Method Article

Using Nicotine in a Silica-Exposed Mouse Model to Promote Lung Epithelial-Mesenchymal Transition

1.7K views

⸱

DOI:

10.3791/65127

⸱

March 3rd, 2023

 ,  ,  ,  ,  ,  ,  , 
1Key Laboratory of Industrial Dust Control and Occupational Health of the Ministry of Education, Anhui University of Science and Technology, 2Key Laboratory of Industrial Dust Deep Reduction and Occupational Health and Safety of Anhui Higher Education Institutes, Anhui University of Science and Technology, 3Anhui Province Engineering Laboratory of Occupational Health and Safety, 4School of Medicine, Department of Medical Frontier Experimental Center, Anhui University of Science and Technology

* These authors contributed equally

In This Article

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 mice5,6. 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/kg7,8. 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.

Access restricted. Please log in or start a trial to view this content.

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.

Access restricted. Please log in or start a trial to view this content.

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 sever...

Access restricted. Please log in or start a trial to view this content.

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 importa...

Access restricted. Please log in or start a trial to view this content.

Disclosures

All authors declare they have no conflicts of interest.

Acknowledgements

This study was supported by the University Synergy Innovation Program of Anhui Province (GXXT-2021-077) and the Anhui University of Science and Technology Graduate Innovation Fund (2021CX2120).

Access restricted. Please log in or start a trial to view this content.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
10% formalin neutral fixativeNanchang Yulu Experimental Equipment Co.
alcohol disinfectantXintai Kanyuan Disinfection Products Co.
BSA, Fraction VBeyotime BiotechnologyST023-200g
CD206 Monoclonal antibodyProteintech60143-1-IG
Citrate Antigen Retrieval Solutionbiosharp life scienceBL619A
dimethyl benzeneWest Asia Chemical Technology (Shandong) Co
Enhanced BCA Protein Assay KitBeyotime BiotechnologyP0009
GAPDH Polyclonal antibodyProteintech10494-1-AP
Hematoxylin and Eosin (H&E)Beyotime BiotechnologyC0105S
HRP substrateMillipore CorporationP90720
HRP-conjugated Affinipure Goat Anti-Mouse IgG(H+L)ProteintechSA00001-1
HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L)ProteintechSA00001-2
ImmPACT[R] DAB EqV Peroxidase (HRP) SubstrateVector LaboratoriesSK-4103-100
Masson's Trichrome Stain KitSolarbioG1340
MethanolMacklin
NicotineSigmaN-3876
phosphate buffered saline (PBS) BiosharpBL601A
Physiological saline The First People's Hospital of Huainan City
PMSFBeyotime BiotechnologicalST505
Positive fluorescence microscopeOlympusCorporationBX53+DP74
Prestained Color Protein Molecular Weight Marker, or Prestained Color Protein LadderBeyotime BiotechnologyP0071
PVDF membranesMillipore3010040001
RIPA Lysis BufferBeyotime BiotechnologyP0013B
SDS-PAGE gel preparation kitBeyotime BiotechnologyP0012A
Silicon dioxideSigma#BCBV6865
TGF-βBiossbs-0086R
Vimentin Polyclonal antibodyProteintech10366-1-AP
Name of Material/ EquipmentCompanyCatalog Number
0.5 mL TubeBiosharpBS-05-M
Oscillatory thermostatic metal bathAbson
Paraffin Embedding MachinePrecision (Changzhou) Medical Equipment Co.PBM-A
Paraffin SlicerJinhua Kratai Instruments Co.
PipettesEppendorf
Polarized light microscopeOlympusBX51
Precision BalanceAcculabALC-110.4
RODI IOT intelligent multifunctional water purification systemRSJRODI-220BN
Scilogex SK-D1807-E 3D ShakerScilogex
Small animal anesthesia machineAnhui Yaokun Biotech Co., Ltd.ZL-04A
Universal Pipette TipsKIRGENKG1011
Universal Pipette TipsKIRGENKG1212
Universal Pipette TipsKIRGENKG1313
Vortex Mixers VWR
Name of Material/ Equipment
Adobe Illustrator
ImageJ
Photoshop
Prism7.0

References

  1. Wonnacott, S. Presynaptic nicotinic ACh receptors. Trends in Neurosciences. 20 (2), 92-98 (1997).
  2. Berry, K., et al. Association of electronic cigarette use with subsequent initiation of tobacco cigarettes in US youths. JAMA Network Open. 2 (2), 187794(2019).
  3. Masso-Silva, J., et al. Chronic e-cigarette aerosol inhalation alters the immune state of the lungs and increases ACE2 expression, raising concern for altered response and susceptibility to SARS-CoV-2. Frontiers in Physiology. 12, 649604(2021).
  4. Cisneros-Lira, J., Gaxiola, M., Ramos, C., Selman, M., Pardo, A. Cigarette smoke exposure potentiates bleomycin-induced lung fibrosis in guinea pigs. American Journal of Physiology. Lung Cellular and Molecular Physiology. 285 (4), 949-956 (2003).
  5. Sager, T., et al. Tobacco smoke exposure exacerbated crystalline silica-induced lung toxicity in rats. Toxicological Sciences. 178 (2), 375-390 (2020).
  6. Zhou, L., et al. Cigarette smoking aggravates bleomycin-induced experimental pulmonary fibrosis. Toxicology Letters. 303, 1-8 (2019).
  7. Liu, J., et al. Behavior and hippocampal Epac signaling to nicotine CPP in mice. Translational Neuroscience. 10, 254-259 (2019).
  8. Cao, J., et al. RNA deep sequencing analysis reveals that nicotine restores impaired gene expression by viral proteins in the brains of HIV-1 transgenic rats. PLoS One. 8 (7), 68517(2013).
  9. Zhao, Y., et al. Silica particles disorganize the polarization of pulmonary macrophages in mice. Ecotoxicology and Environmental Safety. 193, 110364(2020).
  10. Tang, Q., et al. Pirfenidone ameliorates pulmonary inflammation and fibrosis in a rat silicosis model by inhibiting macrophage polarization and JAK2/STAT3 signaling pathways. Ecotoxicology and Environmental Safety. 244, 114066(2022).
  11. Hinz, B., et al. The myofibroblast: One function, multiple origins. The American Journal of Pathology. 170 (6), 1807-1816 (2007).
  12. Liu, Y., et al. Long non-coding RNA-ATB promotes EMT during silica-induced pulmonary fibrosis by competitively binding miR-200c. Biochimica et Biophysica Acta. Molecular Basis of Disease. 1864 (2), 420-431 (2018).
  13. Li, B., et al. A suitable silicosis mouse model was constructed by repeated inhalation of silica dust via nose. Toxicology Letters. 353, 1-12 (2021).
  14. Chellian, R., et al. Rodent models for nicotine withdrawal. Journal of Psychopharmacology. 35 (10), 1169-1187 (2021).

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Tags

Nicotine ExposureSilica Mouse ModelLung FibrosisPulmonary FibrosisSubcutaneous InjectionNasal Silica ExposureImmunohistochemical StainingCollagen DepositionTGF Beta One

Related Articles