Caveolin‐1 negatively regulates inflammation and fibrosis in silicosis

Abstract Inhalation of crystalline silica causes silicosis, the most common and serious occupational disease, which is characterized by progressive lung inflammation and fibrosis. Recent studies revealed the anti‐inflammatory and anti‐fibrosis role of Caveolin‐1 (Cav‐1) in lung, but this role in silicosis has not been investigated. Thus, this study evaluated Cav‐1 regulatory effects in silicosis. It was found that Cav‐1 levels were significantly reduced in the lung from silicosis patients and silicotic mice. The silicosis models were established in C57BL/6 (wild‐type) and Cav‐1 deficiency (Cav‐1 −/−) mice, and Cav‐1 −/− mice displayed wider alveolar septa, increased collagen deposition and more silicotic nodules. The mice peritoneal‐derived macrophages were used to explore the role of Cav‐1 in silica‐induced inflammation, which plays a central role in mechanism of silicosis. Cav‐1 inhibited silica‐induced infiltration of inflammatory cells and secretion of inflammatory factors in vitro and in vivo, partly by downregulating NF‐κB pathway. Additionally, silica uptake and expression of 4‐hydroxynonenal in silicotic mice were observed, and it was found that Cav‐1 absence triggered excessive silica deposition, causing a stronger oxidative stress response. These findings demonstrate the protective effects of Cav‐1 in silica‐induced lung injury, suggesting its potential therapeutic value in silicosis.

endocytosis. 5 Caveolin-1 (Cav-1) is the caveolae major functional protein, highly expressed in lungs. 6 Cav-1 regulates lung fibrosis and inflammatory response. In lungs from bleomycin-induced mice and radiation-induced rats, Cav-1 deficiency aggravated fibroblast proliferation and collagen deposition. 7,8 Cav-1 inhibited the infiltration of inflammatory cells and the secretion of inflammatory factors in lungs from lipopolysaccharide (LPS)-instilled mice. 9,10 Furthermore, Cav-1 inhibited macrophages activation through multiple pathways. 10,11 The role of Cav-1 in silicosis has not been investigated. Therefore, this study aimed to elucidate the function of Cav-1 in silicosis. The results showed that Cav-1 protected against lung injury in silicosis.
Cav-1 also inhibited silica-induced inflammation, partly by regulating the NF-κB pathway, silica intake and oxidative stress. These results provide new ideas for exploring the silicosis mechanism and propose that Cav-1 is a potential silicosis therapeutic target.

| Subjects
Paraffin-embedded sections of biopsy specimens were collected from nine silicosis subjects. A local pneumoconiosis diagnosis group diagnosed silicosis following the GBZ25-2014 standard issued in

| Animals
Male C57BL/6 mice were purchased from Silaike Laboratory. The

Yuan Lei group from Shanghai Medical College of Fudan University
provided Cav-1 knockout (Cav-1 −/− ) mice free of charge. 12 All animal experiments were conducted following the Animal Care and Use Committee of Central South University guidelines (permit number: 2020sydw0474).
On day 0, the silicosis model mice were intratracheally injected with 25 μl silica (U.S Silica Company; MIN-U-SIL5) suspension (200 mg/ml, equal to 5 mg/mouse). 13 The mice in the sham group were intratracheally injected with 25 μl sterile saline, as previously described. 14 In the first experiment, all mice were euthanized 28 days after treatment, except mice in the silica 7 day group, which were euthanized 7 days after treatment. In the second experiment, all mice were euthanized 28 days after treatment.

| Bronchoalveolar lavage fluid isolation
Bronchoalveolar lavage fluid (BALF) was collected from euthanized mice at designated time points. BALF samples were centrifuged at 1000 g for 10 min at 4℃. The protein concentrations of cell-free supernatants were measured with a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). The cell pellets were re-suspended in 1 ml phosphate-buffered saline for cell counting using Countess 3 Automated Cell Counters (Invitrogen).

| Lung tissues collection
Left lungs were collected and fixed with 4% paraformaldehyde for histopathological analysis. Right lungs were preserved at 80℃ for Western blot (WB) and Reverse transcription real-time polymerase chain reaction (RT-qPCR) analyses.

| Lung histology
Fixed lungs were dehydrated, embedded in paraffin and cut into 4 μm-thick slices. The lung tissue sections were stained with haematoxylin-eosin (HE) and Masson trichrome staining. Histological examination and photomicrography were conducted using a scanner (3DHISTECH).
Pulmonary fibrosis was quantified as eight grades according to the Ashcroft score as previously described. 15 Silicotic nodules were graded as follows: cellular nodules as Stage I, scores 1-point; fibrotic cellular nodules as Stage II, scores 1.2-points; cellular fibrotic nodules as Stage III, scores 1.4-points; and fibrotic nodules as Stage IV, scores 1.6-point. 16 The total silicotic nodules of each sample and the total number of points were counted and calculated, respectively.

| Immunohistochemistry
The mice lung sections were evaluated for immunohistochemical lo- The positive staining area was calculated from five random and noncoincident fields in each section at ×200 using the Image-Pro-Plus software to quantify expression of caveolin-1, collagen I and 4-HNE.
Neutrophil (MPO) and macrophage (CD68 + cell) quantification were determined in 10 random and noncoincident fields in each section at ×100.

| Warthin-Starry silver staining of the silica
The histological sections were stained with 1% silver nitrate in a 43℃-water bath for 30 min. After washing, the enzyme reaction was developed with a colour liquid containing 1.5 ml of 2% silver nitrate, 2 ml of 0.15% hydroquinone and 3.75 ml of 5% gelatin solution (1% citric acid solution, pH 4). When the sections turned brown-yellow, the reaction was terminated using preheated water (54℃). Finally, they were dehydrated and mounted.

| Isolation and culture of peritoneal-derived macrophages
Peritoneal-derived macrophages (PDMs) are commonly used for research in various diseases 17 and simulate the inflammation of alveolar macrophages in silicosis. 18 Therefore, this study employed silica-stimulated PDMs to explore the Cav-1 mechanism in silicosis.
The PDMs were isolated from WT and Cav-1 −/− mice as previously described. 19 After 12 h of serum starvation, cells were exposed to 50 μg/ml silica (Nano-SiO 2 ; Invivogen). 20,21 The cells were harvested for protein extraction after treatment for 3 and 24 h, respectively. Supernatants of 24 h-treated cells were centrifuged at 1800 g for 5 min at 4℃. After that, the cell-free supernatants were preserved at −80℃ for cytokine detection.

| Reverse transcription real-time polymerase chain reaction (RT-qPCR)
Total RNA was isolated from the lung tissues using TRIzol (Invitrogen) following the manufacturer's instructions. A Revert Aid First Strand cDNA Synthesis Kit reverse-transcribed the RNA into DNA (Invitrogen). RT-qPCR was performed using a CFX96 Quantitative PCR Detection System (Bio-Rad). Specific primers were designed from GenBank sequences and synthesized by Sangon Biotech.

| Statistical analysis
The SPSS 25 statistical software was used to analyse the data. All data were expressed as the mean, standard deviation. The twotailed Student t test analysed comparisons between two groups. p < 0.05 was considered statistically significant.

| Caveolin-1 was significantly reduced in lungs from silicosis patients and silicotic mice
Paraffin-embedded sections of lung tissues from nine patients with silicosis and nine cases of tumour-adjacent tissues used as control were collected, and immunohistochemistry (IHC) was performed to measure the Cav-1 expression. The IHC staining showed that the Cav-1 level was high in normal tumour-adjacent tissues but significantly decreased in silicosis lung tissues (p < 0.0001) ( Figure 1A).
The silicosis model was established in WT mice for 7 and 28 days to detect the Cav-1 level in silicosis. The lung tissue damage, silicotic nodule formation and inflammation infiltration progressed with increasing modelling time ( Figure 1B). In mice from the silica 7 day group, part of the alveolar wall was destroyed, causing alveolar fusion. Around the small airway, silicotic nodules and inflammatory cells were observed. In mice from the silica 28 day group, part of the alveolar wall was remodelled into a continuous fibre wall with collagen deposition. The size and amount of silicotic nodules increased, and inflammatory cells diffusely infiltrated ( Figure 1B, Table 1). The formation of silicotic nodules confirmed the silicosis model.

| Caveolin-1 deficiency exacerbated silicotic lung injury and fibrosis
The silicosis model was established in WT and Cav-1 −/− mice for 28 day. Based on HE staining, Cav-1 deficiency significantly aggravated lung structural remodelling and silicotic nodules formation in silicosis (Figure 2A). After 28 days of silica treatment, the Cav-1 −/− mice displayed more severe morphological damage, evidenced by more alveoli fusion, fibroblasts, collagen in the fibrotic walls ( Figure 2A[c,d]), and more confluent silicotic nodules than the WT mice (Figure 2A[a,b]). The higher protein content in BALF further indicated that Cav-1 deficiency aggravated lung injury after silica treatment ( Figure S1). Based on Masson staining and IHC stain-

| Caveolin-1 deficiency exacerbated silicainduced inflammation
CD68 and MPO are the most commonly used markers of macrophage and neutrophil. 22 The IHC detected the accumulation of These results indicate that Cav-1 negatively regulates silica-induced inflammation in PDMs.

| The possible mechanisms of Caveolin-1 regulating silica-induced inflammation
The sections from WT and Cav-1 −/− silicotic mice were treated with Warthin-Starry (W-S) silver staining to display the distribution of dust particles. Macrophages in the lungs from Cav-1 −/− silicotic mice had much more silica dust deposits than WT mice ( Figure 4A[c,d]), indicating that Cav-1 absence caused more silica dust accumulation in the lung tissue, thus causing continuous stimulation.
Silica induces pulmonary inflammation through oxidative stress. 23,24 The IHC of 4-HNE shows that the Cav-1 −/− oxidative stress level is significantly higher than the WT, indicating that the same dose of silica dust causes more severe oxidative damage to cells due to the Cav-1 absence ( Figure 4B).

| DISCUSS IONS
It was firstly found in this study that Cav-1 levels were significantly reduced in the lung from silicosis patients and silicotic mice. Cav-1 deficiency exacerbated the silica-induced lung injury. Cav-1 negatively regulates silica-induced inflammation, in part, by regulating the NF-κB pathway, silica intake and oxidative stress. Therefore, Cav-1 has an essential protective effect in silicosis.
Cav-1 is highly expressed in lung tissue, an anti-inflammatory and anti-fibrosis against lung injury.

| CON CLUS ION
Cav-1 significantly reduced in the lungs of silicosis patients and silicotic mice. Cav-1 deficiency exacerbated silica-induced lung injury and fibrosis. Cav-1 negatively regulated silica-induced inflammation, partly through NF-κB pathway. Cav-1 regulation is a potential treatment approach for silicosis.

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest to declare. Writing -review and editing (lead).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available upon request from the corresponding author.