Effects Of Oxidative Stress Induced By PM_2.5 In Activation And Mitochondrial Damage Of Mast Cells

Background and objectives:To a certain extent,fine particulate matter(PM_2.5)pollution is a by-product of the transformation of global urbanization and industrialization to green development.Driven by globalization,it will accompany the entire process of human society’s technological,economic and cultural development.PM_2.5 is rich in various metal elements,inorganic salts,organics（such as polycyclic aromatic hydrocarbons）and black carbon,which can enter the alveoli with respiratory movement.On one hand,PM_2.5 can directly stimulate the alveolar mucosa and the cells around the capillaries（such as endothelial cells,lymphocytes,lung macrophages and mast cells,etc.）,inducing various types of hypersensitivity or toxic reactions;on the other hand,it can also penetrate the alveolar air-blood barrier into the blood and accumulate in some specific organs or tissue cells through circulatory system,causing a series of negative pathophysiological effects.Research has found that exposure of respiratory tract to PM_2.5 has become an important reason for the increasing incidence of various allergic diseases year by year.PM_2.5 exposure from traffic exhaust is positively correlated with asthma symptoms.Each increase of its atmospheric concentrations by 5μg/m³ will increase the incidence of acute wheezing and shortness of breath by 10%and28%,respectively.The latest research also shows that PM_2.5 can directly cause atopic dermatitis（AD）,eczema,urticaria and other allergic skin diseases.Allergic reaction is an"excessive"response to protect the body when allergens enter and stimulate the immune system.The key part of the reaction is activation of mast cells（MCs）in the body.In addition to classic allergen-specific Ig E allergic reaction pathway,MCs can also be directly activated and degranulated by nicotine,ethanol and other substances,resulting in an allergy-like reaction in vivo and in vitro.Our previous study has confirmed that PM_2.5 can activate MCs（LAD2）and induce degranulation to releaseβ-Hexosaminidase（β-Hex）and IL-4.This process may be related to increasing accumulation of intracellular reactive oxygen species（ROS）.In given conditions,the electrons leak from the electron transport chain（ETC）of mitochondria（ie,“electron leakage”）,and subsequently,oxygen（O₂）is incompletely reduced to superoxide（·O₂^-）in advance.Most of intracellular ROS is generated during this process,which is completed by respiratory chain protein complex I,II and III（Cox I～III）located on the inner mitochondrial membrane.The respiration of eukaryotic cells depends on the mitochondrial membrane potential（MMP）formed by transporting of electrons in mitochondrial ETC and pumping of protons（H⁺）in the matrix.Electrons in ETC pass through Cox I,II,III and finally transmit to O₂ in Cox IV,and drive the H⁺reflux in the mitochondrial inner membrane space from matrix,catalyzing oxidative phosphorylation to generate ATP for energy supplement.In bronchial epithelial cells and keratinocytes,studies have also indicated that PM_2.5 can induce ROS production and rapid depolarization of MMP,which subsequently affect cellular respiration,changing energy supply and causing cell apoptosis.Skin is the largest organ of human body,and external environmental factors,especially atmospheric PM_2.5,have a more direct,rapid and obvious effect on skin allergy-related symptoms.Therefore,the impact of PM_2.5 on the skin and its mechanism have attracted more and more attention in recent years.In this experiment,PM_2.5 was used to treat MCs to analyze the levels of oxidative stress and degranulation;Then,after pre-treatment with N-acetyl-L-cysteine（NAC）of MCs to furtherly interfere with the generation of ROS,the possible relationship between oxidative stress induced by PM_2.5 and activation（degranulation）of MCs was further explored;Finally,Meta-analysis was conducted to quantitatively analyze the odds ratio（OR）of PM_2.5 to allergic skin diseases（AD,eczema and urticaria）,and to macro-evaluate the exposure-response relationship of PM_2.5 pollution on the risk of allergic skin diseases.Experiment 1:Methods:1.PM_2.5 samples were collected using a PM sampler（TH-150A）in the urban area of Chongqing,and suspensions of PM_2.5 in DMEM were prepared;2.MCs（P815）were cultivated and treated with various concentrations of PM_2.5suspensions（0-200μg/ml）.Cell viability,intracellular ATP content and activities of Cox I and Cox III were determined,and then alteration of MMP levels were analyzed by flow cytometry;3.MCs（P815）were cultivated and treated with different concentrations of PM_2.5suspensions（0-200μg/ml）in the absence or presence of NAC（10 m M）for 6 h and 24 h,respectively.Cell viability,ROS generation,β-Hex release rate and IL-4 secretion were measured.Results:1.Treatment of MCs with PM_2.5,cell viability and intracellular ATP content were gradually reduced with the increase of PM_2.5 concentrations;the ratios of normal MMP cells in each group were significantly lower than that of the control group;the activity of mitochondrial respiratory chain Cox III was significantly decreased;the activity of mitochondrial respiratory chain Cox I was significantly reduced only when PM_2.5concentration reached 200μg/ml.2.Co-treatment of MCs exposure to PM_2.5 with 10 m M NAC,cell viability was significantly reduced;ROS production,β-Hex release rate,and IL-4 content were both significantly decreased.Conclusion:1.Cell viability of MCs can be inhibited by PM_2.5 exposure in a concentration-dependent manner;PM_2.5 treatment induces impairment of ATP synthesis,substantial generation of ROS,disruption of MMP level,and suppression of mitochondrial Cox III activity in MCs;Levels of oxidative stress in MCs are exacerbated,and mitochondrial membrane function and energy supply can be impaired by treatment with PM_2.5.We evaluate that the mechanism of cell oxidative stress induced by PM_2.5 may be related to the inhibition of Cox III activity.2.ROS generation can be reduced by co-treatment with antioxidant NAC,which in turn ameliorates oxidative stress in MCs.Release ofβ-Hex and IL-4 significantly increase in MCs exposed to PM_2.5,while co-incubation with NAC shows an increase in resistance to degranulation;the observed inhibitory trends are parallel to those of attenuation in generation of ROS,indicating that elevated oxidative stress plays a critical role in regulating PM_2.5-induced allergic inflammation.Experiment 2:Methods:Pub Med,Web of Science,CNKI,Sino Med,WANFANG DATA and VIP databases were retrieved online on November 10,2019.According to the selected search terms,related domestic and international publications were collected systematically.Meta-analysis was performed on the relationship between exposure of PM_2.5and allergic skin diseases（AD,eczema,and urticaria）.Observational studies（including cohort studies,time-series studies,case-crossover studies,and cross-sectional studies）were included.Results of those studies can be expressed in terms of effect size,which represented the exposure dose-response relationship.Effect size of each study can be converted into OR and its 95%confidence interval（CI）of the occurrence of allergic skin disease for every 10μg/m³increase in atmospheric PM_2.5 concentrations.OR of PM_2.5 to allergic skin diseases was evaluated,and subgroup and sensitivity analysis were performed;Publication bias was assessed by Egger’s and Begg’s test,and OR was adjusted using trim and fill method.Results:1.A total of 15 studies on the relationship between PM_2.5 exposure and allergic skin diseases were included.2.When outdoor PM_2.5 concentrations increased by 10μg/m³,risk for allergic skin diseases was OR=1.0066,95%CI=[1.0033,1.0100];subgroup analysis showed risk for AD was OR=1.0320,95%CI=[1.0056,1.0590];risk for eczema was OR=1.0066,95%CI=[1.0029,1.0103];risk for urticaria was OR=0.9994,95%CI=[0.9852,1.0139].3.Sensitivity analysis:no significant changes were found in the new combined OR,and the estimated values all fell within the CI after excluding studies one by one,indicating that results of Meta-analysis were stable and reliable;the publication bias test and adjust:the adjusted OR=1.0070,95%CI=[1.0030,1.010].Conclusion:PM_2.5is a key risk factor for the occurrence of allergic skin diseases.For every 10μg/m³ increase in atmospheric concentrations,the risk of allergic skin diseases increases by0.70%.