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

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 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. After 2 weeks of ac…

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 sever…

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…

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