2]. Despite advancements in workplace safety measures and personal protective equipment, silicosis remains a significant global health challenge, particularly in developing nations. Epidemiological data indicate a 64.6% increase in incident cases and a 91.4% rise in prevalent cases worldwide from 1990 to 2019 [3]. In 2019 alone, silicosis accounted for 6.557 million disability-adjusted life years (DALYs) worldwide [4]. At present, silicosis remains an incurable occupational lung disease, and lung transplant is the only therapeutic choice for patients at the end-stage of respiratory collapse. This situation highlights the need to clarify the molecular processes that drive silicosis development and pinpoint new therapeutic objectives.

The pathogenesis of silicosis involves complex and interrelated biological processes, including inflammation, immune response, programmed cell death, and tissue remodeling. After inhalation, silica particles are phagocytosed by alveolar macrophages, culminating in the secretion of pro-fibrotic mediators, most notably transforming growth factor-beta (TGF-β). As a central regulator, TGF-β orchestrates fibroblast activation and excessively promotes extracellular matrix (ECM) deposition [5, 6]. Alveolar type II epithelial cells (AEC II) are essential for maintaining alveolar structure and regeneration. Emerging evidence suggests that injury or dysfunction of AEC II contributes significantly to fibrosis in silicosis [7]. Moreover, silica exposure directly induces apoptosis in alveolar epithelial cells, further aggravating lung damage [8, 9]. However, the specific molecular mechanisms mediating silica-induced AEC II apoptosis remain poorly understood.

Among key molecules produced by AEC II, SP-A plays dual roles in pulmonary surfactant regulation and innate immune defense. As a member of the collectin family of C-type lectins, SP-A reduces alveolar surface tension by stabilizing surfactant phospholipids at the air-liquid interface [10, 11]. A decline in SP-A disrupts alveolar stability and may lead to alveolar collapse [12]. Beyond these biophysical functions, accumulating evidence implicates SP-A in the pathogenesis of various lung diseases [13], including idiopathic pulmonary fibrosis associated with SFTPA1 mutations [14, 15]. In experimental silicosis models, SP-A expression is significantly decreased in lung tissues [16]. Additionally, administering exogenous SP-A has been shown to reduce silica-induced rat macrophage cytotoxicity [17]. These findings suggest a potential protective role for SP-A in silicosis. However, most studies have focused on altered SP-A expression or activity rather than its complete loss. Consequently, the mechanistic impact of SP-A deficiency on silica-induced epithelial injury and fibrogenesis remains largely unknown.

Our research investigated the functional involvement of SP-A in silicosis pathogenesis, focusing on its effect on AEC II apoptosis and fibrotic progression. Findings revealed a significant decrease in SP-A levels in both silicosis patients and silica-exposed rodents, correlating with disease severity. SP-A knockout exacerbated silica-induced pulmonary fibrosis and AEC II apoptosis in vivo. In vitro, SP-A silencing exacerbated silica-induced apoptosis in AEC II by altering the Bcl-2/Bax/Caspase-3 pathway. These results reveal a novel protective role of SP-A in silica-induced lung fibrosis and highlight its potential as a therapeutic target for silicosis.

Materials and methods

Clinical samples

Paraffin-embedded human lung tissue samples were obtained post-mortem from forensic cases handled by the School of Forensic Medicine, Southern Medical University. The collection included specimens from five healthy individuals and five patients with stage III silicosis, all acquired with informed consent from the donors’ immediate family members. The research protocol received ethical clearance from the Medical Ethics Committee of Guangdong Province Hospital for Occupational Disease Prevention and Treatment (No. GDHOD MEC 2010026).

Animals and treatments

Male wild-type (WT) C57BL/6 mice (6–8 weeks old) were obtained from the Guangdong Medical Laboratory Animal Center, and Sftpa1 gene knockout (Sftpa1^-/-) mice were generated through CRISPR/Cas-mediated genomic engineering by Cyagen Biosciences Inc. (Cyagen, China). All animals were procured with a certificate of qualification (NO. 44007200079719; NO. 44826100000578). All mice were housed under specific pathogen-free (SPF) conditions with ad libitum access to standard laboratory chow and filtered water throughout the study period. Before initiation of any animal experiments, full ethical clearance was obtained from the accredited Experimental Animal Ethics Committee of the Guangdong Province Hospital for Occupational Disease Prevention and Treatment (No. GDHOD MEC 2021003).

Crystalline silicon dioxide (SiO₂) particles with a purity exceeding 99% and an average diameter of 0.5–10 μm (Sigma-Aldrich, USA) were used in this study. Before use, the particles were thoroughly ground, autoclaved for sterilization, and dried. A suspension was then meticulously mixed to 250 mg/mL in sterile saline, followed by ultrasonic agitation to ensure homogeneous dispersion. WT and Sftpa1^-/- mice were randomly allocated into four experimental groups: saline WT, saline Sftpa1^-/-, silica WT, and silica Sftpa1^-/-. On the first day, the silica groups received an intratracheal instillation of 20 μL silica suspension (250 mg/mL) based on our preliminary studies [18, 19], while the saline groups received 20 μL of saline. Ten mice per group were sacrificed at 14 (14D), 28 (28D), and 56 (56D) days post-exposure. In each group, five mice were designated for lung function measurements, after which blood and lung tissue were collected, and the remaining five were used for bronchoalveolar lavage fluid (BALF) collection.

BALF collection and enzyme-linked immunosorbent assay (ELISA)

BALF was collected by performing three lavages with 500 μL sterile saline each. Cellular elements were pelleted by centrifugation (3000 ×g, 10 min). Following the manufacturer's protocol, SP-A concentration in the clarified BALF was measured using a mouse SP-A ELISA kit (Huamei, China). Briefly, 100 µl of the sample was dispensed into an anti-SP-A pre-coated ELISA plate and incubated at 37℃. After removing unbound fluid, biotinylated detection antibodies were added and incubated, followed by horseradish peroxidase (HRP)-conjugated streptavidin, and substrate solution was added. Reactions were terminated with 50 μL stop solution, and absorbance (OD450) was immediately measured using a Multiskan GO plate reader (Thermo Fisher Scientific, USA).

Pulmonary function

Before respiratory mechanics testing, mice received pentobarbital sodium anesthesia. Lung mechanics were evaluated using a forced pulmonary test system (Buxco Research Systems, USA) following standardized protocols provided by the manufacturer and validated research method [20]. Following trachea exposure, a cannula was inserted and sutured to prevent air leakage. The instrument was checked for airtightness and calibrated before setting the parameters: both positive and negative pressure at 50 cm H2O, with an inspiration of 0.8 ml/sec and a slow expiration rate of 0.5 ml/sec. Each mouse underwent three consecutive measurements per test to assess inspiratory capacity (IC), vital capacity (VC), forced vital capacity (FVC), forced expiratory volume at x seconds (FEVx), as well as dynamic (Cdyn) and static (Cst) pulmonary compliance parameters.

RNA sequencing analysis

Total RNA extraction from mouse lung samples was conducted with the TRIzol Reagent (Invitrogen, USA). The concentration and purity of the extracted RNA were assessed spectrophotometrically using a NanoDrop instrument (Thermo Scientific, USA). Complementary DNA (cDNA) synthesis was performed in two sequential steps: (1) first-strand generation employing a commercial reverse transcriptase system, followed by (2) second-strand synthesis incorporating end-repair and poly-A tailing modifications. Following adapter ligation, size selection was conducted with Hieff NGS® DNA Selection Beads. PCR amplification was then employed for library construction prior to high-throughput sequencing on the Illumina Novaseq X Plus platform. Detailed experimental protocols and data analysis procedures were provided by GENE DENOVO (Guangzhou, China). Raw sequencing data underwent quality control processing through fastp (version 0.18.0), followed by alignment to the mouse reference genome GRCm39 (Ensembl database version: Ensembl 113, species: Mus musculus) using HISAT2 (version 2.2.0). DESeq2 (v1.20.0) identified differentially expressed genes, applying thresholds of |log₂FC| > 1.5 and a false discovery rate (FDR) < 0.05. Differentially expressed genes (DEGs) were then matched to the Gene Ontology (GO) database to classify their functions, and significantly enriched GO terms were identified using a threshold of P ≤ 0.05. Functional pathway enrichment analysis of differentially expressed genes was performed through the Kyoto Encyclopedia of Genes and Genomes (KEGG) database using the Omicshare platform, with a significance threshold set at P < 0.05.

Histological analysis

Lung samples were collected immediately after sacrifice, fixed in 4% paraformaldehyde (Biosharp, China), and processed for paraffin sectioning. To assess any pathological changes and the degree of lung fibrosis, we performed hematoxylin and eosin (H&E) staining and Masson’s trichrome staining. Specifically, H&E staining involved incubating sections in hematoxylin (Sigma, Germany) for 10 minutes, followed by eosin (Guangfu, Tianjin, China) for 3 minutes. Masson’s trichrome staining was performed using a commercial kit (MXB Biotech, Fujian, China), strictly following the manufacturer’s instructions. H&E-stained sections were evaluated for general pathological changes and fibrosis severity, with pathology scores assigned based on the method detailed by Li et al. [21] and fibrosis quantified using the Ashcroft scoring system [22] (Tables S1 and S2). The collagen volume fraction (%) was determined using Image-Pro Plus 6.0 software [23].

Immunohistochemistry (IHC) was performed to evaluate the expression of SP-A in lung samples. Briefly, after deparaffinization and antigen retrieval, endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 minutes. Sections were then incubated with a primary anti-SP-A antibody at 37 °C for 1 hour, followed by incubation with the corresponding secondary antibody at 37 °C for 30 minutes. Detection was achieved using DAB chromogen for 3 minutes at ambient temperature. Finally, slides were counterstained with hematoxylin, dehydrated, and sealed with coverslips. All images were acquired using the Motic digital pathology scanning system (EasyScan, Motic, China). For each group, five randomly selected high-power fields per sample were analyzed. Staining intensity was quantified using the H-score method: (3 × % strongly stained cells) + (2 × % moderately stained cells) + (1 × % weakly stained cells) [24].

Hydroxyproline (HYP) assessment

The HYP concentration was calculated according to the manufacturer's protocol (Jiancheng, Nanjing, China) [25]. Mouse lung tissue samples (50 mg) were homogenized in 1 mL of hydrolysis solution and incubated in a boiling water bath for 20 minutes to ensure complete hydrolysis. Following cooling to room temperature, the pH of the digest was adjusted to 6–8 and decolorized with activated charcoal. The mixture was then centrifuged at 3500 rpm for 10 minutes. A 200 μL aliquot of the supernatant was transferred to a 96-well plate, and the absorbance was measured at 550 nm.

Terminal dUTP Nick End Labeling (TUNEL) immunofluorescence label

Following deparaffinization and antigen retrieval, lung sections were permeabilized using 10% Triton X-100. TUNEL staining was performed using a TMR (Red) TUNEL Cell Apoptosis Detection Kit (Servicebio, China) according to the manufacturer’s protocol. Briefly, sections were incubated with a reaction mixture containing TdT enzyme, TMR-5-dUTP, and equilibration buffer (1:5:50) for 1 hour at 37 °C in the dark. Immunostaining was conducted with rabbit anti-SP-C primary antibody and Alexa Fluor® 488-conjugated secondary antibody. Nuclei were counterstained with DAPI-containing mounting medium (Solarbio, China) and samples were preserved at 4°C. Fluorescence imaging was performed using an Olympus BX61 microscope (Olympus, Japan) and a Zeiss LSM 980 system (Zeiss, Germany), with quantitative analysis of positive cells conducted using Image-Pro Plus 6.0 [26].

Cell culture

The human alveolar epithelial cell line A549 (American Type Culture Collection source) was maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in 5% CO₂, using standard 25 cm² tissue culture flasks for propagation.

Cell viability assay

Cell viability assessment was performed using the Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories, Japan) following SiO₂ exposure. A549 cells were plated in 96-well plates (5 × 10³ cells/well, in triplicate) and allowed to adhere for 24 hours. Cells were treated with SiO₂ at varying concentrations (0, 1.0, 2.5, 5.0, 7.5, 10, 15.0, 20.0, and 30.0 μg/cm2) for 12, 24, and 48 hours. After incubation with CCK-8 reagent (37°C, 1 h), absorbance at 450 nm was measured using a Multiskan GO microplate reader (Thermo Scientific, USA). Results were normalized to untreated controls (100% viability).

Cell transfection

SP-A expression in A549 cells was knocked down using a small interfering RNA (siRNA) transfection kit (Santa Cruz, USA) strictly following the manufacturer's protocols. SP-A siRNA (h) pool consisted of three distinct siRNA duplexes (sequences provided in Table S3). Briefly, when the cell density in the 6-well plate reached 70–80%, the transfection reagent was mixed with the transfection medium and then added to the cells. After 2 hours of incubation in the cell incubator, an equivalent amount of DMEM supplemented with 20% FBS was introduced. After 24 hours of additional culture, silencing efficiency was validated by western blotting and RT-qPCR analysis.

Flow cytometry analysis

For apoptosis analysis, cells were trypsinized and collected after treatment with 10 μg/cm2 SiO₂ for 12, 24, and 48 hours. Next, 5 μL of Annexin V and 2.5 μL of PI fluorescent dye (Multi Science, China) were added to the cell suspensions. After thorough mixing and 15-minute dark incubation at room temperature. Samples were analyzed using a DxFLEX flow cytometer (Beckman Coulter, USA) with CytExpert software for data acquisition and processing.

Real-time quantitative PCR (RT-qPCR)

Total RNA isolation from pulmonary tissues and A549 cell cultures was performed with TRIzol Reagent (Invitrogen, USA). cDNA was then synthesized according to the manufacturer's protocol for the One-Step RT kit (TransGen Biotech, China) using a thermal cycler (Analytik Jena, Germany). Quantitative PCR analysis was performed on a LightCycler®96 instrument (Roche, Germany) with PerfectStart Green qPCR Super Mix (TransGen Biotech, China). Relative gene expression was computed through the 2^-∆∆Ct approach, normalizing the gene expression level to β-actin in A549 cells and GAPDH in lung tissue samples. Primer sequences were listed in Table S4.

Western blotting assay

Protein extraction was conducted from lung tissue and A549 cell cultures using a mammalian protein extraction kit (KeyGEN, China). Protein quantification was achieved through bicinchoninic acid assay with a BCA Protein Assay Kit (Solarbio, China). For immunoblotting analysis, aliquots containing 20–40 μg protein lysates were resolved by discontinuous SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (8% or 12% gels) and subsequently transferred onto PVDF membranes (Millipore, USA). Following a 2-hour blocking with 5% skim milk (Biofroxx, Germany), membranes were incubated with specific primary antibodies (4°C, 16 hours) and corresponding HRP-conjugated secondary antibodies (room temperature, 90 min). Signal detection employed an enhanced chemiluminescence detection kit (KeyGEN, China), with band intensity quantification performed using ImageJ software. The antibodies were detailed in Table S5.

Statistical analysis

All statistical analyses were conducted utilizing R statistical software (v4.2.1) and GraphPad Prism (v8.0). Quantitative data are expressed as mean ± standard deviation (SD). Comparisons between two groups were conducted using Student's t-test, while comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc multiple comparisons test. A two-tailed P <0.05 was considered statistically significant.

Result

Silica exposure reduces SP-A levels in the lungs of silicosis patients and experimental silicosis mice

To investigate the expression of SP-A in silicosis, immunohistochemical staining was performed on lung tissues from five patients with confirmed silicosis and five healthy controls. Compared to the healthy group, SP-A positive cells along the alveolar wall were significantly diminished in the silicosis group, with brownish-yellow granules primarily observed in AEC II and some macrophages (Fig. 1A and B). Notably, SP-A–positive macrophages likely reflect ligand binding and internalization rather than endogenous production [27–29]. Similarly, mice exposed to silica for 14, 28, or 56 days exhibited a progressive decline in SP-A expression, primarily in AEC II along the alveolar wall, with weak SP-A signals present in terminal bronchiolar Clara cells (Fig. 1C and D). Consistently, SP-A levels in BALF also decreased over time following silica exposure (Fig. 1E). Western blot analysis confirmed the downregulation of SP-A protein levels (Fig. 1F), which was corroborated by decreased Sftpa1 mRNA levels as quantified by real-time PCR in silica-treated murine lungs (Fig. 1G). Collectively, our findings demonstrate that silica-induced pulmonary injury leads to marked downregulation of SP-A expression across species, as observed in both clinical specimens and experimental animal models of silicosis.

Fig. 1.

SP-A levels in the lungs of silicosis patients and experimental silicosis mice. A Representative SP-A immunohistochemistry in lung tissues from healthy human controls and silicosis patients (arrows: positive cells). B Quantification of SP-A IHC scores (n = 5 cases per group). C Representative SP-A immunohistochemistry in saline-/silica-treated WT mice lung tissues (arrows: positive cells). D Quantification of SP-A IHC scores (n = 5 mice per group). E Quantification of SP-A levels in BALF from saline- and silica-treated WT mice (n = 5 mice per group). F Western blot analysis of lung SP-A expression with quantification (n = 5 mice per group). G Relative mRNA expression of Sftpa1 (n = 5 mice per group)

Sftpa1-/- mice exhibit increased susceptibility to silica-induced lung injury

Given the significant downregulation of SP-A in both human and experimental silicosis models, we hypothesized that SP-A may confer protective effects during silicosis progression. SP-A is a secreted protein with a complex oligomeric structure of approximately 630 kDa [30], enabling multivalent binding to diverse ligands and mediating various physiological functions [31, 32]. However, recombinant SP-A proteins expressed in vitro or in vivo often display impaired oligomerization and diminished biological activity [33, 34]. To elucidate the mechanistic contributions of SP-A in silicosis pathogenesis, we conducted a reverse validation experiment using Sftpa1⁻/⁻ knockout mice. Genotyping and Western blotting confirmed successful knockout of Sftpa1 in mouse lungs (Fig. S1A and B). Under saline treatment, neither Sftpa1⁻/⁻ nor WT mice exhibited mortality or lung pathology throughout the study period (Table S6 and Fig. S2). Following baseline assessment, WT and Sftpa1⁻/⁻ mice were humanely euthanized at predetermined time points (14, 28, and 56 days) following silica exposure (Fig. 2A).

Fig. 2.

Silica-induced pulmonary histopathology and fibrosis in Sftpa1^-/- mice. WT and Sftpa1^-/- mice were exposed to saline or silica for 14, 28, and 56 days (n = 5 mice per group). A Schematic diagram illustrating in vivo administration of silica particles or saline. B H&E staining of lung sections. C Histological scoring of H&E-stained section. D Masson’s trichrome staining of lung sections. E Quantification of collagen volume fraction. F Hydroxyproline contents in lung tissues. G Western blot analysis of α-SMA, Collagen I, and Fibronectin. H Corresponding quantification of protein expression levels. I Relative mRNA expression of Acta2, Col1a1, and Fn1.

H&E staining revealed that silica-exposed Sftpa1^-/- mice developed more severe alveolar damage, thickened alveolar septa, and significantly higher histological scores compared to WT mice (Fig. 2B and C). Masson’s staining and quantification of collagen volume fraction further demonstrated markedly increased collagen deposition in Sftpa1^-/- mice, as indicated by more intense blue staining (Fig. 2D and E) and elevated hydroxyproline levels (Fig. 2F). Fibrotic pathway activation was also significantly enhanced in Sftpa1^-/- mice following silica exposure, as demonstrated by coordinated elevation of α-SMA, collagen I, and fibronectin expression, evident through both protein quantification and mRNA analysis when compared to WT mice (Fig. 2G-I). These results suggest that SP-A deficiency aggravates silica-induced pathological remodeling and promotes pulmonary fibrosis.

Deficiency of SP-A accelerates pulmonary function decline and increases alveolar surface tension in experimental silicosis mice

As the most prevalent protein in pulmonary surfactant, SP-A is essential for alveolar stability and lung function [11, 35]. To investigate the effects of SP-A deficiency on pulmonary function following silica exposure, pulmonary function assessments were performed using the Buxco forced vital capacity system (Fig. 3A). Compared to WT mice, Sftpa1⁻/⁻ mice exhibited significantly reduced IC, VC, and FVC, indicative of more severe ventilatory impairment (Fig. 3B). FEV₀.₁ and FEV₀.₂, the murine equivalents of human FEV₁ and FEV₂, were also markedly reduced in Sftpa1⁻/⁻ mice. Notably, the FEV₀.₁/FVC ratio was elevated at day 56, suggesting a greater degree of obstructive ventilatory dysfunction associated with SP-A deficiency (Fig. 3C). Furthermore, both Cdyn and Cst, parameters reflective of lung elasticity and alveolar surface tension, were significantly reduced in Sftpa1⁻/⁻ mice compared to WT controls (Fig. 3D). Taken together, these results indicate that loss of SP-A accelerates silica-induced pulmonary dysfunction, as evidenced by impaired ventilatory mechanics and increased alveolar surface tension.

Fig. 3.

Pulmonary function and alveolar surface tension in Sftpa1^-/- mice following silica exposure. WT and Sftpa1^-/- mice were exposed to silica particles for 14, 28, and 56 days (n = 5 mice per group). A Pulmonary function test schematic. B Pulmonary function parameters measured: IC, VC, FVC, (C) FEV_0.1, FEV_0.1/FVC, and FEV_0.2. D Alveolar surface tension indices: Cdyn and Cst.

SP-A deficiency exacerbates pulmonary cell apoptosis in experimental silicosis mice

To elucidate the molecular mechanisms underlying SP-A’s protective role, transcriptomic analysis was performed on lung tissues collected at days 28 and 56 post-exposure, based on the more pronounced pathology observed at these time points. Principal component analysis (PCA) (Fig. S3A), DEGs bar charts (Fig. S3B), and heatmaps (Fig. S3C) indicated distinct transcriptomic profiles between silica-exposed WT and Sftpa1^-/- mice. A total of 822 DEGs were consistently identified at both time points (Fig. 4A). GO enrichment classification of these DEGs demonstrated predominant enrichment in the "biological process" category, particularly "cellular process", which contained the highest gene count (n=720) (Fig. 4B). KEGG pathway enrichment highlighted apoptosis as a significantly involved pathway (Fig. 4C). TUNEL staining showed no significant apoptosis under saline treatment between groups (Fig. S3D). However, silica-exposed Sftpa1⁻/⁻ mice demonstrated substantially increased apoptotic cell counts compared to WT mice at both experimental timepoints (Fig. 4D and E). This suggests that SP-A deficiency promotes apoptosis and may contribute to silicosis progression.

Fig. 4.

Transcriptomic and apoptosis analysis of Sftpa1^-/- mouse lungs after silica exposure. WT and Sftpa1^-/- mice were exposed to silica particles. A Venn diagram illustrating the number of DEGs (n = 3 mice per group). B Top enriched GO terms. (n = 3 mice per group). C Significant KEGG pathways (n = 3 mice per group). D Representative TUNEL staining of lung tissues, nuclei counterstained with DAPI. E Quantification of TUNEL fluorescence intensity per field (n = 5 mice per group).

SP-A deficiency enhances intrinsic apoptotic pathway activation in the lungs of experimental silicosis mice

To further delineate apoptotic pathways involved, transcriptomic data were analyzed for apoptosis-related genes. Heatmap revealed pronounced changes in genes associated with the intrinsic pathway, with only one gene showing differential expression in the extrinsic pathway (Fig. 5A). These observations suggest that SP-A may regulate the silicosis progression through modulation of the intrinsic apoptosis pathway. We then assessed the expression of key molecules in the canonical intrinsic apoptosis pathway, which includes Bcl-2, Bax, caspase-3, and caspase-9. In silica-exposed Sftpa1⁻/⁻ mice, expression levels of Bcl2 (anti-apoptotic) were significantly reduced at both transcriptional and protein levels, while Bax, Casp9, and Casp3 (pro-apoptotic) were markedly upregulated (Fig. 5B and C).

Fig. 5.

SP-A deficiency promotes intrinsic apoptotic pathway activation after silica exposure. WT and Sftpa1^-/- mice were treated with saline or silica. A Apoptosis-related gene clustering (n = 3 mice per group). B Relative mRNA expression of Bax, Bcl2, Casp9, and Casp3 (n = 5 mice per group). C Western blot analysis of Bax, Bcl-2, Caspase-9, and Caspase-3with quantification (n = 5 mice per group). D Representative immunofluorescence images showing SP-C and TUNEL co-staining in Mouse lung tissues and quantification of TUNEL fluorescence intensity per field in AEC II (n = 5 mice per group). Nuclei were counterstained with DAPI.

Given that SP-A is predominantly secreted by AEC II [36], we hypothesized that its deficiency affects AEC II apoptosis. Immunofluorescence demonstrated co-localization of TUNEL-positive signals with SP-C, a specific marker of AEC II, indicating increased apoptosis in this cell type in Sftpa1⁻/⁻ mice (Fig. 5D). Collectively, these findings demonstrate that SP-A deficiency promotes activation of the intrinsic apoptotic pathway via the Bcl-2/Bax/Caspase-3 signaling axis.

Silica exposure reduces SP-A synthesis and activates intrinsic apoptosis in vitro

In selecting an AEC II model for our experiments, we prioritized A549 cells owing to their endogenous SP-A production and extensive literature supporting their use as type II pneumocyte equivalents [37, 38]. CCK-8 assays revealed that silica exposure (1–30 μg/cm²) induced a dose- and duration-related reduction in A549 cell viability (Fig. 6A). Based on these results, 10 μg/cm2 was chosen for further analysis. At this dose, SiO₂ treatment reduced SP-A protein and SFTPA1 mRNA levels in a time-dependent manner (Fig. 6B-D). Meanwhile, fibrotic markers were upregulated, confirmed by Western blot and qPCR analyses (Fig. 6E-G). Flow cytometry revealed increased apoptosis over time (Fig. 6H), accompanied by elevated expression of intrinsic apoptotic markers at both protein and mRNA levels (F 6g. 6I and 6).

Fig. 6.

SP-A expression and intrinsic apoptotic pathway activation in silica-exposed A549 cells. A Cell viability of A549 cells exposed to varying silica doses for 12, 24, and 48 hours, assessed by CCK8 assay. "a" indicates significant differences in cell viability at all three time points. B and C Western blot analysis of SP-A expression with quantification. D SFTPA1 mRNA levels following 10 μg/cm² SiO₂ exposure for 12, 24, and 48 hours. E and F Western blot analysis of α-SMA, Collagen I, and Fibronectin with quantification. G Relative mRNA expression of ACTA2, COL1A1, and FN1. H Flow cytometry analysis of Annexin V/PI staining after 10 μg/cm² SiO₂ exposure for 12, 24, and 48 hours. I Western blot analysis of Bax, Bcl-2, Caspase-9, and Caspase-3 with quantification. J Relative mRNA expression of BAX, BCL2, CASP9, and CASP3. n = 3 independent cell experiments for panels (A, C, F, and I). n = 5 independent cell experiments for panels (D, G, H, and J).

SP-A regulates AEC II apoptosis via the Bcl-2/Bax/Caspase-3 signaling pathway

To explore SP-A’s regulatory role in apoptosis, SFTPA1 expression was silenced using siRNA. Knockdown efficiency ranged between 60–80% based on RT-qPCR and Western blot analyses (Fig. 7A). A 24-hour SiO₂ treatment was selected as the optimal time point for mechanistic studies. Flow cytometry revealed enhanced silica-induced apoptosis following SP-A knockdown (Fig. 7B and C). Concomitant alterations in apoptosis-related molecules were observed, characterized by downregulation of the anti-apoptotic BCL2 alongside upregulation of pro-apoptotic mediators (BAX, CASP9, and CASP3), evident at both translational and transcriptional levels (Fig. 7D-F).

Fig. 7.

SP-A regulates A549 cell apoptosis via the Bcl-2/Bax/Caspase-3 signaling pathway. A SP-A expression at protein (n = 3 independent experiments) and mRNA (n = 5 independent experiments) levels after siRNA treatment in A549 cells. B and C Flow cytometry analysis of Annexin V/PI staining in A549 cells following SP-A silencing, with quantification of apoptosis (n = 5 independent experiments). D and E Western blot of Bax, Bcl-2, Caspase-9, and caspase-3 expression in A549 cells with quantification (n = 3 independent experiments). F Relative mRNA expression of BAX, BCL2, CASP9, and CASP3 in A549 cells (n = 5 independent experiments). G Flow cytometry analysis of Annexin V/PI staining following SP-A silencing, with or without 20 μM Q-VD-OPh and 10 μg/cm² SiO₂ treatment (n = 5 independent experiments)

To determine whether SP-A modulates apoptosis via the intrinsic pathway, the broad-spectrum caspase inhibitor Q-VD-Oph was used [39, 40], and the concentration used in this study (20 μM) has been demonstrated to be non-cytotoxic to A549 cells [41, 42]. The pro-apoptotic effects of SP-A-silencing were diminished by Q-VD-Oph treatment (Fig. 7G). These findings support the conclusion that SP-A regulates AEC II apoptosis through modulation of the intrinsic apoptotic pathway through Bcl-2/Bax/Caspase-3 signaling.

Discussion

As a critical regulator of pulmonary homeostasis, SP-A has been implicated in diverse respiratory disorders [16, 17]. However, its specific role in silicosis pathogenesis has remained unclear. This study is, to the best of our knowledge, the first to integrate human lung tissues, genetically engineered mouse models, and mechanistic in vitro assays to demonstrate that SP-A deficiency accelerates AEC II apoptosis and fibrotic progression following silica exposure.

Accumulating evidence has demonstrated a significant inverse association between decreased SP-A levels and disease severity in various pulmonary fibrosis conditions [13, 43, 44]. Most past research endeavors have focused on serum SP-A concentrations as diagnostic or prognostic biomarkers [13, 45], while few have investigated SP-A expression in lung tissue, particularly in human samples. Existing tissue-level data are largely derived from animal models. For instance, Liu et al. [16] observed significant SP-A downregulation in a rat model of silicosis, and Guan et al. [46] observed a marked reduction in SP-A–positive cells following prolonged silica exposure. Consistent with these experimental findings, our histological analysis revealed significantly lower SP-A expression in the lungs of silicosis patients compared to healthy controls. This trend was recapitulated in a murine model, wherein SP-A levels declined progressively following silica instillation. Complementary in vitro experiments further showed that AEC II secreted decreasing amounts of SP-A with increasing silica exposure, reinforcing the notion that silica disrupts SP-A homeostasis at both tissue and cellular levels.

SP-A deficiency has been previously linked to impaired surfactant structure and heightened susceptibility to pulmonary inflammation and fibrosis [47, 48]. Beyond its structural role in surfactant regulation, SP-A modulates innate immunity responses and protects against immune-mediated pulmonary injury [49, 50]. Mutations in SFTPA1 and SFTPA2 genes impair protein folding and secretion, leading to intracellular aggregation, endoplasmic reticulum stress, and heightened susceptibility to pulmonary fibrosis [14, 15]. However, the contribution of SP-A deficiency to silicosis has not been directly studied. Here, we established a silicosis model using Sftpa1⁻/⁻ mice and found that SP-A deletion significantly exacerbated silica-induced lung fibrosis, pulmonary dysfunction, and AEC II apoptosis. Similarly, silencing SP-A in A549 cells in vitro promoted apoptosis, supporting a protective role for SP-A against silica-induced epithelial injury. While our global Sftpa1 knockout model cannot fully distinguish epithelial-autonomous effects from those mediated by macrophages or other immune cells, future studies employing AEC II-specific conditional knockout mice could clarify the cell-type-specific role of SP-A in silicosis.

Transcriptomic analysis and subsequent validation revealed that SP-A deficiency exacerbated silica-induced intrinsic apoptosis. Silica alters the Bax/Bcl-2 equilibrium, enhancing mitochondrial membrane permeability and subsequent cytochrome c efflux, a pivotal step in apoptosis initiation. This, in turn, facilitates apoptosome formation via caspase-9 and Apaf-1, ultimately activating caspase-3 to execute apoptosis [51, 52]. Our findings indicate that SP-A deficiency amplifies this intrinsic apoptotic cascade. Notably, similar pro-apoptotic effects of SP-A deficiency have been reported in other epithelial cell types, including intestinal and renal tubular cells [53, 54]. SP-A appears to regulate apoptosis both through direct interactions with target cells and via receptor-mediated signaling (e.g., SP-R210, SIRPα, and TLRs), with the relative contribution depending on cellular context and SP-A oligomeric form [11, 55, 56]. Treatment with the pan-caspase inhibitor Q-VD-OPh abolished SP-A silencing–induced apoptosis, confirming that the observed effects are largely mediated through caspase-dependent intrinsic apoptosis pathway. Nevertheless, the pan-caspase inhibitor may affect multiple caspase-mediated processes, and other regulated cell death modalities, such as necroptosis, pyroptosis, and ferroptosis, could also contribute to silica-induced injury.

Despite these insights, several limitations should be acknowledged. First, exogenous SP-A administration would provide the most direct evidence of its protective effects, but functional SP-A exists as high-order oligomers (~630–650 kDa) purified from human BALF [57, 58], whereas recombinant human SP-A (~26–36 kDa) lacks proper oligomerization and exhibits reduced or incomplete biological activity [59, 60]. Future studies employing structurally intact SP-A are essential to establish therapeutic potential in vivo. Second, while our data implicate the Bax/Bcl-2/caspase-3 axis, the precise mechanisms by which SP-A regulates apoptosis remain to be elucidated, including potential roles of mitochondrial dysfunction, receptor-mediated signaling, or other regulated cell death pathways. The pathway we describe likely represents one facet of a broader and more complex pathophysiology. Third, although A549 cells were used as an in vitro model of AEC II due to their endogenous SP-A expression, their carcinoma origin limits physiological relevance. Further studies using primary human AEC II are warranted to validate these findings under more physiologically relevant conditions.

Conclusion

In summary, we demonstrate that SP-A expression is consistently reduced in silicosis patients, experimental silicosis mice, and epithelial cell models, correlating with disease severity. SP-A deficiency aggravates silica-induced pulmonary fibrosis, partly through enhanced epithelial apoptosis involving the Bax/Bcl-2/Caspase-3 pathway. Beyond suggesting a novel disease mechanism, our findings highlight SP-A contributes to disease development and provide a foundation for future work, including cell-type-specific validation and functional rescue studies, to more rigorously assess the therapeutic relevance of SP-A in silicosis and related fibrotic lung diseases.

Abbreviations

SP-A

Surfactant protein A

AEC II

Alveolar type II epithelial cells

Sftpa1^-/-

Sftpa1 gene knockout

DALYs

Disability-adjusted life years

TGF-β

Transforming growth factor-beta

ECM

Excessively promotes extracellular matrix

WT

Wild-type

SPF

Specific pathogen-free

HYP

Hydroxyproline

DMEM

Dulbecco's Modified Eagle Medium

FBS

Fetal bovine serum

CCK-8

Cell Counting Kit-8

SiO₂

Crystalline silicon dioxide

BALF

Bronchoalveolar lavage fluid

ELISA

Enzyme-linked immunosorbent assay

IC

Inspiratory capacity

VC

Vital capacity

FVC

Forced vital capacity

FEVx

Forced expiratory volume at x seconds

FDR

False discovery rate

DEGs

Differentially expressed genes

GO

Gene Ontology

KEGG

Kyoto Encyclopedia of Genes and Genomes

H&E

Hematoxylin and eosin

IHC

Immunohistochemistry

TUNEL

Terminal dUTP Nick End Labeling

RT-qPCR

Real-time quantitative PCR

Cdyn

Dynamic pulmonary compliance

Cst

Static pulmonary compliance

PCA

Principal component analysis

Authors’ contributions

YL, PW, ZH and WS conducted the experiments and performed data analysis. YO, JF, HY, ZZ, ZD, and CZ collected animal samples. KC and XT provided resources. YL and PW drafted the manuscript. JS and YQ provided comments on the study. YQ and NZ provided funding support. NZ supervised the project, designed the experiment. All authors approved the final version of the manuscript.

Fundings

This study was supported by the Natural Science Foundation of Guangdong Province, China (No. 2023A1515010085 and No. 2025A1515010794); Biomedical Industry Innovation Subsidy of Guangzhou Science and Technology Bureau (No. 202302002); Zhongnanshan Medical Foundation of Guangdong Province (No. ZNSXS-20250029); Guangdong Medical Scientific Research Foundation (No. A2025148).

Data availability

The data and materials that support the findings of this study are available on request from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

The clinical human specimens were approved by the Medical Ethics Committee of Guangdong Province Hospital for Occupational Disease Prevention and Treatment (GDHOD MEC 2022002). All specimens were acquired with informed consent from the donors’ immediate family members. Animal experiments were approved by the Experimental Animal Ethics Committee of the Guangdong Province Hospital for Occupational Disease Prevention and Treatment (No. GDHOD MEC 2021003).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.