Long-term Respiratory Cadmium Exposure Induces Chronic Obstructive Pulmonary Disease-like Lung Lesions In A Mouse Model

Background Chronic obstructive pulmonary disease（COPD）is a common chronic inflammatory respiratory disease characterized by persistent respiratory symptoms and progressive decline in lung function.More and more studies have found that exposure to air pollutants and atmospheric fine particles is an important risk factor for COPD,but it is not clear which specific substances are related to the cause of COPD caused by air pollutants and atmospheric fine particles.Cadmium（Cd）is one of the key components of air pollutants and fine particles.Several studies have shown that there is a specific relationship between environmental Cd exposure and COPD,but whether Cd exposure induces COPD has never been confirmed in animal experiments.Objective The objectives of this study are as follow:To establish a COPD animal model of long-term respiratory Cd exposure by respiratory aerosol exposure;To dynamically observe the influences of respiratory Cd exposure on alveolar epithelial surfactants;To observe the impacts of Cd exposure on cellular senescence in mouse lungs and human lung epithelial cells,and to explore the role of senescence-mediated integrative stress in Cd-induced downregulation of surfactants in alveolar epithelial cells;In vitro and in vivo animal experiments were used to observe the effect of respiratory Cd exposure on the expression of cysteine-rich 61（CYR61）in alveolar epithelial cells,and explore the role of pulmonary CYR61 up-regulation in the progression of COPD.Methodology In vivo,the method of respiratory aerosol exposure was used.The mice were divided into two groups:short-term Cd group and long-term Cd group.In short-term Cd group,mice were exposed to Cd Cl₂（10 mg/L）aerosol through the respiratory tract for 4 hours a day and 10 weeks continuously;In long-term Cd group,mice were exposed to Cd Cl₂（10 mg/L）aerosol through the respiratory tract for 4 hours a day and 6 months continuously.The following experiments were performed 24 hours after the last exposure:some mice（6 in each group）were used to detect lung function and calculate the values of FEV0.1,FVC,FEV0.1/FVC,RL,Re and Cdyn;Some mice（6 in each group）were used to undergo alveolar lavage,and the number and classification of inflammatory cells in alveolar lavage fluid were detected and calculated;The remaining mice（6 in each group）were used to collect blood serum for measurement of serum Cd content,inflammatory cytokines and CYR61 level.Mouse lungs were collected to analyze pulmonary Cd content,expression of inflammatory factors and epithelial-interstitial transformation,detect lung histopathology,and evaluate airway inflammation and collagen deposition around small airways.In vitro experiments,MLE-12 cells and BEAS-2B cells were used as the model,which was composed of seven independent experiments.Experiment 1.The effect of Cd on the expression of surfactant protein in MLE-12 cells;Experiment 2:Cd induced down-regulation of surfactant protein in MLE-12 cells:time-effect relationship;Experiment 3.The influence of short-term exposure to high concentration of Cd on cellular senescence in MLE-12 cells;Experiment 4.The influence of long-term exposure to low concentration Cd on cellular senescence in human alveolar epithelial cells;Experiment 5.Cd-induced integrated stress in MLE-12 cells:concentration-effect relationship;Experiment 6.Cd-induced integrated stress in MLE-12 cells:time-effect relationship;Experiment 7.The impacts of Cd on CYR61 in BEAS-2B cell.MLE-12cells and BEAS-2B cells were cultured in a culture medium containing a specific concentration of Cd Cl₂for a specific time,and then harvested and tested for specific indicators.The population study was carried out using a case-control design.All patients with COPD diagnosed for the first time were randomly selected from COPD cohort study（AHCC）established by the Second Affiliated Hospital of Anhui Medical University.The subject adopts a matched case-control study design.150 COPD patients were recruited from AHCC,and the lung function of all recruited COPD patients was tested according to the standardized method;150 sex and age-matched control subjects were selected from the physical examination center of the Second Affiliated Hospital of Anhui Medical University.COPD was confirmed according to GOLD standard;The numbers of pulmonary CYR61-and P65-positive cells were detected by immunohistochemistry;Serum CYR61 and inflammatory cytokines were detected by ELISA;The quantitative data were expressed by mean±standard deviation,and the statistical difference between the two groups was tested by Student t test;Analysis of variance was used to test the statistical differences between different groups,and SNK was used to compare the two groups.The classification variables are expressed in frequency and percentage,usingχ²Test or Fisher’s exact probability method for statistical analysis.Pearson correlation analysis and linear regression analysis were used to evaluate the correlation between CYR61 and inflammatory cytokines.Logistic regression analysis was used to evaluate the correlation between serum CYR61 and hospital stay.P<0.05 indicates that the difference is statistically significant.Results The results of this study were composed of three parts:（1）Establishment of COPD animal model of respiratory Cd exposure.This study observed pulmonary histopathological damage and lung function changes caused by long-term respiratory Cd exposure.The main results were as follows:long-term respiratory Cd exposure caused COPD-characteristic pathological changes in mouse lungs,mainly manifested as the destruction of alveolar structure,increase of alveolar chord length,thickening of small bronchial wall and inflammatory cell infiltration;Long-term respiratory Cd exposure increased BALF inflammatory cells,especially macrophages and neutrophils;Long-term respiratory Cd exposure up-regulated inflammatory cytokines in mouse lungs and promoted the epithelial-mesenchymal transformation and collagen deposition in small airways of mouse lungs;Long-term respiratory Cd exposure caused COPD-characteristic lung function decline.（2）The role of cellular senescence mediated integration stress in Cd-induced down-regulation of surfactant protein in alveolar epithelial cells.This study observed Cd-induced down-regulation of surfactant protein in mouse lung and human lung epithelial cells and evaluated the influence of Cd exposure on cellular senescence in mouse lungs and human alveolar epithelial cells.The main results were as follows:In vivo animal experiments found that respiratory Cd exposure down-regulated the expression of mouse alveolar surfactant protein（SPA）,and short-term and long-term respiratory Cd exposure caused the up-regulation of mouse alveolar aging index（SIRT3 and P21）;In vitro cell experiments showed that Cd exposure down-regulated surfactant protein in human alveolar epithelial cells,and Cd exposure up-regulated cellular senescence in human alveolar epithelial cells and caused integrative stress.（3）The impacts of respiratory Cd exposure on CYR61 in alveolar epithelial cells and its correlation with COPD.This study examined the impacts of cadmium exposure on the expression of CYR61 in mouse lungs and human lung epithelial cells,and evaluated the correlation between serum CYR61 content,lung inflammation and lung function index in patients with COPD.The main results were as follows:Cd exposure up-regulated the expression of CYR61 in mouse lungs and human alveolar epithelial cells;The levels of serum CYR61 and a series of inflammatory cytokines in patients with COPD were increased;The elevation of serum CYR61 has a relationship with lung function decline and the prolongation of hospital stay in COPD patients;The elevation of serum CYR61 was positively correlated with pulmonary NF-κB activation and serum MCP-1 increase in COPD patients;Mediation analysis showed that inflammatory chemokine MCP-1 was a mediator between CYR61 and pulmonary function decline in COPD patients.Conclusions Based on the above results,the conclusions are as follow:（1）Long-term respiratory Cd exposure induces COPD-like lung lesions,including COPD-characteristic alveolar structure destruction,airway inflammation,pulmonary EMT and ECM collagen deposition around small airways and COPD-characteristic lung function decline.（2）Respiratory Cd exposure promotes cell senescence and down-regulates the secretion of surfactant protein by alveolar type II cells.Cell senescence-evoked integrative stress might play an important role in Cd-induced down-regulation of surfactant protein in alveolar epithelial cells;（3）Respiratory Cd exposure up-regulates the expression of CYR61 in mouse lungs and human alveolar epithelial cells.The up-regulation of CYR61 is associated with lung inflammation and lung function decline in COPD patients.This study provides evidence for the first time that long-term respiratory Cd exposure causes COPD-like lung injury in mice,and heavy metal Cd may be one of the key components of COPD caused by air pollutants and atmospheric fine particles.