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Summary

This study describes a mouse model to study the synergistic effect of nicotine on the progression of pulmonary fibrosis in experimental silicosis mice. The dual-exposure mouse model simulates the pathological progression in the lung after simultaneous exposure to nicotine and silica. The methods described are simple and highly reproducible.

Abstract

Smoking and exposure to silica are common among occupational workers, and silica is more likely to injure the lungs of smokers than non-smokers. The role of nicotine, the primary addictive ingredient in cigarettes, in silicosis development is unclear. The mouse model employed in this study was simple and easily controlled, and it effectively simulated the effects of chronic nicotine ingestion and repeated exposure to silica on lung fibrosis through epithelial-mesenchymal transition in human beings. In addition, this model can help in the direct study of the effects of nicotine on silicosis while avoiding the effects of other components in cigarette smoke.

After environmental adaptation, mice were injected subcutaneously with 0.25 mg/kg nicotine solution into the loose skin over the neck every morning and evening at 12 h intervals over 40 days. Additionally, crystalline silica powder (1-5 µm) was suspended in normal saline, diluted to a suspension of 20 mg/mL, and dispersed evenly using an ultrasonic water bath. The isoflurane-anesthetized mice inhaled 50 µL of this silica dust suspension through the nose and were awoken via chest massage. Silica exposure was administrated daily on days 5-19.

The double-exposed mouse model was exposed to nicotine and then silica, which matches the exposure history of workers who are exposed to both harmful factors. In addition, nicotine promoted pulmonary fibrosis through epithelial-mesenchymal transformation (EMT) in mice. This animal model can be used to study the effects of multiple factors on the development of silicosis.

Introduction

Silica exposure in workers is inevitable in some occupational settings, and once exposed to silica, the deterioration progresses even after removal from the environment. In addition, most of these workers smoke, and traditional cigarettes contain thousands of chemicals, with the key addictive component being nicotine¹. E-cigarettes are becoming increasingly popular in younger age groups²; these e-cigarettes act as a nicotine delivery system and increase nicotine access, thus increasing lung susceptibility and pneumonia³. Cigarette smoke also accelerates pulmonary fibrosis in bleomycin-exposed mice⁴ and increases pulmonary toxicity and fibrosis in silica-exposed mice^5,6. However, whether nicotine can affect the inflammatory and pulmonary fibrosis process caused by silica remains to be investigated.

The silicosis mouse model established by the one-time inhalation of a high dose of silica into the trachea is traumatic to mice. Although this method quickly provides a silicosis model, it does not match the reality of an environment where workers are repeatedly exposed to silica. Therefore, we established a silica-exposed mouse model by repeatedly giving a low dose of silica suspensions via a nasal drip; this dose can cause inflammation and fibrosis in mice.

To circumvent the effects of other cigarette components, this mouse model was subcutaneously injected with nicotine into the loose skin of the neck for determining the effect of the addictive component, nicotine, on silicosis. By administering subcutaneous injections, accurate dosing can be achieved, thus making it possible to create nicotine exposure models and observe dose-toxicity responses, as well as addiction. A nicotine addiction model has been developed in male mice, with a nicotine injection dose of 0.2-0.4 mg/kg^7,8. In that model, to meet the drug-seeking needs of the addicted mice, two subcutaneous injections were administered at intervals of 12 h. This mouse nicotine addiction model is useful for simulating human smoking habits and exposure to silica.

Single-factor animal models have limitations in disease studies, whereas the method described here involves a two-factor mouse model of nicotine and silica co-exposure. Prior to the silica exposure, the mice were pre-exposed to nicotine to replicate nicotine exposure in people who smoke. Subsequently, silica exposure took place from day 5 to day 19 to imitate silica exposure in a working environment for individuals with a history of smoking.

Alveolar macrophages are known to play a significant role in the regulation of lung inflammation and fibrosis. Macrophages cannot break silica down upon its inhalation of silica, leading to macrophage polarization or apoptosis⁹ and the release of cytokines such as tumor necrosis factor-alpha (TNF-α) and transforming growth factor beta (TGF-β). M1 macrophages, which are identified by the presence of the surface marker CD86, are the primary instigators of the inflammatory response in silicosis, while M2 macrophages, which are marked by CD206, are responsible for the fibrotic phase of the condition¹⁰. In dual-exposed mice, nicotine induced the polarization of macrophages toward the M2 phenotype in silica-injured lungs, thus promoting pulmonary fibrosis. Furthermore, TGF-β1 is key to the induction of fibrosis and EMT¹¹; the increased expression of TGF-β1 accelerated the progression of lung fibrosis through EMT. This model successfully analyzed the effects of nicotine on silicosis and further highlighted the importance of nicotine cessation.

Protocol

All procedures were conducted according to the guidelines issued by the National Institutes of Health's Guide for the Care and Use of Laboratory Animals (the 8th edition of the NRC) and were approved by Anhui University of Science and Technology Animal Ethics Committee.

1. Animal preparation

1. House 32 male C57/BL6 mice aged 8 weeks in a laboratory with a 12 h light/dark cycle. Ensure that the mice have free access to food and water.
2. After 2 weeks of acclimation to the environment, when the 10 week old mice weigh 23-26 g, use random numbers generated by a computer to randomly group the animals without partitioning, resulting in four groups of mice: the control group (Con), the nicotine group (Nic), the silica group (SiO₂), and the nicotine combined with silica group (Nic+ SiO₂).

2. Nicotine preparation

NOTE: The nicotine density is 1.01 g/mL.

1. Dissolve the nicotine in anhydrous ethanol, and dilute 20x to prepare a nicotine stock solution of 50 mg/mL.
2. Dilute the nicotine stock solution 1,000x with sterile normal saline to make a working solution at a concentration of 0.05 mg/mL.
   ​NOTE: Store nicotine away from light. The stock solution can be stored at 4 °C for up to 1 week.

3. Silica preparation

1. Suspend sterile crystalline silica in saline to prepare a 20 mg/mL silica suspension, and oscillate it in an ultrasonic shaking water bath for 25 min.
2. Shake the silica suspension with a vortex oscillator for 3 min before the mice receive the nasal drip. Mix the silica suspension by pipetting up and down 2x-3x with a 200 µL pipette, and then take up 50 µL for the nasal drip.
   NOTE: The silicon dioxide suspension should be mixed and administered via nasal drip as soon as possible.

4. Mouse capture and nicotine exposure

1. Weigh the mice, and record the weights daily before exposure.
2. On days 1-40, subcutaneously inject nicotine twice daily (12 h apart) into the mice in the Nic and Nic+ SiO₂ groups. Use a nicotine dose of 0.25 mg/kg; for example, ensure that a mouse weighing 25 g receives 6.25 µg of nicotine. For this mouse, the injection volume would be as follows: 6.25/0.05 = 125 µL. Simultaneously, inject the mice in the Con group with an equal volume of saline.
3. Prepare the required 1 mL syringes for each group, and use them to aspirate the injection volume of nicotine or saline. Before taking up the nicotine, first draw 0.1-0.2 mL of air into the syringe, and then draw up 125 µL of nicotine. Carefully tap the syringe to fill the needle and the front end of the syringe with the nicotine. Place the nicotine-containing syringe in a tray protected from light.
   NOTE: At the time of injection, 0.1-0.2 mL of air is situated behind the nicotine, guaranteeing that all the liquid is successfully administered to the mouse.
4. Grasp the tail of the mouse with the right hand, and when the animal relaxes, use the left thumb and forefinger to press the skin on the back of the neck from the tail to the head up to the edge of the ear, applying moderate pressure. After that, release the right hand, and use the left small thumb and ring finger to pinch the tail and hind limbs, at which point the mouse will be completely immobilized by the left hand.
   NOTE: Grasp the mouse with appropriate force. Insufficient strength makes it easy for mice to bite, whereas excessive strength may result in the asphyxiation of the mice.
5. Using the right hand to inject, puncture the skin on the back of the neck near the ear edge of the mice in a head-to-tail direction with the syringe, and inject with nicotine at a uniform rate. Look for a semicircular skin bulge that indicates a successful injection.
   NOTE: When the needle has punctured into the skin, there will be a feeling of resistance; the resistance disappears after the needle has been inserted. At this point, the needle needs to be slightly withdrawn to inject slowly and evenly.

5. Silica exposure in vivo

1. On days 5-19, instill the silicon dioxide suspension into the nasal cavity of the mice in the SiO₂ and Nic+ SiO₂ groups. Rapidly anesthetize each mouse with 2% isoflurane in an anesthesia machine at a dose of 3.6 mL/h. After anesthesia, place the mouse on the palm of one hand, and expose the nasal cavity of the mouse. Instill 50 µL of silica suspension into the nasal cavity within 4-8 s.
2. To allow the silica suspension to enter the lungs as soon as possible, after instillation, press the mouse's heart gently with the index finger for 3-5 s, 3x-5x per second, to promote its respiratory rate.
3. After the respiration of the mouse gradually becomes uniform, place the mouse in a cage to observe for 3 min.
4. For all mice receiving the silica suspension, repeat steps 1-3. In the control group, instill 50 μL of sterile saline into the nasal cavity on days 5-19.

6. Acquisition of fresh and fixed lung tissues

1. On day 41, use 50 mg/kg of sodium pentobarbital to anesthetize the mice intraperitoneally at a dose of 0.1 mL/20 g according to their body weight. Fix the limbs on a foam test plate, and spray the fur with 75% alcohol.
2. Perform cardiac perfusion to obtain fresh lung tissue, cut the midline of the abdomen to expose the chest cavity, and make a small opening at the apex of the right heart. Then, inject 20 mL of pre-cooled phosphate-buffered saline (PBS) slowly and evenly from the apex of the left heart, causing the blood to flow out from the opening created at the apex of the right heart. Take out the entire lung lobe, place it in a pre-cooled 1.5 mL centrifuge tube, and transfer to −80 °C for storage pending protein extraction.
3. Perfuse other mice with 20 mL of pre-cooled PBS followed by 10 mL of 4% paraformaldehyde at the same location to fix whole lungs; preserve each lung in 30 mL of 4% PFA.
4. After 72 h, embed the fixed lung tissues in paraffin.
   1. Immerse the lung tissue in the prepared fixative in an ultrasonic water bath at 40 °C for 30 min, followed by a dehydration process in 75% ethanol, 95% ethanol, and anhydrous ethanol for 50 min each.
   2. Wash 2x with xylene for 50 min each.
   3. Place the tissue in melted paraffin wax at 55-60 °C for 2-3 h so that the xylene in the lung tissue is gradually replaced by paraffin wax.
   4. Fix the lung tissue in the embedding frame with paraffin, and wait for the wax block to harden. After the wax block has hardened, use the paraffin sectioning machine to slice the lung at 4-5 µm.
5. Place the lung sections in ultrapure water at 45 °C. Once the paraffin has melted, load the lung section onto an adherent microscope slide.

7. Hematoxylin and eosin (HE) staining

1. Bake the slides at 60 °C for 2 h, and place the slides in xylene to dewax for 2 x 30 min. Subsequently, place the slides in anhydrous ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5 min each. Finally, wash them for 3 x 5 min with ultrapure water.
2. Drop 50 µL of hematoxylin staining solution onto the lung tissue with a pipette gun, and stain for 5 min; then, wash the slices with running water for 5 min.
3. Add 50 µL of 2% hydrochloric acid in ethanol onto the section, and rinse with distilled water for 30 s. Then, add 50 µL of eosin staining solution onto the lung tissue to stain for 1 min.
4. Dehydrate, transparentize, and seal the paraffin sections.
   1. Add 100-150 mL of 75% ethanol, 85% ethanol, 95% ethanol, anhydrous ethanol, and xylene in a combined plastic dyeing tank. Place the slides in 75%, 85%, 95%, and anhydrous ethanol for 5 min each to dehydrate.
   2. Then, place the slides in xylene for 5 min to transparentize.
   3. Finally, drop 20 µL of neutral gum onto the lung section, and place a coverslip over the slide to seal it.
      ​NOTE: The above steps are carried out at room temperature.

8. Masson staining

1. Dewax, hydrate, and wash the paraffin sections as described in step 7.1.
2. Stain the sections with 50 µL of configured Weigert's hematoxylin staining solution for 7 min, followed by 50 µL of acidic ethanol fractionation solution for 10 s, and wash with running water to turn the nuclei blue.
3. Stain the sections with 50 µL of Masson trichrome solution for 4 min, and wash with water.
4. Rinse the sections with distilled water for 1 min, and then stain with Lichun red acid magenta for 7 min.
5. Wash the sections with a weak acid working solution (30% hydrochloric acid) for 1-2 min, phosphomolybdic acid solution for 1-2 min, and weak acid working solution for 1-2 min.
6. Stain the sections in aniline blue staining solution for 2 min, and then wash with a weak acid working solution for 1 min.
7. Dehydrate the sections with 95% ethanol followed by anhydrous ethanol for 1 s, transparentize with xylene for 1 s, and finally seal with neutral resin as described in step 7.4.

9. Immunohistochemistry

1. Bake the slices at 60 °C for 6 h. Dewax, hydrate, and wash the paraffin sections as described in step 7.1.
2. Antigen retrieal: place the slices in a heat-resistant plastic slicing box containing enough citrate antigen retrieval solution to submerge the slices, and boil for 20-30 min.
3. Cool to room temperature, remove the sections, and rinse with deionized water. Subsequently, soak for 2 x 5 min in PBS containing 0.5% Tween-20 (PBST).
4. Draw a loop near the lung section on the slide with a hydrophobic pen to form a hydrophobic circle.
5. Add 50 µL of 3% hydrogen peroxide solution (3% H₂O₂) dropwise to the slices, and incubate for 15 min at room temperature protected from light for the inactivation of endogenous peroxidase.
6. Soak the slices in PBST for 3 x 5 min.
7. Add 50 µL of 5% bovine serum protein (BSA)-PBST dropwise to the slices, and block for 1 h at room temperature.
8. Discard the blocking solution, add 50 µL of the diluted primary antibodies CD206 (dilution: 1:1,500), TGF-β1 (dilution: 1:200), and vimentin (dilution: 1:2,000) dropwise to the slices, and incubate overnight at 4 °C.
   NOTE: In this work, all the antibodies were diluted in 5% BSA.
9. The next day, return the slices to room temperature (40 min). Wash the slices as described in step 9.6.
10. Dilute the secondary antibody in 5% BSA. Add 50 µL of horseradish peroxidase-labeled secondary antibody (dilution: 1:500) dropwise, and incubate for 1 h at room temperature. Wash the slices as described in step 9.6.
11. Drop 50 µL of 3,3'-diaminobenzidine (DAB) solution corresponding to the enzyme-labeled secondary antibody onto the lung tissue, and incubate for 5-10 min in a black immunohistochemical wet box. Observe under the microscope until the color develops to a strong brown. Rinse the slices with distilled water to terminate the reaction.
    NOTE: Brown staining is considered positive staining.
12. Stain with 50 µL of hematoxylin stain for 30 s, and wash with running water. Next, soak the slices in 75%, 85%, 95%, and anhydrous ethanol for 3 min each and then in xylene for 2 x 10 min. Seal the slides as described in step 7.4.

10. Western blot analysis

1. Take the lung tissue out of the freezer, and weigh it. Next, add 150 µL of RIPA lysis buffer containing PMSF per 20 mg of lung tissue. Lyse the mouse lungs on ice for 3-5 min using a handheld homogenizer, shake gently for 2 h at 4 °C to allow adequate lysis of the lung tissue, centrifuge at 4 °C and 14,800 × g for 15 min, and collect the supernatant.
   NOTE: Add 100 mM phenylmethanesulfonylfluoride (PMSF) a few minutes before using the RIPA lysis buffer. The final concentration of PMSF is 1 mM (e.g., 10 µL of PMSF + 990 µL of RIPA).
2. Use a BCA protein concentration kit to determine the protein concentration of the sample by following the manufacturer's instructions. After determination, using the smallest concentration of protein lysate as a base, dilute the same batch of protein lysate with RIPA to the smallest concentration to achieve the same mass and volume.
   NOTE: The total amount of lung tissue loading is 30 µg.
3. Add 40 µL of 5x loading buffer to 160 µL of protein lysate, and heat it at 100 °C for 10 min.
4. Assemble the prepared 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel in the western blotting electrophoresis system, add the SDS-PAGE electrophoresis buffer, gently pull out the electrophoresis comb, and add 20 µL of protein sample per well slowly and uniformly. Switch on the power, and start the electrophoresis at 80 V for 30 min. After the proteins have reached the lower gel layer, switch to 100 V, and continue the electrophoresis for 60-70 min.
   NOTE: Electrophoresis needs fresh electrophoresis buffer.
5. Transfer the proteins to a PVDF membrane by wet transfer. Activate the PVDF membrane with methanol in advance (5-30 s). Take the electrophoresis gel plate out, carefully pry it open, place the separator gel into the wet transfer buffer, make the electric transfer sandwich, and assemble the wet transfer system. Fill the electrophoresis tank with transfer buffer, and transfer at 400 mA for 90 min.
   NOTE: The transfer buffer is cooled in advance to avoid the effects of high temperatures during the wet transfer.
6. Wash the PVDF membrane with Tris-buffered saline with 0.5% Tween-20 (TBST) for 3 x 5 min. Block the PVDF membrane with 5% skim milk diluted in TBST, and incubate on a shaker at room temperature for 60 min; then, wash with TBST for 2 min. After washing, incubate the membrane overnight at 4 °C with the primary antibody vimentin (1:5,000) and GAPDH (1:10,000) diluted in 5% BSA.
7. Place the membrane at room temperature for 30 min, and wash with TBST for 5 x 5 min. After washing, incubate the membrane with the secondary antibody (1:10,000) diluted in 5% skim milk for 60 min at room temperature.
8. Place the membrane, protein-side up, drop the prepared ECL working solution, incubate for 1-2 min, and observe the results with a gel imager. Use the greyscale values measured by the software in the gel imager to evaluate the protein expression levels.
   NOTE: Here, GAPDH served as an internal reference.

Representative Results

A mouse model to study nicotine combined with silica exposure was established to investigate the potential role of nicotine in the progression of silicosis in mice. Figure 1 depicts the experimental procedure for using a dual-exposure mouse model, which paired a nicotine injection with the nasal instillation of a silica suspension. The pathological changes of the mice in each group were observed using HE staining. The mice exposed to nicotine combined with silica had significantly more severe lung damage than those exposed to nicotine or silica alone. Lymphocytes increased near the lymphatic vessels in the lungs of the nicotine-exposed mice, forming inflammatory cell clusters. Masson staining revealed a significant increase in collagen fiber deposition in the lungs exposed to nicotine combined with silica compared to the lungs in the other groups, and this was supported by the Masson staining quantification (Figure 2). The alveolar structure was destroyed in the silica-exposed mice, and the number of macrophages increased. However, after exposure to nicotine combined with silica, there was significant inflammatory cell infiltration, and fibroblast nodules appeared. In addition, accumulated macrophages, especially M2 macrophages, present in the double-exposed group. M2 macrophages are essential for the advanced fibrotic stage of silicosis.

Additionally, a significant increase in the pro-fibrotic factor TGF-β1 was observed by immunohistochemical (IHC) staining in the lungs of dual-exposed mice, especially in inflammatory cells near lymphatic vessels (Figure 3). TGF-β1 secreted by macrophages promotes the EMT of epithelial cells and pulmonary fibrosis¹². Compared with mice exposed to silica alone, vimentin levels were significantly elevated in the lungs of dual-exposure mice. Both IHC staining and protein quantification indicated severe EMT in dual-exposed mice (Figure 4). The combined evidence suggests that chronic silica exposure promotes EMT after the upregulation of TGF-β1, leading to an increase in fibroblasts and progressive fibrosis. At the same time, the addition of nicotine accelerates the process of pulmonary fibrosis by aggravating EMT, allowing lung fibrosis to appear earlier. This model is designed to explore the impact of nicotine on pulmonary fibrosis, which is a consequence of  chronic silica exposure in humans.

Figure 1: Design of an experimental model of combined exposure to nicotine and silica in mice. Continuous injection of nicotine for 1-40 days and continuous nasal instillation of silica for 5-19 days. Please click here to view a larger version of this figure.

Figure 2: Nicotine promotes the formation of fibroblastic masses in the lungs of silica-exposed mice. (A) HE staining was used to visualize pathological changes in the lungs. The dual-exposed mice had severe inflammatory cell infiltration and fibrosis. The green arrows indicate inflammatory cell masses. Scale bar = 50 µm. (B) Masson staining was used to show collagen fibers in the lungs. Scale bar = 50 µm. (C) Quantification of collagen fibers in the lungs. *p < 0.05, **p < 0.01, ***p < 0.001. Please click here to view a larger version of this figure.

Figure 3: Increased CD206-positive cells and TGF-β1 in the lungs of nicotine and silica dual-exposed mice. (A) IHC staining of CD206 was used to observe the distribution and expression of macrophages in the dual-exposed mice. CD206-positive macrophages increased in the lungs of mice after combined nicotine and silica exposure. The short arrows represent CD206-positive cells. Scale bar = 50 µm. (B) TGF-β1, a promoter of fibrosis, was elevated in dual-exposed mice. The long arrows represent TGF-β1-positive cells. Scale bar = 50 µm. (C,D) AOD of CD206 and TGF-β1. AOD = IOD (integrated optical density)/area. The AOD reflects the concentration of CD206 and TGF-β1 per unit area. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Abbreviations: TGF-β1 = transforming growth factor-beta; IHC = immunohistochemistry; AOD = average optical density. Please click here to view a larger version of this figure.

Figure 4: Promotion of lung fibrosis by nicotine in silica-exposed mice through the aggravation of EMT. (A) The expression of vimentin in each group was observed by IHC staining. Vimentin was strongly expressed in the lungs of the mice exposed to nicotine and silica. Scale bar = 50 µm.(B) Western blot of vimentin expression in each exposure group, with an increase in vimentin in the lung tissues of double-exposed mice. (C) The AOD value of vimentin was significantly more elevated in the double-exposed group compared to the other groups. (D) The relative protein expression level of vimentin compared to GAPDH. The vimentin expression in the double-exposure group was significantly higher than in the nicotine- or silica-exposure groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Abbreviations: EMT = epithelial-mesenchymal transition; AOD = average optical density. Please click here to view a larger version of this figure.

Discussion

A dual-exposure animal model is necessary to investigate the role and the potential mechanisms of concurrent exposure to nicotine and crystalline silicon dioxide. This model was achieved in this work through the subcutaneous injection of nicotine and the nasal drip of silica. To ensure a successful nicotine injection, the operator has to become familiar with grasping the mice, as grasping the skin at the back of the neck could be painful for them. Therefore, allowing the mice to adapt gradually to the grasping is important. In addition, a critical step in silica exposure in mice is the nasal drip. To increase the success of the procedure and the survival rate of the mice, it is essential to practice the nasal drip beforehand.

When performing the injection, the syringe should be skimmed over the mouse’s head to avoid a strong struggle when the mouse sees the syringe. The mouse’s tail should be firmly grasped with the right hand, while the left hand should be used to push the skin up to the ear margin at the back of the neck carefully and gently and to pinch the skin there. After releasing the mouse’s tail with the right hand, the tail and hind limbs should be stabilized using the left thumb and ring finger to prevent biting. Using a 1 mL syringe in the right hand, the needle should be inserted into the loose skin above the neck at a 30° angle in a head-to-tail direction. The needle should be withdrawn slightly after the resistance is lost, and the injection should be administered slowly and evenly. On days 5-19, mice in the dual-exposed group were exposed to nicotine and silica. It is recommended that the nicotine be injected at 08:00 a.m., followed by the nasal drip of silicon dioxide at 14:00 p.m., and another injection of nicotine at 20:00 p.m. The two nicotine injections must be given 12 h apart to avoid the effects of repeated grasping on the mice.

Performing subcutaneous injections and nasal drip presents several technical challenges. For subcutaneous injections, the mice should be grasped with appropriate force. If the skin at the back of the neck is pinched too tightly, the mouse’s airway will be blocked, quickly leading to suffocation. For optimal strength, the skin at the back of the neck should be pinched until the eyeballs protrude slightly. At this time, the mouse feels the lowest pain and breathes smoothly. In addition, a 1 mL syringe that is emptied of air should be used to draw the nicotine injection from the tube, followed by a further 0.1-0.2 mL of air. Before injecting, the bubbles should be gently flicked out. Due to the small volume of the injection, the nicotine left at the tip of the needle end may lead to an insufficient dose. The key parts of the injection are inserting the needle quickly, injecting the nicotine slowly, and removing the needle gently. For nasal drip, in this study, the nasal cavity of the mice was fully exposed under deep anesthesia, followed by the slow and uniform instillation of a silicon dioxide suspension. It is also important to gently press the thoracic cavity after silica exposure to avoid coughing or suffocation.

This model has certain limitations. The 1-5 µm silica particles used in the experiments were not collected from the environment and were pure silica particles. In contrast, in actual occupational settings, workers may be exposed to a variety of hazardous materials, not just silica particles, such as a mixture of coal and silica dust in a ceramic factory or a coal mine. We used nasal instillation silica to achieve low doses and repeated multiple exposures to simulate the chronic exposure of workers. In the dual-exposure mouse model, although the nicotine does not come from cigarette smoke exposure, the injected nicotine directly enters the bloodstream, allowing for a more focused exploration of the role of nicotine in the silicosis process and the avoidance of the effects of other components of cigarette smoke.

Scientific research on silicosis has typically utilized single-factor mouse models, either through the inhalation or bronchial perfusion of silica, to assess the role of silica in the development of disease. An alternative approach is the nasal instillation of silicon dioxide, which is simple and easy and allows for the establishment of repeated and chronic silica exposure models¹³. The well-established silicon dioxide nasal drip model causes minimal damage to the mice and could be combined with other factors to create a multi-factorial-exposed animal model. Additionally, the main methods of nicotine exposure in mice include nicotine in drinking water, subcutaneous injection of nicotine, nicotine infusion via osmotic minipumps, and tobacco smoke exposure¹⁴. The subcutaneous injection used in the dual-exposure model allows the dosage and timing of nicotine to be precisely set, ensuring that each mouse is administered the same dose.

In short, the animal model of nicotine in combination with silica exposure replicates a realistic chronic exposure pattern, and this model can be utilized for further investigations on the impacts of nicotine on inflammation and fibrosis in the development of silicosis. Additionally, this model serves as a foundation for forming a dual-exposure animal model with multiple doses and time frames.

Materials

10% formalin neutral fixative	Nanchang Yulu Experimental Equipment Co.
alcohol disinfectant	Xintai Kanyuan Disinfection Products Co.
BSA, Fraction V	Beyotime Biotechnology	ST023-200g
CD206 Monoclonal antibody	Proteintech	60143-1-IG
Citrate Antigen Retrieval Solution	biosharp life science	BL619A
dimethyl benzene	West Asia Chemical Technology (Shandong) Co
Enhanced BCA Protein Assay Kit	Beyotime Biotechnology	P0009
GAPDH Polyclonal antibody	Proteintech	10494-1-AP
Hematoxylin and Eosin (H&E)	Beyotime Biotechnology	C0105S
HRP substrate	Millipore Corporation	P90720
HRP-conjugated Affinipure Goat Anti-Mouse IgG(H+L)	Proteintech	SA00001-1
HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L)	Proteintech	SA00001-2
ImmPACT[R] DAB EqV Peroxidase (HRP) Substrate	Vector Laboratories	SK-4103-100
Masson's Trichrome Stain Kit	Solarbio	G1340
Methanol	Macklin
Nicotine	Sigma	N-3876
phosphate buffered saline (PBS)	Biosharp	BL601A
Physiological saline	The First People's Hospital of Huainan City
PMSF	Beyotime Biotechnological	ST505
Positive fluorescence microscope	OlympusCorporation	BX53+DP74
Prestained Color Protein Molecular Weight Marker, or Prestained Color Protein Ladder	Beyotime Biotechnology	P0071
PVDF membranes	Millipore	3010040001
RIPA Lysis Buffer	Beyotime Biotechnology	P0013B
SDS-PAGE gel preparation kit	Beyotime Biotechnology	P0012A
Silicon dioxide	Sigma	#BCBV6865
TGF-β	Bioss	bs-0086R
Vimentin Polyclonal antibody	Proteintech	10366-1-AP
Name of Material/ Equipment	Company	Catalog Number
0.5 mL Tube	Biosharp	BS-05-M
Oscillatory thermostatic metal bath	Abson
Paraffin Embedding Machine	Precision (Changzhou) Medical Equipment Co.	PBM-A
Paraffin Slicer	Jinhua Kratai Instruments Co.
Pipettes	Eppendorf
Polarized light microscope	Olympus	BX51
Precision Balance	Acculab	ALC-110.4
RODI IOT intelligent multifunctional water purification system	RSJ	RODI-220BN
Scilogex SK-D1807-E 3D Shaker	Scilogex
Small animal anesthesia machine	Anhui Yaokun Biotech Co., Ltd.	ZL-04A
Universal Pipette Tips	KIRGEN	KG1011
Universal Pipette Tips	KIRGEN	KG1212
Universal Pipette Tips	KIRGEN	KG1313
Vortex Mixers	VWR
Name of Material/ Equipment
Adobe Illustrator
ImageJ
Photoshop
Prism7.0