| Backgrouds:Chronic obstructive pulmonary disease（COPD）is one of the most common respiratory diseases,a progressive disease characterized by persistent respiratory symptoms and incomplete reversible airflow limitation.Epidemiological studies show that people over 40 years old are at a high risk of COPD,and the prevalence of COPD in people over 40 years old reaches 9%-10%and 13.7%in China.COPD is the third leading cause of death in the world and has become one of the priorities of global chronic disease prevention and treatment.The incidence and mortality of COPD are high and it is difficult to cure the disease.Elucidating the etiology and pathogenesis of COPD and preventing the occurrence and development of COPD are important topics in the research field of respiratory diseases.Studies have shown that many factors,such as cigarette smoke,occupational environment,air pollution,respiratory tract infection,increased airway responsiveness,and genetic factors,are all risk factors for COPD.Among them,the occurrence and development of COPD caused by environmental factors are the hot and difficult points in the research of respiratory diseases.Cadmium（cadmium,Cd）is a widespread toxic heavy metal in the environment.Environmental cadmium exposure is strongly associated with respiratory diseases.Population epidemiological studies found that exposure to cadmium-containing dust in occupational environments increased the incidence of COPD.Environmental cadmium exposure may be one of the risk factors for the occurrence and development of chronic obstructive pulmonary disease.Epithelial mesenchymal transition（EMT）is the transformation of epithelial cells into a stromal cell phenotype.EMT is associated with airway remodeling,airway inflammation,as well as airway obstruction,and plays a key role in the development and progression of COPD.At the same time,it was found that the micro RNA-30（mi RNA-30,mi R-30）family,as an important member of the tiny RNA family,is involved in the process of EMT.The hypothesis is proposed that mi R-30may play a role in COPD caused by cadmium exposure through EMT,but the association of cadmium exposure with the occurrence and development of COPD and its mechanism are not currently clear,requiring further demonstration in case-control study as well as laboratory studies.Objective:A case-control study t study and in vitro and in vivo experiments were used to explore the association between environmental cadmium exposure and EMT or COPD.A mouse model of chronic obstructive pulmonary disease（COPD）induced by cadmium exposure was established,and the changes of lung function and lung pathology induced by cadmium exposure were observed in vivo.An in vitro cell model of EMT induced by cadmium exposure was established to observe the effect of cadmium exposure on the migration and invasion of human alveolar epithelial cells.Western blot and RT-PCR were used to detect EMT markers and mi R-30 levels in mouse lung and human alveolar epithelial cells,and to explore the role and mechanism of mi R-30 and lung EMT in COPD induced by cadmium exposure.This study provides a theoretical basis for the causal association between environmental cadmium exposure and COPD,and provides new ideas and strategies for the clinical prevention and treatment of COPD.Methods:Case-control study:The second affiliated hospital of Anhui medical university,respiratory and critical care medicine in Anhui province since 2017,as of January 2020,has successfully enrolled 512 first diagnosed COPD patients,and completed the study baseline data questionnaire,collected demographic information and clinical characteristics,collected and preserved serum samples,completed the total plasma RNA extraction and mi R-30 detection,with graphite furnace atomic absorption spectrophotometry.The study population and specimens were from the Anhui COPD specialty cohort.The 400 subjects who met the entry criteria were screened,and the clinical data and specimens of the 400 subjects in the control group were all obtained from the health examination center of our hospital.Complete blood routine and inflammatory factor testing in all subjects,Pulmonary function levels in COPD patients by pulmonary function instrument,including the FVC（L）,FEV1%,FEV1/FVC%.Biochemical laboratory tests of blood routine and inflammatory factor levels in COPD patients,including CRP,TNF-α,IL-6,MCP-1.COPD patients were divided into four grades according to pulmonary function:Gold 1,FEV1≥80%;Gold2,50%≤FEV1≤79%;Gold 3,30%≤FEV1≤49%;Gold 4,FEV1<30%.The serum cadmium,plasma mi R-30 levels and EMT markers were tested in all COPD patients and healthy controls;12 non-COPD patients and non-COPD lung tissues were collected,and EMT markers were detected by immunohistochemistry;the association between serum cadmium,mi R-30,EMT and COPD progression were analyzed.Animal experiment:The pathological changes and lung function of mice induced by environmental cadmium exposure were observed.A COPD mouse model induced by cadmium exposure was established.The mice were intratracheal instillation of cadmium chloride solution（0.2μg/50μL/mouse）once every other day for 8 weeks.At the end of modeling,the left lung tissues of mice were fixed in paraformaldehyde and embedded in paraffin for pathological experiments,HE staining and immunohistochemistry detection of EMT markers.The remaining lung tissues were placed in 1.5m L enzyme-free EP tubes and stored in an ultra-low temperature refrigerator at-80℃for a long time.EMT markers were detected by Western blot and mi R-30 levels were detected by RT-PCR.Cell experiment:Human lung epithelial cells（BEAS-2B）were selected to establish a model in vitro,and the effect of cadmium exposure on the migration and invasion of BEAS-2B cells was detected.The protein expression levels of EMT markers and mi R-30 gene expression levels were detected.A logistic regression,scatter plot or linear correlation method were used to investigate the role and mechanism of mi R-30 mediated lung EMT in environmental cadmium exposure.Results:This study included 400 COPD patients（73.2%male）aged between 58.5and 82.0 years（median age is 72.0 years）,including 293 males,107 females,and 400healthy control subjects.There were no differences in age and gender between COPD patients and controls.More number of previous smokers in COPD patients（n=254,63.4%）than the control group（n=174,43.5%）.Routine blood tests showed increased white blood cells（WBC,median 6.69×109/L）,neutrophils（NEU,median 4.69×109/L）and monocytes（MON,median 0.46×109/L）counts,lymphocytes（LYM,median1.06×109/L）and eosinophils（EOS,median 0.21×109/L）,and basophils（BAS,median0.021×109/L）counts not different from the control group.Compared with the control group,Serum CRP（86.5±10.5μg/m L）,TNF-α（93.5±5.9 ng/m L）and MCP-1（212.5±32.5 pg/m L）were higher in COPD patients than in controls（40.2±6.4μg/m L,36.5±7.8 pg/m L,65.3±8.8 pg/m L）（P<0.001）,IL-6（43.5±12.3 pg/m L）Also elevated compared to the control group（20.3±8.4pg/m L）,However,it was not significant（P>0.05）.COPD patients were divided into three groups based on low serum Cd concentration（<0.77μg/L）,medium（0.771.01μg/L）and high（>1.01μg/L）.There was no difference in age or sex between the low Cd（n=133）,medium Cd（n=133）and high Cd（n=133）.Among them,the number of COPD patients who had never smoked was negatively correlated with serum Cd concentration（19/14.5%,15/11.5%,12/9.3%）.The length of hospital stays in COPD patients gradually increased with increasing serum Cd concentration（9.0 days,11.0 days,13.0 days）.Furthermore,leukocytes（6.32×109/L,6.91×109/L,7.12×109/L）,neutrophils（4.35×109/L,4.70×109/L,4.95×109/L）,lymphocytes（0.92×109/L,1.02×109/L,1.18×109/L）and eosinophils（0.11×109/L,0.123×109/L,0.031×109/L）from COPD patients with different circulating Cd concentrations are significant different（P<0.01）.In COPD patients,CRP（67.8±8.6μg/m L,101.5±12.1μg/m L,122.3±15.6μg/m L）and inflammatory cytokines such as TNF-α（78.6±7.8 ng/m L,122.6±15.3 ng/m L,155.3±20.5 ng/m L）and MCP-1（165.3±8.9 pg/m L,198.6±22.1 pg/m L,256.6±32.5pg/m L）were increased simultaneously with serum Cd（P<0.01）.Inflammatory factor IL-6（32.8±4.2 pg/m L,44.6±5.5 pg/m L,56.3±4.6 pg/m L）was also increased simultaneously with serum Cd,but it was not statistically significant（P>0.05）.With the increase of serum Cd concentration in COPD patients,FVC（1.85L,1.61L,1.42L）,FEV1%（40.2%,34.5%,30.5%）,FEV1/FVC%（53.4%,47.5%,42.5%）gradually decreased,statistically significant（P<0.01）.Compared with the control group,（1）the level of serum Cd in COPD patients was increased,and the level of serum Cd was negatively correlated with the level of lung function（FVC,FEV1%,FEV1/FVC%）;（2）Patients with COPD have reduced serum mi R-30 levels,which is positively correlated with pulmonary function（FVC,FEV1%,FEV1/FVC%）levels.（3）In COPD patients with increased serum Cd concentration,the lung epithelial marker protein E-cadherin was decreased,and the lung interstitial marker proteinsα-SMA,N-cadherin and Vimentin were increased,which indicated that EMT occurred in lung tissue of COPD patients.Serum Cd in COPD patients was negatively correlated with mi R-30.It is related to the occurrence of lung EMT.（4）Cd exposure induced lung tissue structural damage and reduced pulmonary function in mice.（5）EMT occurred in the lungs of Cd-exposed mice,and the level of mi R-30was decreased.（6）Cd exposure promoted the migration and invasion of BEAS-2B cells.（7）Cd exposure induced EMT in BEAS-2B cells,while mi R-30 was decreased.Conclusion:（1）The retrospective case-control study showed that the serum cadmium content of COPD patients was significantly higher than that of normal control population,and showed a significant negative correlation with lung function level;the plasma mi R-30 level of COPD patients was significantly lower than that of normal control population,showing a significant positive correlation with lung function level.Therefore,serum Cd was negatively correlated with pulmonary function and negatively correlated with plasma mi R-30 levels,clarifying the role of mi R-30 in the decreased pulmonary function of COPD due by environmental cadmium exposure.（2）Compared with the control group,COPD patients have EMT in the lungs,and it is positively correlated with the degree of Cd exposure,which provides a theoretical basis for the down-regulation of mi R-30 to participate in the progression of COPD induced by Cd exposure by affecting EMT.（3）By constructing a COPD mouse model induced by cadmium chloride instillation,it was observed that the mice exposed to Cd had obvious lung injury and significantly decreased pulmonary function.Molecular experiments showed that mi R-30 in the lung of the exposed mice was decreased,and EMT occurred in the lung of the mice.This study lays the foundation for elucidating the role of mi R-30 down-regulation in Cd exposure-induced COPD in mice through EMT at the animal level in vivo.（4）In vitro cell experiments showed that EMT occurred in BEAS-2B cells after Cd exposure,accompanied by the enhancement of cell migration and invasion.At the same time,it was found that the level of mi R-30 in the exposed group was decreased,suggesting that mi R-30 down-regulation plays an important role in EMT of BEAS-2B cells induced by Cd exposure.In conclusion,mi R-30 downregulation-mediated lung EMT plays an important role in the pathogenesis of COPD due to Cd exposure,providing new targets for the prevention and treatment of COPD. |
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