The Expression And NGF/TrkA Regulation Mechanism Of SH2-Bβ In Alveolar Macrophages In Guinea Pig Models Of Asthma

Bronchial asthma is a kind of complex disease,airway inflammation and airway hyperresponsiveness represent important pathophysiological features.Airway inflammation and airway hyperresponsiveness are interconnected through a bidirectional signaling mechanism between the immune cells and the neural cells,and bidirectional signaling molecules that mediate the crosstalk between the immune and neural cells has becomethe focus in asthma research.Recent studies have demonstrated that nerve growth factor(NGF)is bidirectional signaling molecule.Biological functions of NGF are often fulfilled by binding to its high-affinity specific receptor:the tyrosine kinase receptor A(TrkA).SH2-Bβis a membrane protein which located in the cytoplasm, it is a TrkA-bind protein,after activation by NGF,TrkA binding to SH2-B to cause tyrosine phosphorylation in SH2-Bβ,which action on AKT/PKB to allow AKT/PKB activation further.But SH2-Bβis involved in the pathogenesis of asthma and whether it is through this pathway through this pathway its role is unclear.Alveolar macrophages represent one of the element that links the immune and the nervous systems.In this study,we esamine whether SH2-Bβtake part in the pathogenesis of asthma and investiganted the possible pathway of its action in this process.Thus we clarity the molecular biology mechanism of AMs in the pathogenesis of asthma from the perspective of signal tramsduction,and to provide an important scientific basis for treatmen of asthma and explore new targets or more targetedMaterials and methods1.Animals 60 specific pathogen-free albino guinea pigs weighing 250-300 grams were provided by the Laboratory Animal Center,College of Basic Medicine,Jilin University. These guinea pigs were randomly divided into 5 groups:(1)namely the phosphate buffer solution(PBS)control(n=10);(2)asthmatic group(n=12);(3)anti-NGF group(n=14);(4)anti-TrkA group(n=14);(5)Budesonide group(n=10).At the first day of experiment,(2)(3)(4)(5)were injected pertioneally with 1ml of the mixture containing 10%chicken ovalbumin and equivalent volume of aluminium hydroxide gel prepared with PBS,in control group,the animals received only intraperitoneal PBS injection(1ml).Followed 14 days later by daily 5-min PBS aerosol inhalation for 5 consecutive day in control group;in other groups,aerosol inhalation of 0.5%ovalbumin in PBS for 2～5 min were administered with for 5 consecutive days.The guinea pigs in the anti-NGF group,anti-TrkA group;Budesonide group were administered 3h before OVA atomization inhalation.But control group with PBS atomization inhalation and intraperitoneal injection,the same time with the asthma group.2.Determination of airway hypersensitivityThe guinea pigs were measure airway resistance in last inhalation OVA after 24h,pentobarbital anesthesia.Measure the airway hypersensitivity(including inspiratory resistance and expiratory resistance)with AniRes 2005 animal lungs function analysis (Beijing Beilanbo Technology Go.Ltd.).3.Vascular perfusion,sacrifice of guinea pigs,sample preparationCell numbers in BALF were detected,the lever of IL-4 and IL-1βwere measured using ELISA kits,pathological changes of the bronchi and lung tissues of different groups.4.AMs purificationThe small fracdtion of cell after centrifuged were resuspended in RPMI-1640 meduium supplemented with 10%calf serum,the cells were plated onto a 24-well plate with small glass slides placed in the wells.The culture flask and the 24-well plate were incubated for 2 h at 37℃in 5%CO₂ with saturated humidity.The small slides were taken and washen clean for immunofluorescence assay and immunocytochemistry.The adherent cells(AMs)in the cell culture flask were digested with 0.02%EDTA-Na₂ harvested and stored at -70℃. 5,Hematoxylin-Eosin(HE)staining of bronchial and lung tissue pathologyThe bronchial and lung tissue pathology from different groups were embedded with paraffin and were sectioned into 5μm for HE staining to observe and taken pictures using light microscope.6.Immunofluorescence detection of SH2-Bβin the AMsIn the control and asthmatic groups,the AMs immobilized on the small slides were fixed for 30 min using 4%paraformaldehyde,washing with PBS and staining with immunlfluoreacence(TRITC-SH2-Bβ),we observed and took pictures by fluorescence microscope.7.Detecting the expressions of SH2-Bβand TrkA in AMs by using immunocytochemical technique.SH2-Bβprotein was observed in groups of(1)(2)(3)(4)(5),referencing to two-step immunocytochemical kits.Detection of TrkA protein with the same approach in groups of(1)(2)(3).8.Western blot method to detect protein level of SH2-Bβ,TrkA in AMsAfter extraction and quantification of protein from AMs,the samples were electrophoresed with SDS-PAGE.The samples were culture with primary and secondary antibody and detected.9.Statistical analysisThe data were analyzed with SPSS 11.5 software.The resures were represented as Mean±SD,and comparisons between the groups were performed using independent sample t test and LSD test in univariate analysis of variance(ANOVA).P value less than 0.05 was considered to denote significant difference.Results1.Pathological changes of the bronchi and lung tissuesBronchial lumen stenosis and many inflammatory cells such as lymphocy tes and eosinophils infiltrating the lung tissues and the bronchiolar lumen and walls were found. Constitution of bronchial and lung tissues in control group is normal,other groups are releaser than asthma group.2.Airway resistance measurement showedSignificantly shife of the concentration-response curves in airway resistance to methacholine(Ach)was observed in the asthmatic group compared with the normal control group,its inspiratory resistance and expiratory resistance were increased(P＜0.01 or P＜0.05).It is indicating that experimental model of asthma were successfully established.3.Results of immunofluorescenceIn the control and asthmatic groups,SH2-Bβin the AMs mainly on the cell membrance and in the cytoplasm in the AMs of BALF with control and asthma groups,but asthmatic group with a much greater intensity.4.Immunocytochemical analysis for SH2-Bβand TrkASH2-Bβof AMs was expressed in the five groups respectively,but their strengths are different,asthmatic group was significantly higher than the other four groups (P＜0.05).TrkA of AMs was expressed in the control,asthma,anti-NGF groups,and asthmatic group is the highest than the other groups.5.Detecting the expression of SH2-Bβand TrkA in AMs with Western blot experimentWestern blotting revealed significantly more intense expression of SH2-Bβin the AMs from asthmatic guinea pigs than in the control,anti-NGF,anti-TrkA,budesonide groups(P＜0.01).The AMs of the asthmatic guinea pigs also significantly more intense TrkA expression than those of control and anti-NGF groups(P＜0.05).6.Enzyme-linked immunosorbent assay(ELISA)detect the levels of IL-1βand IL-4 in BALFThe levels of 1L-1βand IL-4 in BALF of OVA-sensitized asthma guinea pigs were significantly higher than in other groups,(P＜0.05 or P＜0.05).There has statistical significance in two groups Conclusion1.SH2-Bβexpression in the AMs of control and asthma groups,and found increased SH2-Bβexpression in the AMs of asthma,suggest that SH2-Bβparticipates in the pathogenesis of asthma.2.NGF and TrkA can accommodate expression of SH2-Bβ,and NGF also can affect the expression of TrkA,based on these finding,suggest that SH2-Bβparticipates in pathogenesis of asthma through the NGF-TrkA signaling pathway,and it is a important signaling molecule in this pathway3.Budesonide suspension could affect expression of SH2-Bβin the AMs,this may be suggested that another way of glucocorticoid treatment asthma...