| Background:Bronchial asthma(referred to as asthma)is a heterogeneous disease affecting the respiratory airway,characterized by variable airflow obstructions and airway hyperreactivity.Airway inflammation runs through the occurrence and development of disease.At present,the number of people with the asthma is already close to 300 million worldwide,and the prevalence rate is continuously increasing.It is estimated that the number of people with the disease will reach 400 million by 2025.With the sustaining development of molecular biology,the hypothesis of pathogenesis in asthma also have been constantly updated by the researchers.It is generally believed that the development of asthma is associated with a variety of mechanisms,mainly including airway allergy,the imbalance airway neuromodulation,airway remodelling and airway inflammation,which plays a very important role in regulation for the process of asthma.Some reaschers have shown that the development of asthma is closely related to the key molecules of immunology and the transmission of inflammatory signaling pathways.Information transmission between cells plays an important role in many physiological functions of human body.In recent years,exosomes,which can be released by a variety of cells,have been attracted extensive attention.They are vesicular bodies including proteins,lipids,mRNA,miRNA and other bioactive substances,which can mediate the communication between cells and participate in a variety of pathophysiological processes such as immune response,antigen presentation,inflammation and cell migration.There are more and more evidences that exosomes are closely related to asthma,and it is believed that exosomes can promote the inflammatory response of asthma.In our previous findings,we found that the development of asthma may be related to the increased release of exosomes,which may be one of the key factors leading to the deterioration of airway inflammation in asthma.Therefore,we prepare to serve exosomes isolated from serum of guinea pigs model of allergic asthma as object in this study.To explore whether exosomes could mediate the airway inflammation and pathogenesis of asthma from the level of cell and molecular and provide theoretical value for the diagnosis and treatment of asthma.Objective:To explore serum-derived exosomes can play a pro-inflammatory role on BEAS-2B cells and mediate the pathogenesis of asthma.Methods:1.The female guinea pigs(250±5g)in the HDM group and the control group were sensitized and challenged with HDM and Phosphate buffers,respectively.Then,bronchoalveolar lavage fluid(BALF)and blood were collected,and finally were identified for the model of asthma.2.Exosoems were isolated and extracted from the serum of guinea pigs with asthma by ExoQuick kit.Their morphological characteristics were observed by transmission electron microscope(TEM)and the marker proteins CD63 and HSP70 of exosomes were determined by western blotting(WB).The exosomes and the nucleus were stained by fluorescent dye PKH67 and DAPI respectively.The uptake of exosomes by BEAS-2B cells were observed.3.Subsequently,different concentrations of serum-derived exosomes were added to BEAS-2B cells at different periods.The expression of inflammatory cytokines were detected by RT-PCR and ELISA.4.Serum-derived exosomes were added to BEAS-2B cells and the expression of TLR4,IKK-α/β and p-65 were detected by WB.The expression of p-65 was detected by immunofluorescence.5.After the treatment of BEAS-2B cells with TLR4 blocker TAK242 and NF-kB blocker bay11-7082,they were co-cultured with serum-derived exosomes,and RT-PCR and ELISA were used to determine the effects on the secretion of inflammatory cytokines of BEAS-2B.6.After the treatment of BEAS-2B cells with TLR4 blocker TAK242 at different concentrations,they were co-cultured with serum-derived exosomes and the expression of TLR4,IKK-α/β and p-65 were detected by WB.7.Finally,we screened which components of serum-derived exosomes had an effect on the secretion of inflammatory cytokines in BEAS-2B cells,and verified whether they could change the function of cells by blockers and recombinant proteins in vitro.Results:1.The behavior of the HDM guinea pigs showed dyspnea,pruritus,irritability,urinary and fecal incontinence,hair pricking and so on.2.The results showed that the HDM group presented submucosal edema of bronchial,hyperplasia of mucosal gland,and infiltration of a large number of neutrophils,lymphocytes and a small number of eosinophils in the surrounding of lung tissues.3.Compared with the control group,the total number of cells,lymphocytes,eosinophils and neutrophils in BALF of HDM group were significantly increased(P<0.001).The levels of IL-4 and IL-13 in BALF supernatant of HDM group were significantly increased(P<0.001)compared with the control group.4.The concentrations of exosomes in serum from HDM group were significantly increased(P<0.001)compared with the control group.From the result of transmission electron microscope,we could observe that the diameter of serum-derived exosomes in the two groups was 30-100 nm.Serum-derived exosomes can be absorbed by BEAS-2B cells,which could increase the expression of pro-inflammatory factor IL-6 and growth factor NGF in BEAS-2B cells.5.In addition,the results indicated that serum-derived exosomes from the model of guinea pigs with asthma could activate the TLR4/NF-kB signaling pathway,leading to the increase of the expression of proteins,such as TLR4,p-IKK-α/β and p-P65.Therefore,the P65 protein went through the nucleus resulting in the expression of pro-inflammatory factor IL-6 and growth factor NGF.After blocked the TLR4/NF-kB signaling pathway could inhibit the activation of p-65,leading to the decrease of the expression of IL-6 and NGF.6.HSP70 of exosomal membrane from serum may participate in the TLR4/NF-kB signaling pathway to promote the expression of IL-6 and NGF.The pro-inflammatory functions of exosome in serum were inhibited by HSP70 blocker HSP70-IN-1.In addition,the recombinant protein HSP70 in vitro has the similar pro-inflammatory role compared with serum-derived exosomes.Conclution:The allergic asthma model in guinea pigs could be established successfully by HDM sensitization and challenged.Serum-derived exosomes can play a pro-inflammatory role,and may activate the expression of TLR4/ NF-kB signaling pathway by inducing the expression of membrane protein HSP70 on their surface to participate in the pathogenesis of asthma. |
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