Role Of Endoplasmic Reticulum Stress In PM2.5-induced Airway Inflammation And Apoptosis

Objective: To investigate the role and molecular mechanism of endoplasmic reticulum stress in inflammation and apoptosis of human airway epithelial cells induced by fine particulate matter(PM2.5),with the hope of identifying new therapeutic strategies.Methods: The two parts of this research were clinical tissue specimen verification and in vitro cell experiment:1.Clinical tissue specimen verification: Samples of surgically removed excess lung tissue(no tumor cells confirmed by immunohistochemistry)were collected from 24 patients,including 14 patients with a history of chronic obstructive pulmonary disease(COPD)and 10 patients in the control group(no history of COPD).The expressions of GRP78 and PERK proteins in human bronchopulmonary tissue were observed by immunohistochemistry.2.In vitro cell experiment: Human airway epithelial cells,16-HBE,were cultured in vitro.In order to select a reasonable intervention concentration and time,the CCK8 assay was performed to evaluate the cell viability of each group before and after stimulation with PM2.5 at different times.16-HBE cells were then divided into three groups: the untreated control group,the PM2.5 stimulation group,and the PM2.5 +selective PERK inhibitor GSK2606414 group,in which the cells were pretreated with GSK2606414 before the treatment with PM2.5.Western blot was utilized to measure the expression levels of endoplasmic reticulum(ER)stress-related proteins,such as GRP78,CHOP,PERK,p-PERK,e IF2α and p-e IF2α,before and after the interventions.ELISA assay was used to determine the concentrations of tumor necrosis factor(TNF)-α,interleukin(IL)-6 and mucin(MUC)5AC.Images of GRP78 and MUC5 AC protein expression in each group were observed by immunofluorescence chemistry.The apoptosis of each group was detected by flow cytometry.Result: 1.Clinical experiment results showed that compared with non-COPD patients,the expression levels of GRP78 and PERK proteins in the lung tissues of COPD patients were significantly increased.2.Cell experiment results:(1)CCK8 results revealed that 100μg/m L PM2.5 for 24 hours was the optimal concentration and time.Further experimental results illustrated that after PM2.5 stimulation for 24 hours,the content of reactive oxygen species,the number of apoptotic cells and the relative concentrations of TNF-α,IL-6 and MUC5 AC in cell supernatant were significantly increased compared to the control groups(all P < 0.05).Immunofluorescence detection of GRP78 and MUC5 AC protein expression in the PM2.5 groups was also stronger than the control groups.Besides,the expression levels of endoplasmic reticulum stress-related proteins in the PM2.5 groups,including GRP78,CHOP,PERK,e IF2α,p-PERK and p-e IF2α,were also increased compared to the control groups(all P < 0.05).(2)After the intervention of the inhibitor GSK2606414,compared with the PM2.5 groups,the contents of the above ER stress-related proteins,the inflammatory factors,MUC5 AC and reactive oxygen species in the cell supernatant were markedly relieved.The number of apoptosis and the fluorescence expressions of GRP78 and MUC5 AC proteins were also prominently decreased(all P< 0.05).Conclusion: PM2.5 could promote airway epithelial oxidative stress,apoptosis,inflammation and mucus hypersecretion through ER stress.In addition,PERK/e IF2α,the ER stress-related signaling pathway,may play an important role in regulating oxidative stress,apoptosis,inflammation and mucus hypersecretion of airway epithelial cells.