| Purpose:With the acceleration of China’s industrialization and urbanization,more and more people pay attention to environmental problems.Air pollution affects human health.Fine particulate matter(PM2.5)in the air is characterized by small diameter,large surface area,organic compounds,heavy metals and pathogenic bacteria,and is an important factor causing body diseases due to air pollution.In clinical work,the season of severe air pollution is often the season of acute exacerbation of chronic obstructive pulmonary disease and high incidence of pulmonary infectious diseases.Therefore,it is very important to explore effective ways to deal with the adverse effects of air pollution on respiratory health.Nuclear factor E2-related factor 2(Nrf2)is a major regulator of antioxidant response,and many of its downstream target genes are involved in correcting intracellular REDOX imbalance.The respiratory system is continuously exposed to various pollutants and oxidants,and the REDOX balance maintained by Nrf2 is critical for maintaining airway health.To date,researchers have found that Nrf2 deletion leads to a variety of respiratory diseases,including respiratory infections,chronic obstructive pulmonary disease(COPD),acute respiratory distress syndrome(ARDS),asthma,idiopathic pulmonary fibrosis(IPF),etc.,and activation of Nrf2 has a protective effect on these lung diseases.Ferroptosis is a new type of programmed cell death,which is mainly characterized by excessive accumulation of reactive oxygenspecies(ROS)in an iron-dependent manner.Ferroptosis is involved in the occurrence and development of many pulmonary diseases,and plays a key role in the pathogenesis of chronic obstructive pulmonary disease,pulmonary fibrosis,pulmonary infection,asthma and acute lung injury.It has been reported that PM2.5 can induce iron death in epithelial cells,mouse lung tissues and human bronchial epithelial cells.However,the exact mechanism and the role of Nrf2 in this process are not clear.This study aims to explore the mechanism of Nrf2 signaling pathway in PM2.5-induced lung injury and iron death,and to explore a new target for alleviating respiratory diseases caused by air pollution.Methods:In this study,the mechanism of Nrf2 signaling pathway alleviating PM2.5-induced lung injury by regulating iron death was studied by using PM2.5-induced C57BL/6 mouse model and human bronchial epithelial cell(BEAS-2B)model.1.Animal experiments: C57BL/6 Wild-type(WT)mice and Nrf2 knockout(Nrf2-/-)mice were randomly divided into blank group and PM2.5 exposure group(a total of 4 groups of 8 mice in each group).Operation: Feed for 3 days under SPF condition.The final concentration of PM2.5 was 220μg/50 μ L with sterile normal saline at room temperature.For the PM2.5 exposed group,50 ul of PM2.5 normal saline solution was nasal dropped every day,and for the non-exposed group,50 ul of normal saline was nasal dropped every day,and PM2.5 stimulation was given for 7 days.The mice were sacrificed 24 h after the last stimulation and bronchial lavage fluid(BALF),blood and lung tissue were collected.Nrf2-/-mice and WT mice were purchased from Jackson Laboratory and Beijing(Certificate SYXK(JI)2019-0012).(1)The changes of lung histopathology,inflammatory factors Ll-6 and TNFα in bronchial lavage fluid of mice induced by PM2.5 were detected.(2)Lung tissues of mice were stained with HE,PAS and DAB.(3)The contents of iron ion,MDA and GSH in lung tissues of mice were detected byusing corresponding kits.(4)The protein expressions of Nrf2,HO-1,NQO1,x CT,GPX4,TFR,FTH1 and FTL in lung tissues of mice were detected by Western Blot.2.Cell experiments: Be AS-2B cells were transfected with Nrf2 si RNA for 72 h,and then Western blotting was performed with anti-NRF2 antibody(1:1000)to evaluate the transfection efficiency.The si RNA group with the highest transfection efficiency was selected for subsequent cell experiments.Normal BEAS-2B cells and Nrf2 knockdown BEAS-2B cells were pretreated with fer-1,an iron death inhibitor,for 1 h.Then the cells in the two groups were induced with PM2.5 for 18 h,respectively,and collected.Beas-2b cells were pretreated with Nrf2 activator sulforaphane(SFN)for 1 h and then induced with PM2.5for 18 h to collect cells.(1)CCK-8 assay was used to detect the toxicity of PM2.5 induced normal BEAS-2B cells and Nrf2 knockdown BEAS-2B cells.(2)Confocal iron orange staining was used to detect iron ion levels in PM2.5 induced normal BEAS-2B cells and NRF2-knocked down BEAS-2B cells.(3)ROS production in PM2.5 induced normal BEAS-2B cells,NRF2-knockdown BEAS-2B cells and NRF2-activated Be AS-2B cells was detected.(4)The contents of iron ion,MDA and GSH in PM2.5 induced normal BEAS-2B cells,NRF2-knockdown BEAS-2B cells and NRF2-activated Be AS-2B cells were detected by using corresponding kits.(5)Western Blot was used to detect Nrf2,ho-1,NQO1,x CT,GPX4,TFR,FTH1 and FTL protein expressions in HBEC cells induced by PM2.5,nrf2-knockdown beas-2b cells and nrf2-activated beas-2b cells.Results:1.Animal experiment:(1)In the PM2.5 induced mouse lung injury model,HE staining of the lung tissue of mice in the PM2.5 group showed that the alveolar wall was damaged and inflammatory cells gathered around the airway.PAS staining showed increased production of bronchial mucus.The increase of il-6 and TNFα in bronchial lavage fluid(BALF).(2)In the PM2.5 induced lung injury model of mice,iron ion in serum of mice in PM2.5 group increased,iron deposition was observed by DAB staining,MDA production and GSH consumption in lung tissues increased.(3)In the PM2.5 induced mouse lung injury model,the expression of Nrf2 and its downstream HO-1 and NQO1 were slightly activated,while the protein expression of x CT and GPX4 decreased in PM2.5 group.(4)In the PM2.5 induced lung injury model,the expression of TFR,FTH1 and FTL proteins in the lung tissues of mice in PM2.5 group increased.(5)Compared with WT group,Nrf2-/-group mice showed more severe inflammatory response,as HE staining showed more damage to alveolar wall,PAS staining showed more obvious goblet cell proliferation,and the increase of inflammatory factors IL-6 and TNFα in bronchial lavage fluid was also more significant.(6)Compared with WT group,Nrf2-/-group mice showed more severe iron ion accumulation and iron metabolism disorder,which was manifested by more significant increase of iron ion in serum,more severe iron deposition and more significant increase of ferritin TFR,FTH1 and FTL expression by DAB staining.(7)Compared with WT group,Nrf2-/-group mice showed more severe lipid peroxidation,showing more obvious increase of MDA and consumption of GSH,and more obvious decrease of protein expression levels of x CT and GPX4.2.Cell experiment:(1)Compared with normal BEAS-2B cells,Nrf2 knockdown BEAS-2B cells showed more severe cell death in PM2.5 induced in vitro model.(2)In the PM2.5 induced in vitro model,compared with normal BEAS-2B cells,be AS-2B cells with low Nrf2 knockdown showed more obvious accumulation of iron ions,increased ROS,MDA and CONSUMPTION of GSH.(3)In PM2.5 induced in vitro model,compared with normal BEAS-2B cells,the expression of Nrf2,HO-1,NQO1,x CT and GPX4 in BEAS-2B cells with Nrf2 knockdown was more significantly decreased,while the expression of TFR,FTH1 and FTL was more significantly increased.(4)In pm2.5-induced in vitro model,compared with normal BEAS-2B cells,the protein expressions of Nrf2,HO-1,NQO1,x CT and GPX4 in NRF2-activated BEAS-2B cells were increased;The production of ROS and MDA decreased,and the consumption of GSH decreased.Conclusion:PM2.5 induced ferroptosis in vivo and in vitro.Nrf2 signaling pathway regulates ferroptosis,improves lipid peroxidation,alleviates PM2.5 induced bronchial epithelial cell death and lung tissue injury in mice... |
PDF Full Text Request