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.
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.
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.
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…
The authors have nothing to disclose.
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).
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 |