Activation Of The Nrf2-ARE Pathway Improves The Phagocytosis Of Alveolar Macrophages Under The Cigarette Smoke Extract

Chronic obstructive pulmonary disease(COPD)is a common preventable and treatable disease characterized by persistent airflow limitation.It is associated with increased chronic inflammatory reactions of airways and lungs to toxic particles or gases.According to the report of the World Health Organization,COPD is the third largest cause of death in the world,and the death toll in 2015 was 3.2 million.The occurrence and development of COPD is mainly related to smoking.The mortality rate of COPD patients is closely related to smoking.Studies have shown that reduced function of immune cells in the lower respiratory tract increases the probability of pathogen colonization,thereby promoting the development of COPD [1].Alveolar macrophages serve as major members of the lower respiratory immune cells.Its mechanism in COPD has been recognized by scholars at home and abroad [2].Cigarette smoke is one of the major risk factors for COPD.Foreign scholars have pointed out that the composition of cigarette smoke will promote the infiltration of alveolar macrophages(AM)by mast cell-promoting expression of serine serine member S31(Prss31)and polarize to M2 type.It promotes the progression of COPD disease.And also cigarette smoke induces alveolar macrophages autophagy [3].Therefore,finding a corresponding anti-oxidative stress signaling pathway to defense oxidative stress caused by cigarettes and improving phagocytosis of alveolar macrophages can be used as a new research direction in the treatment of COPD.The Nrf2-ARE pathway is a classical anti-oxidative stress pathway.However,whether the activation of Nrf2-ARE pathway can improve the phagocytosis of alveolar macrophages and the intracellular reactive oxygen species under the influence of cigarette smoke is not yet clear.Therefore,this study investigated the phagocytosis of Nrf2-ARE signaling pathway in alveolar macrophages against fluorescein isothiocyanate(FITC)-Staphylococcus aureus.Objective:To explore whether the Nrf2-ARE pathway activators,sulforaphane and terbutyl hydroquinone,can repair the phagocytosis and antioxidant stress ability of alveolar macrophages after cigarette smoke stimulation.Methods:1.Cell Culture: Alveolar macrophages(NR8383)were cultured using F-12 K containing 20% fetal bovine serum and 1% double antibodies(penicillin and streptomycin).2.Preparation of Cigarette Smoke Extracts: Light the cigarettes on the cigarette smoke extract device.The smoke generated by the combustion of the three cigarettes was dissolved in 20 ml of F-12 K broth using the negative pressure generated by the suction of a 50 ml syringe.3.Intervention of drugs and cigarette extracts on alveolar macrophages: A blank control group was established: normal medium treatment for 16 h +24 h.CSE stimulation group: normal medium treatment 16 h + cigarette extract treatment 24 h.Drug intervention group: 2.5?mol/ml sulforaphane and 20?mol/ml tert-Butylhydroquinone(T-BHQ)treatment for 16 h + cigarette extracts for 24 h.4.Detection of phagocytic function of alveolar macrophages: The difference in phagocytosis of alveolar macrophages was examined by flow cytometry.5.Detection of reactive oxygen species in alveolar macrophages: Flow cytometry was used to detect reactive oxygen species in alveolar macrophages from each group.6.Western blot was used to detect the expression level of nuclear protein Nrf2 in alveolar macrophages.7.Detection of cytotoxicity: The cytotoxic effects of drugs and cigarette extracts on alveolar macrophages were examined using a CCK-8 kit(Cell Counting Kit-8,CCK8).Results:1.Compared with the blank group,the phagocytosis function of alveolar macrophages stimulated by cigarette smoke extract was significantly decreased(7001.66±559.47)VS(1508±349.6),P<0.05).2.The effect of 2.5?mol / ml sulforaphane and 20?mol/ml t-butyl hydroquinone could significantly improve the phagocytosis of alveolar macrophage(3034±272;2753±160.1,P<0.05)and reduce the intracellular reactive oxygen species(67.93%±2.05%)VS(38.53%±1.22%);(34.03%±2.27%),P<0.05).3.Western blotting confirmed that after 16 h intervention,sulforaphaneand t-butylhydroquinonecanpromote alveolar macrophages Nrf2 nuclear protein expression via activatingthe Nrf2-ARE pathway(0.87±0.06)VS(2.12±0.03);(2.80±0.06)?P<0.05)?4.Under the action of cigarette smoke extract,we have found that the phagocytosis of alveolar macrophage and the expression of Nrf2 nucleoprotein was positively correlated(r=0.93,P<0.05).The intracellular reactive oxygen species and the expression of Nrf2 nucleoprotein was negatively correlated(r=-0.942,P<0.05).The phagocytosis of alveolar macrophage and the intracellular reactive oxygen species is negatively correlated(r=-0.883,P<0.05).Conclusion:Nrf2-ARE pathway activatorscan improve the phagocytosis of alveolar macrophages caused by cigarette smoke extract and have protective effects on antioxidant stress.