The Upregulative Effect Of PKB/Akt On IL-1β And VEGF In The Lower Respiratory Tract And IL-1β In Viscerosensory Afferent Sites Of The Asthmatic Mice

Bronchial asthma is a kind of immunogenic inflammatory airway disease,and is characterized by airway smooth mucle spasm and increased airway hyperresponsiveness. But the pathogenesis of the asthma is unclear.Recently,neurogenic inflammation is increasingly recognized.However it is known little about the interaction between the nervouse system and inflammation process.Recently we found that NGF is a kind of important mediator.The level of NGF in the serum of asthmatic patient increase,the study about the bronchial asthma model shows that NGF regulates the airway hyperresponsiveness,exogenous NGF can increase the airway hyperresponsiveness of the guinea pig caused by the histamine,antibody of NGF can decrease the airway hyperresponsiveness.In recent years,our previous results confirmed that the expression of NGF is increased remarkably in the lower respiratory tract and viscerosensory afferent sites of the asthmatic guinea pigs.PBK/Akt is a kind of regulative kinase which play an significant role in glycometabolism,protein synthesis and cell Apoptosis course.Akt can be activated by SH2-Bβin signal transduction pathway when PC12 cell differentiate.Recently we found that Akt/PKB is possibly involved in asthmatic pathogenesy mediated by NGF,but it is uncertain that Akt/PKB how to react..The research show that IL-1βpositive product in blood serum increased signifycantly in the asthmatic period of onset and paracmasis than in normal control.Pre-in-flammatory cytokine IL-1βcan up-regulate the expression of TGF-β1 mRNA and protein in bronchial epithelial cell.The ability of IL-1βto modulate TGF-β1 release may contribute to both abnormal airway repair and development of airway remodeling. IL-1ra indirectly decreased the level of IL-5 in serum,improved the apoptosis of eosinophil(EOS)in lung,then decreased the level of ECP in serum and BALF.All these reveal that IL-1βplay an important role in asthmatic pathogenesy,however,it is a valuable question that what effects of Akt/PKB on the expression of IL-1β.vascular endothelial growth factor(VEGF)which is a member of platelet derived growth factor family,is made up from homodimer glucoprotein forming by two same polypeptide chains through the disulphide bond.VEGF could be secreted by many kinds of cells,and specifically effect on the endothelial cell,adjust endothelial cell mitosis,increases the blood vessel permeability and tension,promote the embryonic period vasiformation.The expressions of VEGF and Flk-1 were increased in the lung tissues of asthmatic rats and positively correlated with the proliferation of airway smooth muscle.VEGF and Flk-1 may take part in the process of airway smooth muscle proliferation in the airway remodeling of asthma.Wang Xiuzhen et al also proved that leukotriene receptor antagonist can postponed the airway remodeling in asthma by reducing the expression level of VEGF and inhibiting vascularization.Some research demonstrate that the dexamethasone possibly regulate the Angiogenesis growth factor /inhibiting factor proportion by degrade VEGF to delay airway remodel -ing.All reveal that IL-1βplay an important role in asthmatic pathogenesy。To study the regulatory effect of PKB/Akt on the expression of VEGF in the lung of the asthmatic mice,The expression of VEGF were observed by Immunofluorescence as well as Western blot in the lung.To study the regulatory effect of PKB/Akt on the expression of IL-1βin the lung,C7-T5 spinal ganglia and the corresponding spinal dorsal horn of the asthmatic mice,The expression of IL-1βwere observed by Immunohistochemical method in the lung,C7-T5 spinal ganglia and the corresponding spinal dorsal horn of all the groups.The changes of interleukin-1β(IL-1β)were investigated by means of Western blot in the lung,C7-T5 spinal cord. Answer for these questions will make the machnism of asthma clear and provide evidence for novel treatment.Materials1.Experimental animals:the BALB/c mice(18～25g)were used,which were purchased from the center for Expeimental Animals of Capital Medical University.The total number was 30.All animal experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laborarory animals.2.Experimental regents:rabbit polyclonal antibody anti-IL-1βand rabbit VEGF polyclonal antibody(BosteCompany,Wuhan);goat anti-habbit IgG conjugated with horseradish peroxidase and SABC immunohitochemistry kit(Boster Company,Wuhan); anti-β-actin antibody,goat polyclonal antibody anti-mouse IgG conjugated with horseradish peroxidase,and goat anti-rabbit IgG conjugated with FITC(Zhongshan Company,Beijing).3.Experimental instruments:low temperature refrigeration centrifuge,ultroviolet spectro photometer,automatic running gel imaging system,GP electrophoresis apparatus,upright fluorescense microscope,-80℃deep freeze refrigerator, homeothermia freezing microtome,Motic Images Advanced 3.2 image analysis system.Methods1.Prepare the asthma model,Asthma was induced in BALB/c mice by intraperitoneal injection chicken egg ovalbumin(OVA)and challenged by exposure to OVA.2.The expression of VEGF were observed by Immunofluorescence as well as Western blot in the lung of all the groups.3.The expression of IL-1βwere observed by Immunohistochemical method in the lung,C7-T5 spinal ganglia and the corresponding spinal dorsal horn of all the groups. The changes of IL-1βwere investigated by means of Western blot in the lung,C7-T5 spinal cord.4.Image analysisThe datas obtained from Western blot were scanned with Chemi Imager 5500 V2.03 software;Integrated Density Value(IDV)of the IL-1βand VEGF protein was calculated with Fluor Chen 2.0 software.The data of Immunochemistry were obtained and analysed semiquantitatively with Motic Images Advanced 3.2 image analysis system.The data of Immunolocalization of IL-1βandVEGF protein were analysed with SPOT Advanced 4.0.2 software.5.Statistics analysisData were disposed with SPSS13.0 statistic software and they were expressed as mean±standard deviation,t test,P＜0.05 was considered statistically significant. Results1.IL-1βImmunohistochemistry showed that the mean optic density(MOD)of IL-1βpositive product in the lung,spinal ganglia and dorsal horn of C7-T5 increased significantly in the asthmatic mice than in normal control(P＜0.01);but decreased in the asthmatic mice blocked with LY294002 than in the asthmatic mice(P＜0.05).Western blot results showed that:compared with normal saline control group,the ratios between integrated density value(IDV)for IL-1βprotein band and IDV forβ-actin protein band increased markedly in the lung,C7-T5 spinal cord of asthmatic group(P＜0.01);however,the ratios between IDV for IL-1βprotein band and IDV forβ-actin protein band of the asthmatic mice blocked with LY294002 group reduced markedly in comparison with asthmatic group(P＜0.05).2.VEGFImmunofluorescence showed that airway smooth muscle(ASM)and vascular density increased significantly in the asthmatic mice than in normal control;but decreased in the asthmatic mice blocked with LY294002 than in the asthmatic mice.Western blot results showed that the ratios between integrated density value(IDV) for VEGF protein band and IDV forβ-actin protein band in the lung increased significantly in the asthmatic mice than in normal control(P＜0.01);but decreased in the asthmatic mice blocked with LY294002 than in the asthmatic mice(P＜0.05).DiscussionIL-1 was called lymphocyte-activating factor,playing an important role in asthma.The research show that IL-1βpositive product in blood serum increased signify- cantly in the asthmatic period of onset and paracmasis than in normal control.Pre-in-flammatory cytokine IL-1βcan up-regulate the expression of TGF-β1 mRNA and protein in bronchial epithelial cell.IL-1ra indirectly decreased the level of IL-5 in serum,improved the apoptosis of eosinophil(EOS)in lung,then decreased the level of ECP in serum and BALF.The ability of IL-1βto modulate TGF-β1 release may contribute to both abnormal airway repair and development of airway remodeling.All these reveal that IL-1βplay an important role in asthmatic pathogenesy.The current experiment showed:IL-1βpositive product in the lung, spinal ganglia and dorsal horn of C7-T5 increased significantly in the asthmatic mice than in normal control;but decreased in the asthmatic mice blocked with LY294002 than in the asthmatic mice.The result reveal that one of the mechanisms for Akt participate the pathogenesis of asthma induced by NGF might be up-regulate the expression of IL- 1βin the asthmatic mice.VEGF could be secreted by many kinds of cells,and specifically effect on the endothelial cell,adjust endothelial cell mitosis,increases the blood vessel permeability and tension,promote the embryonic period vasiformation.The expressions of VEGF and Flk-1 were increased in the lung tissues of asthmatic rats and positively correlated with the proliferation of airway smooth muscle.VEGF and Flk-1 may take part in the process of airway smooth muscle proliferation in the airway remodeling of asthma.Wang Xiuzhen et al also proved that leukotriene receptor antagonist can postponed the airway remodeling in asthma by reducing the expression level of VEGF and inhibiting vascularization.All reveal that VEGF play an important role in asthmatic pathogenesy.The current experiment showed:VEGF positive product in the lung increased significantly in the asthmatic mice than in normal control;but decreased in the asthmatic mice blocked with LY294002 than in the asthmatic mice.The result reveal that One of the mechanisms for Akt participate the pathogenesis of asthma induced by NGF might be up-regulate the expression of VEGF in the lung of the asthmatic mice.Take together,the current study demonstrate that Akt can up-regulate the expression of IL-1βand VEGF in the lower respiratory tract and IL-1βinviscerosen-sory afferent sites of the Asthmatic mice.The conclusion will make the machnism of asthma clear and provide evidence for novel treatment.Conclusions1.The up-regulation of IL-1βby the activated NGF-mediated Akt pathway might be involved in the asthma airway inflammation.2.One of the mechanisms for Akt participate the pathogenesis of asthma Induced by NGF might be up-regulate the expression of VEGF in the lung of the asthmatic mice.