OLA1 Gene Regulates Endoplasmic Reticulum Stress In The Pathogenesis Of Chronic Obstructive Pulmonary Disease

BackgroundChronic obstructive pulmonary disease(COPD)is a common disease and frequently-occurring disease that seriously endangers human health.It manifests as persistent respiratory symptoms and airflow limitation.Respiratory symptoms and airflow limitation are caused by toxic particles or Gas-induced airway and / or alveolar abnormalities.One of the main causes of 85%-90% of COPD patients is smoking.Injury / apoptosis of lung structural cells under smoking-induced oxidative stress(OS)is a pathological basis for the development of emphysema in COPD resulting in limited airflow.There is ample evidence that high oxidative stress in cigarette smoke exposure triggers endoplasmic reticulum stress(ERS)-mediated lung injury / apoptosis and plays an important role in the pathogenesis of COPD,but for cigarette exposure The mechanism of specific regulation of ERS intensity changes and cellular damage in the lower lung tissue cells is unclear.Therefore,the temporal and spatial variation of ERS transition from cytoprotective mechanism to cellular injury / apoptosis mechanism under cigarette exposure to hyper-oxidative stress were further elucidated.It is expected that the fate of cell injury / apoptosis under cigarette exposure will be altered from the OS / ERS pathway,thereby preventing / reversing the incidence of smoking-related COPD.Obg-like ATPase 1(OLA1)gene is a newly identified endogenous negative regulator of antioxidant stress,which is widely expressed in different types of tissues and cell lines including lung tissue.The study of its function found that OLA1 may be an important target of regulating ERS under oxidative stress OS.Previous studies showed that down-regulation of OLA1 gene has protective effect on cells treated with sulfhydryl oxidant and H2O2,and its mechanism depends on the regulation of OLA1 on ERS.Since OS / ERS pathway is closely related to the injury / apoptosis of lung structural cells during the development of smoking-related COPD,OLA1 is an important regulator of ERS activation under oxidative stress.Therefore,we propose a scientific hypothesis: "OLA1 May be involved in the pathogenesis of COPD;down regulation of OLA1 can prevent the occurrence of COPD by inhibiting the injury / apoptosis of lung structural cells under the oxidative stress of cigarette exposure by regulating OS / ERS.”Objectives1.To elucidate the correlation between OLA1 expression level and oxidative stress level,ERS activation degree and impaired lung function in lung tissue of smoking COPD patients.2.To elucidate the correlation between OLA1 expression level and oxidative stress level,ERS activation and pulmonary function in lung tissue of rat COPD model.3.To elucidate the molecular mechanism of OLA1 regulating the injury of lung epithelial cells under ERS exposure to cigaratte smoke.Methods1.The lung tissue samples collected from thoracic surgery were divided into three groups: normal lung function non-smoking group,normal lung function smoking group and smoking COPD group.The clinical data and pulmonary function were collected.mmunohistochemistry was used to detect OLA1,Oxidative stress markers SOD,MDA and ERS-related proteins GRP78,CHOP expression levels,and to explore the correlation between OLA1 expression level and oxidative stress level,ERS activation degree and impaired lung function.2.The rats were divided into control group and cigarette COPD group.The lung function of the rats was collected.the levels of OLA1,oxidative stress markers of SOD,MDA and ERS-related proteins GRP78 and CHOP in lung tissue were detected by immunohistochemistry.The expression of OLA1 and oxidative stress,the degree of activation of ERS,the degree of impaired lung function Correlation.3.A549 cells were cultured in vitro and stimulated by A549 cells at 5% CSE to detect the effect of OLA1 expression on the oxidative stress of cells exposed to cigarettes(SOD,MDA).The expression of OLA1 and ERS-related protein(GRP78,CHOP)was detected by Western blot.4.siRNA down-regulated the expression of OLA1 in A549 cells,A549 cells were stimulated with 5% CSE at different time points,and the levels of OS and MDA were detected.The expression of ERS-related protein(GRP78,CHOP)was detected by Western blot.Results1.Semi-quantitative analysis of immunohistochemistry showed that OLA1,the indicators of oxidative stress SOD,MDA and endoplasmic reticulum stress-related proteins GRP78,CHOP were expressed in human lung epithelial cells and alveolar epithelial cells,OLA1 expression in smoking COPD group and normal lung function smoking group was significantly higher than normal lung function non-smoking group(p <0.05),smoking COPD group was higher than that in normal lung function smoking group(p <0.05);Oxidative stress marker MDA was significantly higher in smoking COPD group and normal lung function smoking group was significantly higher than normal lung function non-smoking group(p <0.05),smoking COPD group was higher than that in normal lung function smoking group(p <0.05),The expression of SOD in smoking COPD group and normal lung function smoking group was significantly lower than that in normal lung function non-smoking group(p <0.05),smoking COPD group was lower than normal lung function smoking group(p <0.05);The expression of endoplasmic reticulum stress-related protein GRP78,CHOP in smoking COPD group and normal lung function smoking group was significantly higher than normal lung function non-smoking group(p <0.05),smoking COPD group was higher than that in normal lung function smoking group(p <0.05);2.The expression of OLA1 in human lung tissue was negatively correlated with pulmonary function(FEV1 / FVC%)(r=-0.667,p <0.01),which was positively correlated with the expression of MDA and was negatively correlated with SOD in oxidative stress markers(r=0.857,p<0.01;r=-0.793,p<0.01),and positively correlated with ERS-related protein GRP78 and CHOP(r = 0.815,p <0.01;r = 0.843,p <0.01);3.Semi-quantitative analysis of immunohistochemistry showed that OLA1,the indicators of oxidative stress SOD,MDA and endoplasmic reticulum stress-related proteins GRP78,CHOP were expressed in the lung tissue of rat bronchial epithelial cells and alveolar epithelial cells,OLA1 expression in cigarette COPD group was significantly higher than that in control group(p <0.05);The expression of MDA in cigarettes COPD group was significantly higher than that in control group(p <0.05).The expression of SOD in cigarette COPD group was significantly lower than that in control group(p <0.05);The expression of ERS-related protein GRP78 and CHOP were significantly higher in cigarette COPD group than in control group(p <0.05);4.The expression of OLA1 in rat lung tissue was negatively correlated with pulmonary function(FEV0.3 / FVC%)(r=-0.861,p<0.01),which was positively correlated with the expression of MDA(r=0.886,p<0.01)and was negatively correlated with SOD(r=-0.934,p<0.01)in oxidative stress markers,and positively correlated with ERS-related protein GRP78 and CHOP(r=0.817,p<0.01;r=0.737,p<0.01);5.Compared with the control group,A549 cells were stimulated with 5% CSE and 1μg / ml LPS for 0,15 min,30min,120 min,120min and 240 min.The cell viability of A549 cells in LPS group decreased with the increase of stimulation time;6.5% CSE stimulated A549 cells at 0,15 min,30min,60 min,120min,240 min,15min after the OS markers SOD activity decreased(p <0.05),MDA expression gradually increased(p <0.05);5% CSE stimulated down-regulation of OLA1 expression in A549 cells at 0,15 min,30min,60 min,120min,240 min,SOD activity also decreased,but the degree of decline was significantly reduced,MDA expression level 15 min,30min rise gradually decreased;7.5% CSE stimulated A549 cells for 0,15 min,30min,60 min,120min,240 min respectively,the expression of CHOP and GRP78 in A549 cells increased at 15,30 and 60 min.5% CSE stimulated OLA1-knockown A549 cells for 0,15 min,30min,60 min,120min,240 min respectivelythe,The changes of ERS-associated protein CHOP and GRP78 in A549 cells treated with 5% CSE decreased,but no significant increase or decrease in CHOP and GRP78 expression was observed.Conclusions1.Compared with normal lung function non-smoking group,high expression of OLA1 expression could be found in bronchial epithelial cells and alveolar epithelial cells in smoking group with and without COPD.group.OS,ERS levels also significantly increased in smoking group with and without COPD.OLA1 levels and OS,ERS levels have significant correlation;rat COPD model lung tissue also showed similar results,suggesting that OLA1 upregulation may be involved in the pathogenesis of smoking-related COPD.2.5% CSE induced the expression of OLA1,the levels of OS and ERS in A549 cells.The down-regulation of OLA1 expression by siRNA could reduce the level of OS and ERS in A549 cells,thereby reducing the injury of A549 cells.