Induction Of Lung Structure Cell Apoptosis By Cigarette Smoke Via Endoplasmic Reticulum Stress To Promote The Development Of COPD

Objective:To investigate whether induction of lung structure cell apoptosis by cigarette smoke (CS)via endoplasmic reticulum stress will promote the development of COPD.Methods: The whole experiment was divided into two parts: animal research and clinical research.ⅠAnimal researchForty adult male Wistar rats were randomly divided into Control group, 2 month smoking exposed group(2-M-S-G) , 4 month smoking exposed group(4-M-S-G)and ex-smoking group(exposed to CS for 4 month and then quit smoking for 1 month)respectively. The COPD model was established by passive exposing with CS. To evaluate the COPD model by detecting pulmonary functions and observing pathological changes of lung tissues at each time point.Collecting lung tissue of control and experimental group,Tunel was used to detect the apoptotic cell. hybridization in situ and Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine the mRNA expression of CHOP. Immuno- histochemistry and Western blot were used to determine the protein expression of PERK,P-PERK and CHOP.ⅡClinical researchLung tissue specimens of the control group, smoking group, COPD group were collected from patients accepted lobectomy because of lung cancer for 10 specimens in each group (the smoking index of smoking group had no significant difference with COPD group). Tunel was used to detect the apoptotic cell. hybridization in situ and Reverse transcriptase-polymerase chain reaction(RT-PCR) was used to determine the mRNA expression of CHOP. Immunohistochemistry and Western blot were used to determine the protein expression of PERK,P-PERK and CHOP.[Results]:Ⅰ1 Results of animal research(1) The pulmonary function (FEV0.3 and PEF)greatly decreased in rats exposed to 2 month CS in comparison with control group(FEV0.3 75.48±2.57 (%) VS 83.47±4.98(%),PEF33.42±3.21 VS40.11±3.66(ml/s) P <0.05),markedly decreased in rats exposed to 4 month compared with rats after exposed to2 month CS (FEV0.3 66.18±4.12 (%) VS 75.48±2.57 (%),PEF25.89±2.22(ml/s)VS33.42±3.21(ml/s),P <0.05),and improved little in ex-smoking rats(P>0.05).(2) the lung sections from the control rats maintained normal alveolar structure, while upon exposure to cigarette smoking for 2 months, the lung structure appears damaged, with some alveoli developing into bulla. In rats exposed to 4 months CS, a histological evaluation demonstrated that the pulmonary structure was badly impaired, with thinned or ruptured alveolar walls, enlarged airspaces. In ex-smoking rats, the destruction of the alveolar structure was also obvious.(3) The results of TUNEL show that the apoptotic cell is alveolar epithelial cellⅠ(ACEⅠ) and alveolar epithelial cellⅡ(ACEⅡ), vascular endothelial cell(VEC) and bronchial epithelial cell.The apoptosis rate is low in control rats(11.442±1.724(%)),significantly increased in rats exposed to 2 month CS(22.400±1.615(%) VS 11.442±1.724 (%),P <0.05),and markedly elevated in rats exposed to 4 month CS(34.830±3.201 (%) VS 22.400±1.615(%),P <0.05),and slightly decreased in ex-smoking rats(P>0.05).(4) In situ hybridisation showed that CHOP mRNA expression levels was mainly located in AEC,VEC and bronchial epithelial cell. The RT-PCR results demonstrated that the expression of CHOP mRNA was low in the control group, highly increased in rats exposed to 2 months CS(P <0.05), even higher in rats with 4 months CS exposure(P <0.05), and then only slightly altered after smoking cessation(P >0.05), which parallels the in situ hybridisation results.(5)Immunohistochemistry results showed that the expression of P- PERK is mainly stained in cytoplasm of alveolar epithelial cell,vascular endothelial cells and bronchia epithelial cell. while the expression of CHOP was mainly located in nucleus of alveolar epithelial cell.the expression of CHOP is low in control group,greatly elevated in rats exposed to 2 month CS,and markedly increased in rats exposed to 4 month CS, and slightly decreased in ex-smoking rats. Unlike the expression of p-PERK, the total PERK level did not change between the control rats and those exposed to CS.The result of western blotting is consistent with immunohistochemistry results.(6) The correlation analysis show that apoptosis rate ,CHOP mRNA, CHOP protein and P-PERK protein were in negativly correlated with pulmonary function(FEV0.3/FVC (%) and FEV0.3 (ml)) .apoptosis rate showed significant positive correlations with CHOP mRNA and CHOP protein. CHOP protein was positively correlated with P-PERK protein.ⅡResults of clinical research(1) The pulmonary function (FEV1.0/FVC(%) and FEV1(%))greatly decreased in smoking group in comparison with control group(FEV1.0/FVC(%)79.46±6.62 (%) VS 88±6.82 (%),FEV1(%)81.4±6.83 (%) VS89.31±6.06 (%) P<0.05),markedly decreased in COPD group(FEV1.0/FVC(%)58.54±6.23(%) VS79.46±6.62(%) ,FEV1(%)63.50±7.20 (%) VS81.4±6.83 (%) P <0.05).(2)Compared with control group, Compared with control group, COPD group showed thickened bronchial epithelium and smooth muscle, with shedding epithelial cells and the influx of imflammatory cells in the airway, expanded alveolar ducts ,alveolar capsule and alveolar, ruptured alveolar walls and enlargement of airspaces which partly merged into bulla.There is no significance between control group and non-COPD smoker group.(3) The results of TUNEL show that the apoptotic cell is alveolar epithelial cellⅠ(ACEⅠ) and alveolar epithelial cellⅡ(ACEⅡ), vascular endothelial cell(VEC) and bronchial epithelial cell.the apoptosis rate is low in control group(13.5±2.5(%)),significantly increased in smoking group(25.6±1.7 (%) VS 13.5±2.45(%),P <0.05),and markedly elevated in COPD group(34.6±3.0 (%) VS 25.6±1.7 (%),P <0.05).(4) In situ hybridisation showed that CHOP mRNA expression levels was mainly located in AEC. In situ hybridisation demonstrated that the expression of CHOP mRNA was low in the control group, greatly increased in rats exposed to 2 months CS(P <0.05), even higher in rats with 4 months CS exposure(P <0.05), and then only slightly altered after smoking cessation(P >0.05), which parallels the in situ hybridisation results.(5)Immunohistochemistry results showed that the expression of P- PERK is mainly stained in cytoplasm of alveolar epithelial cell,vascular endothelial cells and bronchia epithelial cell. while the expression of CHOP was mainly located in nucleus of alveolar epithelial cell.the expression of CHOP is low in control group,greatly elevated in smoking group,and markedly increased in COPD group. Unlike the expression of p-PERK, the total PERK level did not change between the control group and those smoking group.(6) The correlation analysis show that apoptosis rate ,CHOP mRNA, CHOP protein and P-PERK protein were in negatively correlated with pulmonary function(FEV0.3/FVC (%) and FEV0.3 (ml)) .Apoptosis rate showed significant positive correlations with CHOP mRNA and CHOP protein. CHOP protein was positively correlated with P-PERK protein.[Conclusion]:1.PERK/CHOP signal pathway involved the lung structure cell apoptosis endoplasic reticulum stress induced.2 . Endoplasmic reticulum stress induced apoptosis plays important roles in mechanism of COPD.