| Objective:To clarify the role and mechanism of endoplasmic reticulum stress CHOP signaling pathway in cigarette smoke-induced COPD and COPD related pulmonary hypertension.Methods: Forty-two wild-type C57BL/6J male mice were randomly divided into 7 groups: 4 months blank group,4 months smoking group,4months PBA smoking intervention group(smoking 4 months,starting with gavage plus PBA),6 months blank group,smoking 6 months early intervention group(smoking 6 months,starting with gavage plus PBA),smoking 6 months late intervention group(smoking 6 months,after 4months with gavage plus PBA).The pulmonary ventilation function of mice in each group was detected by small animal lung function instrument,and the pulmonary artery pressure of mice was detected by right ventricular puncture,and the right heart index was measured by mouse heart weighing.The destruction of pulmonary parenchyma andpulmonary vascular remodeling were observed by optical microscope.Detection of endoplasmic reticulum marker protein cadranin by immunofluorescence TUNEL labeling method the proportion of apoptosis in mouse lung tissue.western-blot methods were used to detect endoplasmic reticulum stress marker protein BIP protein levels.Western-blot and RealTime-PCR methods were used to detect CHOP 、Bcl-2 、 Bim 、 Puma 、 DR5 gene protein and mRNA expression levels,respectively.western-blot methods and immunofluorescence methods were used to detect caspase8 and caspase9 protein levels,caspase8 and activity,respectively.Results:1.The results of pulmonary ventilation function in mice : smoking 4months group representing the control group set of peak expiratory flow,peak inhale flow rate,minute ventilation volume declined significantlyThe inspiratory peak velocity(PIF),expiratory peak velocity(PEF),ventilation volume(MV)decreased significantly(PIF2.39±0.16(ML/S)vs4.06±0.35(ML/S),PEF4.08±0.28(ML/S)VS6.55±0.52(ML/S),MV121.5±3.27(ML/S)VS173.78±7.8(ML/S),P<0.05);The4-month smoking intervention group was better than the 4-month smoking group(PIF 3.51 0.08(ml/s)vs 2.39 0.16(ml/s),PEF 5.54 0.12(ml/s)vs 4.08 0.28(ml/s),mv 148.94 4.77(ml/s)121.5 3.27(ml/s),all p< 0.05).Compared with the 6-month control group,the 6-month smokinggroup decreased significantly(PIF 2.08 0.09(mL/s)vs 4.22 0.56(mL/s),PEF 6.54 0.58(mL/s)vs 3.51 0.48(mL/s),MV 174.23 3.29(mL/s)vs103.03 15.02(mL/s));Compared with the group smoking for 4 months,the group smoking for 6 months decreased(PIF 2.08 0.09(ml/s)vs 2.390.16(ml/s),PEF 3.51 0.48(ml/s)vs 4.08 0.28(ml/s),mv103.03 15.02(ml/s)121.5 3.27(ml/s),all p < 0.05).The PBA late intervention group after 6 months of smoking decreased(PIF 3.11 0.08(ml/s)vs 3.84 0.12(ml/s),PEF 5.08 0.07(ml/s)vs 6.06 0.06(ml/s),MV136.77 7.04(ml/s)vs 159.35 7.15(ml/s),all p < 0.05)compared with the PBA early intervention group after 6 months of smoking.The 6-month smoking intervention group was better than the 6-month smoking group(p < 0.05)2.Detection of mean pulmonary artery pressure and right heart index of mice in each group: There was no significant difference between the4-month control group and the 6-month control group.The 4-month smoking group was higher than the PBA intervention group.The 6-month smoking group was higher than the 6-month control group,the 6-month PBA late intervention group and the 6-month PBA early intervention group.The PBA late intervention group smoking for 6 months was higher than the PBA early intervention group smoking for 6 months.The6-month smoking group was higher than the 4-month smoking group(all p < 0.05).3.Pathomorphology of lung tissue and pulmonary vessels in mice ofeach group: The integrity of bronchial goblet cells in mice of smoking 4months group is poor,basement membrane smooth muscle is unevenly thickened or fractured,inflammatory cell infiltration is visible in bronchial lung,and some alveoli are fractured.The above-mentioned situation in the intervention group in 4 months is more obvious than that in the control group in 4 months,but it is less severe than that in the smoking group in 4 months.The integrity of goblet cells,thickening and rupture of basement membrane smooth muscle,infiltration of inflammatory cells in lung and rupture of alveoli were significantly worse in the smoking group in 6 months than in the smoking group in 4 months.Some alveoli were fused and bullae were formed.Pulmonary artery wall was significantly thicker than in the smoking group in 4 months and the control group.This change can also be seen in the intervention group smoking for 6 months,but the degree is less than that in the smoking group smoking for 6 months.Among them,the above changes were more obvious in the PBA late intervention group after smoking for 6 months than in the PBA early intervention group after smoking for 6 months.4.Detection results of endoplasmic reticulum stress marker protein BIP protein expression level in mice of each group: Histone expression level in smoking group for 4 months was lower than that in control group for 4months and PBA intervention group for 4 months,while histone expression level in smoking group for 4 months was lower than that incontrol group for 4 months.Histone expression level of 6 months after smoking was lower than that of 6 months control group,6 months PBA late intervention group and 6 months PBA early intervention group.The PBA late intervention group smoking for 6 months is lower than the PBA early intervention group smoking for 6 months.The 6-month smoking group was lower than the 4-month smoking group(all p < 0.05).5.The expression level of CHOP,Bcl-2,Bim,Puma,DR5 gene protein and mRNA,caspase8,caspase9 protein level and activity test results of mice in each group: smoking for 4 months group is higher than that of 4months control group,smoking for 4 months PBA intervention group,smoking for 4 months PBA intervention group is higher than that of 4months control group;The six-month smoking group is higher than the six-month PBA late intervention group,the six-month smoking PBA early intervention group and the six-month control group.The PBA late intervention group smoking for 6 months is higher than the PBA early intervention group smoking for 6 months.The 6-month smoking group was higher than the 4-month smoking group,all p< 0.05.6.The expression level of cadherin in mice and the detection results of apoptosis in lung structure cells: the smoking group for 4 months was higher than the control group for 4 months,the smoking group for 4months PBA intervention group,and the smoking group for 4 months PBA intervention group was higher than the control group for 4 months.The six-month smoking group is higher than the six-month PBA late intervention group,the six-month smoking PBA early intervention group and the six-month control group.The PBA late intervention group smoking for 6 months is higher than the PBA early intervention group smoking for 6 months.The 6-month smoking group was higher than the4-month smoking group,all p <0.05.Conclusion:1.Endoplasmic reticulum stress CHOP signaling pathway may induces endoplasmic reticulum stress-induced apoptosis through Bcl-2 and DR5 signaling pathways to promote smoking-related COPD and development of pulmonary hypertension in mouse lung structural cells.2.PBA may inhibit endoplasmic reticulum stress CHOP signaling pathway to reduce lung structural damage and lung ventilation function in smoking-related COPD and pulmonary hypertension mice,and improve pulmonary vascular remodeling in mice.PBA plays a therapeutic role in smoking-related COPD and pulmonary hypertension mice. |
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