| ObjectiveAsthma is a chronic inflammatory disease characterized by involving of many kinds of inflammationary cells(eosinophil,mast cell,leukomonocyte,neutrophil, alveolar macrophage),airway composed cells(smooth muscle cell,epithelial cell and so on)and cellular component.Although many works about asthma have been done,the pathogenesis of asthma has not been completely illuminated.Thus,at present,asthma is one of the diseases which can not be cured.AM is the predominant immune effector cell resident in the alveolar spaces and conducting airways,and it is responsible for activating inflammatory responses sufficient to eliminate the interlopers.However,an excessive inflammatory response might perturb gas exchange.This means that the AM must be "ambidextrous"—capable of protecting the organisms and enhancing inflammatory responses.Some studies indicated that AM is over 90%in the all cells of BALF.Our experiments studied the change of the activity of potassium channels and the cytokines production of AM from asthma rats.Then,we analyzed the change of the AM function in asthma and the possible regulatory mechanisms.MethodsRats were sensitized and challenged with ovalbumin(OVA) to make asthma model,the control group used in place of normal saline,HE staining to observe pathological changes in asthmatic rats.24h after the last challenge,rats were anesthetized,trachea intubated and then bronchus alveolar lavaged with cold PBS,each time10ml,repeated 5 times.The BALF was collected and recovery rate was over 90%. AM was isolated from BALF and cultured.K_V(Voltage-dependent potassium channel, K_V) channel current on AM were recorded using whole-cell voltage-clamp system. Expression level of IL-6 and TNF-αin AM were evaluated by western blot and mRNA level of IL-6 and TNF-αin AM were estimated with RT-PCR,respectively,after treated with lipopolysaccharide(LPS)(5ug/ml) 12h or when LPS was absent.Secretion level of IL-6 and TNF-αwere detected with an ELISA kit.Results1.Change of K_V activity on AM membrane:Pathology detected asthma model group significantly increased infiltration of inflammatory cells,red blood cells had substantial exudation,marked thickening of airway smooth muscle and mucus suppository can be seen in airway.Significant increases in total cells in bronchus alveolar lavage fluid(BALF) were found in asthma group compared with control group (P＜0.01).The current altitude which we detected on AM membrane was obviously decreased with the specific blocker of K_V channel(4-AP) confirmed that the current was K_V current.The Kv current density on AM membrane in asthma group[(32.65±18.82) pA/pF,n=11]was significantly lower than control group [(87.57±33.14) pA/pF,n=11](P＜0.01).2.Function of secretion IL-6 and TNF-αof AM:The mRNA and expression level of IL-6 and TNF-αwas increased in asthma group compared with control group regardless of LPS was present or absent.The secretion level of IL-6 and TNF-αof AM without LPS in asthma group were significantly higher than control group(P＜0.01); With LPS,The secretion level of IL-6 and TNF-αof AM in both group were increased (P＜0.01),but there was no significant statistical difference between asthma group and control group(P＞0.05),because the secretion level of IL-6 and TNF-αof AM in control group was more increased than asthma group.Conclusion1.The Kv current density(Kv channel activity) on AM membrane in asthma group markedly decreased.2.The secretion level of IL-6 and TNF-αof AM in asthmatic rats was significantly increased,when AM was activated with LPS,the secretion level of IL-6 and TNF-αof AM in both group were increased,but the control group was more significant. |
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