The Regulation And Mechanism Of Estrogen On Allergic Airway Inflammation In Mice

Asthma is a chronic disease with high prevalence rate worldwide that characterized by airway inflammation,reversible airway obstruction and hyper-responsiveness?AHR?causing dyspnea,cough,and wheezing,affecting more than 300 million patients wordwild.There is a gender disparity in asthma prevalence.Several studies have demonstrated that females have an increased and more severe asthma as compared to males Asthma symptoms and incidence in women appear to change with various stages of life such as puberty,menstruation,pregnancy,and menopause,therefore asthma is closely related to estrogen.Particularly,estrogen modulates the immune function via its receptors,estrogen receptor?,??ER?,ER??and G protein-coupled receptor?GPR?30which expressed by a large amount of immune regulatory cells.Estrogen has many potential effects on inflammation,metabolism,and immunity,especially airway hyper-responsiveness?AHR?.Studies have found that estrogen can reduce airway inflammation,but the impact of estrogen on asthma is still controversial.In the past decades,growing evidence has indicated that the nucleotide-binding domain and leucine-rich repeat protein 3?NLRP3?inflammasome plays a critical role in the airway inflammation such as asthma.Danger-associated molecular patterns?DAMP?or pathogen-associated molecular patterns?PAMP?signals that promote the assembly of the apoptosis-associated speck-like protein containing CARD?ASC?and pro-caspase-1and then trigger NLRP3 inflammasome,meanwhile the other signal,toll-like receptor?TLR?/nuclear factor?NF?-?B pathway,upregulate the expression of pro-inflammatory cytokines interleukin 1?and18?pro-IL-1?and pro-IL-18?.The activated NLRP3inflammasome cleaves the pro-caspase-1 into activated form,which consequently processes the inactivated forms of IL-1?and IL-18 to their activated secreted forms.Recent reports have indicated that the level of NLRP3 inflammasome increased in the challenge with allergen.Moreover,it has been implicated that NLRP3 specific inhibitor reverse the neutrophilic inflammation in model of allergic airways disease.Recently,emerging evidence has showed that estrogen as an inflammatory protective factor can suppress the inflammation mediated by NLRP3 inflammasome.In this study,we mainly observed the effect of estrogen on airway inflammation in asthma mice and the regulation of NLRP3 inflammasome.We used the mouse macrophage cell line RAW264.7 induced by S.aureus to observe the effect of estrogen on the NF-?B/NLRP3signaling pathway at the cellular level.In addition,mice of allergic airway inflammation induced by OVA were used to further investigate the effects of estrogen on airway inflammation and NF-?B/NLRP3 signaling pathway in lung tissue,and to further discuss the mechanism of estrogen ameliorates allergic airway inflammation.Objective1.To study the effects of estrogen on the expression of NF-?B,NLRP3 and ASC proteins of macrophages induced by Staphylococcus.aureus.2.To investigate the regulation of estrogen on allergic airway inflammation and NF-?B/NLRP3 signaling pathway and downstream inflammatory factors in allergic mice.Methods1.Observe the effect of estrogen on the expression of NF-?B,NLRP3 and ASC protein in macrophages induced by Staphylococcus aureus.The mouse macrophage cell line RAW264.7 was planted in a 12-well cell culture dish(density 2.5×10⁵/well,pre-treated with estrogen?E2:17?-estradiol?at a concentration of10^-8mol/l for 24 h,then stimulated with S.aureus for 1 h.After harvesting the cells and extracting proteins,Western Blot was used to detect the expression of NF-?B,NLRP3and ASC proteins.2.Effect of estrogen on allergic airway inflammation in miceC57BL/6 female mice were randomly divided into 5 groups:?1?sham operation goup?sham??2?sham+chicken ovalbumin sensitization group?sham+OVA??3?ovariectomy,group?OVX??4?OVX+OVA?5?OVX+OVA+E2?E2:17?-estradiol?.After establishing ovariectomy and OVA challenge and sensitization models,lung tissue,serum and alveolar lavage fluid were collected from each group.Histology were determined by H&E and PAS staining to observe the difference of airway inflammation in mice.The total number of inflammtatory cells and classification counts in alveolar lavage fluid?BALF?were performed by Wright's staining.The levels of IgE,IL-6 and TNF-?was detected by ELISA kit.3.Evaluate the effects of estrogen on NF-?B/NLRP3 signaling pathway of allergic airway inflammation in miceThe lung tissue and BALF of the above 5 groups of mice were obtained.The mRNA levels of NLRP3 inflammasome and downstream inflammatory factors,IL-1?and IL-18 were detected by RT-PCR.NF-?B,NLRP3,ASC and caspase-1 protein expressions were evaluated by Western blot.Immunofluorescence assay was aslo used to detect NLRP3.ASC and caspase-1 proteins.IL-1?and IL-18 levels were in BALF were investigated by ELISA.Results1.Estrogen inhibits the activation of NF-?B/NLRP3 signaling pathway in macrophages induced by Staphylococcus aureusThe protein expressions of NF-?B,NLRP3 and ASC in RAW264.7 cells was signifiantly increased after S.aureus stimulation.Estrogen ameliorated the expression of NF-?B,NLRP3 and ASC.2.Estrogen significantly inhibits OVA-induced airway inflammationAfter OVA sensitization and stimulation,the bronchial epithelial cells of the mice were edematous and deformed,and the bronchial wall was thickened.A large number of inflammatory cells infiltrated around the tracheal wall,the epithelial goblet cells proliferated obviously,the mucus secretion increased significantly.The level of OVA-specific Ig E extremely raised in OVA-induced mice.Moreover,the total count of leukocytes,eosinophils,lymphocytes,neutrophils and monocytes of BALF were increased significantly in OVA group,as well as IL-6 and TNF-?.In OVX+OVA+E2mice,the airway inflammation and inflammatory cell infiltration was significantly reduced compared with OVX+OVA group,while E2 treatment also significantly decreased the levels of serum IgE levels,inflammatory cells in BALF,IL-6 and TNF-?.3.Estrogen significantly inhibits NF-?B/NLRP3 signaling pathwayAfter OVA sensitization and challenge,the expressions of NF-?B,NLRP3,ASC,caspase-1 and IL-1?in lung tissue of mice were significantly increased.Furthermore,upon OVA-challenge,E2 treatment not only down-regulated the expressions of NF-?B,NLRP3,ASC and caspase-1,but also decreased the level of transcription of the mRNA levels of NLRP3,ASC,caspase-1 in comparison with those in OVX-OVA group.In addition,estrogen replacement reversed OVA-induced increase of the level of mRNA in lung tissue and concentration of IL-1?in BALF.However,E2 treatment did not affect the IL-18 levels in OVX-OVA mice.Conclusions1.Estrogen inhibits the activation of the NF-?B/NLRP3 signaling pathway in the mouse macrophage cell line RAW264.7 induced by Staphylococcus aureus.2.Estrogen ameliorates airway inflammation in allergic mice.3.The NF-?B/NLRP3 signaling pathway is activated in allergic mice,and estrogen improve airway inflammation by inhibiting the activation of the NF-?B/NLRP3signaling pathway.