| Part OneChanges in The expression of Inflammatory Mediators in Human Bronchial Epithelial Cells Stimulated by House Dust Mite ObjectiveTo explore the relationship between IL-29?IL-6?NF-?B and airway inflammation caused by house dust mites through the determination of expression and relationship of IL-29?IL-6 and NF-?B in human bronchial epithelial cells before and after the stimulus of house dust mites and dexamethasone(DEX).Methods and materialsThe same batch of human bronchial epithelial cells in exponential growth phase were randomly divided into five groups:blank group(A),300 ng/mL HDM group(B),1000 ng/mL HDM group(C),3000 ng/mL HDM group(D),and 300 ng/mL HDM+100 ng/mLDEXgroup(E).The relative expression of IL-29 mRNA?IL-6 mRNA and NF-?B/p65 mRNA was detected by real-time fluorescent quantitative polymerase chain reaction.IL-29 and IL-6 protein in cell suspension were detected by ELISA.Results The results showed that after stimulation with HDM for 24 h,the expression of IL-29?IL-6 protein and mRNA?NF-icB/p65 mRNA in group B?C?D were significantly higher than group A,and after co-stimulation with HDM and DEX for 24 h,the expression of IL-29?IL-6 protein and mRNA?NF-?B/p65 mRNA in group E were significantly lower than the groups stimulated by HDM alone but higher than group A.The differences between the different groups were significant(F=191.983;415.311;65.377;283.037;294.367;P<0.01).In addition,the higher the concentration of HDM,the more significant the increase in the IL-29?IL-6 and NF-?B/p65 mRNA expression.There was a positive correlation between NF-?B/p65 mRNA and IL-29 mRNA as well as IL-6 mRNA(r=0.899,P=0.000;r=0.883,P=0.000).ConclusionThe result indicate that house dust mite may increase the expression of IL-29 and IL-6 by activating the NF-?B inflammatory signaling pathways,Which participate in the development of airway inflammation caused by house dust mite possibly.Part TwoExpression mechanism of Inflammatory Mediators in HumanBronchial Epithelial Cells Induced by House Dust MitesObjectiveThrough the determination of the influence of NF-icB/p65 siRNA on expression of IL-29?IL-6 and NF-?B in human bronchial epithelial cells after the stimulus of house dust mites,to explore the relationship between IL-29?IL-6 and NF-?B inflammatory signaling pathways.Methods and materialsThe same batch of human bronchial epithelial cells in exponential growth phase were randomly divided into four groups:blank(group A),300 ng/mL HDM(group B),300 ng/mL HDM +NF?B-siRNA(group C),300 ng/mL HDM + siRNA control(group D).The relative expression of NF-?B/p65mRNA was detected by real-time fluorescent quantitative polymerase chain reaction.IL-29 and IL-6 protein in cell suspension were detected by ELISA.Nucleoprotein of NF-KB/p65 was detected by Western Blot.ResultsThe results showed that after co-stimulation with HDM and NF-?B/p65 SiRNAfor 24 h,the expression of NF-?B/p65mRNA?NF-?B/p65 nucleoprotein?IL-6 and IL-29(mRNA and protein)in group C were significantly lower than group B and group D,but higher than group A.The differences between the different groups were significant(F=193.456;228.565;135.168;55.725;162.279;108.313;P<0.01).ConclusionThe NF-?B signaling pathway is one of the signaling pathways of the expression of IL-29?IL-6 in human bronchial epithelial cells after stimulation of house dust mite.Part ThreeRegulation of Aldose Reductase Inhibitor on Airway InflammationInduced by House Dust MiteObjectiveThrough the determination of the influence of aldose reductase inhibitors on expression of cytokine and NF-?B in airway after the stimulus of house dust mites,to explore new treatment for airway inflammation in the future.Methods and materialsCell experiment:human bronchial epithelial cells were divided into three groups:blank(group A),300 ng/mL HDM(group B),300 ng/mL HDM +aldose reductase inhibitors Zopo(group C),the relative expression of NF-kappa B/p65mRNA?NF-kappa B/p65 nuclear protein and protein of IL-29 and IL-6 in cell supernatant were assayed.Animal experiments:mice were randomly divided into 3 groups:group A(control group),group B(HDM group)and group C(Zopo group),and Mites allergens were used to stimulate mice for establishing airway inflammation model in mice.Changes of airway responsiveness were detected with mouse pulmonary function instrument,hematoxylin and eosin(HE)staining were used to observe inflammatory infiltration of the airway and lung tissue of mice,cell counting plate were used for cell classification and counting in bronchoalveolar lavage fluid(BALF).Then,the relative expression level of IL-6,IL-8,IL-29,TNF-? and NF-kB/p65 in mice lung tissue?protein level of IL-6,IL-8,IL-29,TNF-? in BALF and expression of NF-kB/p65 nuclear protein in lung tissue of mice were detected.ResultsCell experiments:the data showed that after 24 hours of combined action of house dust mites and aldose reductase inhibitors,the NF-kappa B/p65 mRNA level,NF-kappa B/p65 nuclear protein level,IL-6 protein level and IL-29 protein level of human bronchial epithelial cells in group C were significantly lower than those of group B.The differences between the different groups were significant(F=159.909;233.350;167.999;127.196;P<0.01).Animal experiments:pathological examination showed that Zopo group inflammatory cell infiltration significantly reduced compared with the house dust mite group.Bronchoalveolar lavage fluid leukocyte count and eosinophils percentage,lymphocytes percentage were decreased compared with the house dust mite group,and mice airway reactivity significantly decreased compared with the house dust mite group(F=100.179;698.70;157.260;51.899;60.486;P<0.05).Further analysis showed that NF-kB/p65mRNA,NF-kappa B/p65 nuclear protein levels in the lung tissue of mice in Zopo group were significantly lower than that of the house dust mite group(F=124.176;188.517;P<0.05).The relative expression of IL-6?IL-8?IL-29?TNF-? mRNA and protein in BALF of Zopo group were significantly lower than that of the house dust mite group(F=75.162;172.151;59.133;68.042;92.659;133.655;102.864;79.230;P<0.05).ConclusionAldose reductase inhibitor zopolrestat regulates inflammatory signaling mediated by NF-kappa B.The expression of inflammatory factor in human bronchial epithelial cells significantly reduced because of zopolrestat.Hyperresponsiveness and inflammation cells infiltration in airway were significantly reduced by zopolrestat.The expression of inflammatory cytokines in airway of mice reduced because of zopolrestat.Aldose reductase inhibitor zopolrestat reduced the airway inflammation induced by house dust mite. |
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