NGF介导的SH2-Bβ和Akt/PKB信号通路在小鼠气道过敏性免疫反应中的作用
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摘要
前言
支气管哮喘(bronchial asthma,简称哮喘),是由多种炎性细胞特别是巨噬细胞,肥大细胞、嗜酸性粒细胞和T淋巴细胞等参与的慢性气道炎症。国内外支气管哮喘患病率,死亡率逐渐上升,全世界支气管哮喘者约1亿人,成为严重威胁人们健康的主要慢性疾病。我国的哮喘发病率为1%,儿童达3%。神经生长因子(Nerve growth factor, NGF)在炎症反应中起重要作用,被认为是连接神经系统和免疫系统的纽带,许多研究都证明NGF参与哮喘发病,它通过提高气道高反应和诱导气道炎症参与哮喘发病,阻断NGF后可有效减轻哮喘病理变化和降低多种炎性因子的表达。但是NGF参与哮喘发病的机制尚不完全清楚。
NGF促进神经细胞分化、发育和营养等生物学作用主要是通过结合其高亲合力特异性受体TrkA (tyrosine kinase receptor A)实现的。此外,神经生长因子介导SH2-Bβ(Src homology 2β)-Akt/PKB (protein kinase B,逆转录病毒Akt8癌基因V-akt编码的蛋白产物,故也称Akt)是调节嗜铬细胞瘤(PC12)细胞的信号途径之一。SH2-Bβ是广泛表达的高度保守的调节蛋白家族成员之一,能结合非酪氨酸激酶2(Janus kinase 2, Jas2)、胰岛素受体酪氨酸激酶和胰岛素样生长因子、NGF、血小板衍生因子、和成纤维细胞因子。SH2-Bp对NGF依赖的轴突生长和轴突存活非常关键。SH2-Bp加强NGF在PC12细胞中Akt的磷酸化和延长Akt酶活性(NGF激活TrkA使其与SH2-B结合,促使SH2-B发生酪氨酸磷酸化之后作用于丝氨酸／苏氨酸激酶Akt／PKB,Akt进一步激活分叉头转录因子(Forkhead transcription factors),促使PC12细胞分化增殖。研究表明SH2-Bβ在哮喘豚鼠肺组织巨噬细胞中过表达,同时还证明它参与NGF与其受体TrkA介导的哮喘发病。这些研究表明SH2-Bp在NGF介导的细胞功能中发挥基础性作用。基于以上信息,我们推测SH2-Bβ调节Akt磷酸化可能参与NGF介导的哮喘发病。
本课题利用哮喘小鼠模型,以肺组织为研究对象,观察Akt在哮喘肺组织及炎性细胞中的表达情况,观察NGF和SH2-Bβ与气道高反应的关系,观察阻断NGF和SH2-Bβ肺组织病理变化和炎性细胞数量的变化,应用多种方法观察NGF和SH2-Bβ在哮喘发病机制中对Akt及其活性的影响。为探讨NGF介导的哮喘的可能的发病机制和寻找过敏性气道疾病新的治疗靶点提供理论依据。
材料和方法
一、主要试剂
卵清蛋白(OVA, sigma,美国)；乙酰甲胆碱(mAch, sigma,美国)；RPMI1640培养基(GIBCO);定点突变试剂盒(Stratagene,美国)；兔抗鼠NGF抗体(Sigma,美国)；定点突变试剂盒(Stratagene,美国)；鼠多克隆p-Akt抗体(CellSignaling,美国),辣根过氧化酶免疫组化试剂盒(SABC,博士德,中国);ECL (enhanced chemi luminescence)试剂盒(Sigma Chemical,美国);Trizol裂解液(Santa Cruz,美国)；RNA PCR Kit (AMV) Ver.3.0 (Takara Biotechnology,中国大连);RNAout (Takara Biotechnology,中国)。
二、主要仪器
电子天平(BS110S, Startorius,德国)；恒温冷冻切片机(Leice,德国)；-80℃深低温冰箱(Toshiba,日本);低温冷冻超速离心机(3K18, Hitachi, Japan);超声粉碎机(Dr. Hielscher,德国)；通用电泳仪(Bio-Rad,美国)；普通光学显微镜(COIC, XSZ-H);超纯水系统(Millipore A10):MILLIPOREMILLI-B(美国)；超声雾化器(JWC-201D型,北京超声仪器厂)；AniRes2005动物肺功能分析系统(北京贝兰博科技有限公司)；MetaMorph显微图像分析系统(UIC/OLYMPUS); GDS8000凝胶图像处理系统；PCR仪(Biometra,德国)；Motic Images Advanced 3.2 (Motic, Xiamen)。
三、实验方法
1、动物及分组
雌性BALB／c小鼠85只,6-8周龄,体重20.0-22.0 g,清洁级,购自中国科学院实验动物繁育中心。随机分为4组：正常对照组,OVA组(哮喘组),R555E组,NGF阻断组。
2、哮喘模型复制及给药方法
1 mg OVA (Sigma, St Louis, MO, USA)和100mg氢氧化铝溶于0.5 ml PBS中,分别于0,7,14和21天腹腔注射给OVA组,R555E组和anti-NGF组小鼠致敏,从22天到30天雾化吸入10 mg／ml OVA PBS激发哮喘,每天一次,每次30分钟。OVA组继续雾化激发三天,而R555E组和anti-NGF组分别在激发前3小时尾静脉注射以1：10稀释于PBS中的50μl多克隆抗体兔抗鼠R555E和NGF,连续三天。正常组以等量的PBS腹腔注射和雾化吸入。
3、气道反应性测定
采用梯度浓度的mAch激发气道高反应,AniRes2005动物肺功能分析系统进行分析。测定小鼠气道阻力。
4、组织切片的准备
以上各组动物用多聚甲醛固定,取肺,相同位置切片。
5、免疫组织化学方法测定肺组织Akt表达。
6、肺泡灌洗液(BAIF)细胞计数及分类计数。用细胞计数板计数细胞总数,涂片进行Wright Giemsa染色作细胞分类计数。
7、免疫荧光测定p-Akt表达情况。
8、Western blot测定p-Akt蛋白水平,图像分析及数据处理。
9、RT-PCR检测Akt mRNA表达,图像分析及数据处理。
10、苏木素伊红(HE)染色观察肺组织病理变化。常规包埋,冰冻切片,进行苏木素伊红(HE)染色,观察肺组织病理变化。
11、统计学处理
所有数据采用SPSS14.0软件分析,实验数据以x±s表示。P<0.05为差异有显著性。
1、呼吸频率计数
呼吸频率各组无明显差异(P>0.05)。
2、支气管和肺组织病理变化
炎症细胞,粘膜水肿和上皮损伤anti-NGF组和R555E组小鼠没有未加干预的哮喘鼠明显(P<0.01)。在哮喘肺组织肺泡和支气管周围发现许多炎性细胞包括单核细胞,巨噬细胞,淋巴细胞和嗜酸性粒细胞。而正常鼠肺组织没有上述变化。
3、BALF细胞计数和分类
炎症细胞,粘膜水肿和上皮损伤anti-NGF组和R555E组小鼠没有未加干预的哮喘鼠明显(P<0.01)。OVA组小鼠的BALF的总细胞数明显高于正常鼠。巨噬细胞,嗜酸性粒细胞和淋巴细胞数与NGF组和R555E组比较OVA组明显升高。在NGF组和R555E组小鼠肺组织上述变化与OVA组比较明显减轻。
4、气道阻力测定
哮喘鼠对MCH的气道反应性比正常鼠增强(P<0.01)。静脉注射NGF抗体和R555E的OVA致敏鼠气道反应性明显低于OVA哮喘鼠。
5、免疫组织化学染色
哮喘组Akt／PKB在各种炎性细胞表达,包括嗜酸性粒细胞、嗜中性粒细胞、巨噬细胞、淋巴细胞、浆细胞等,并且这些炎性细胞非常多,肺泡内见大量的巨噬细胞,淋巴细胞和浆细胞等。正常组和NGF阻断组炎性细胞表达Akt／PKB很少,并且炎性细胞非常少。
6、免疫荧光分析NGF和SH2-Bβ对Akt活性影响
肺组织免疫荧光染色显示在OVA组p-Akt水平与正常组比较明显高表达。而上述部位过表达的p-Akt水平在NGF组和R555E组又呈降低趋势。
7、免疫印迹法分析p-Akt蛋白表达
在正常鼠的肺组织p-Akt蛋白表达处于低水平,而在OVA组则明显升高。NGF组和R555E组由OVA上调的p-Akt水平反而降低。
8、Anti-NGF和R555E对Akt mRNA的影响
OVA组Akt mRNA水平明显高于正常组,而NGF组和R555E组致敏鼠的Akt mRNA水平与OVA组比较明显降低。
结论
1、哮喘小鼠气道高反应明显增高,NGF参与哮喘小鼠的气道高反应和肺部炎症反应,阻断NGF明显减轻哮喘小鼠的气道高反应和肺部炎症反应。
2、SH2-Bβ参与哮喘气道高反应和肺部炎症反应,阻断SH2-Bβ减轻哮喘小鼠气道高反应和肺部炎症反应。
3、Akt信号分子在哮喘时被激活,NGF调节Akt活性可能是NGF参与哮喘发病的机制之一。
4、NGF介导的SH2-Bβ/Akt/PKB信号通路可能是NGF参与哮喘发病的通路之一
Bronchial asthma (bronchial asthma, referred to as asthma), is a chronic airway inflammation which many inflammatory cells, especially macrophages, mast cells, eosinophils and T lymphocytes take part in. The death rate and prevalence of bronchial asthma increase gradually at home and abroad. Bronchial asthma invalid some 100 million people around the world, becoming a serious threat to people's health a major chronic diseases. China's asthma incidence is 1%, children up to 3%.
Nerve growth factor (NGF) has been specifically implicated in asthma pathogenesis due to its ability to promote sensory neuron hyperreactivity and induce airway inflammation. Blocking NGF can effectively relieve asthmatic pathological changes and reduce a variety of cytokines. While the mechanism of NGF involved in the pathogenesis of asthma not fully understood. In addition, NGF regulates PC 12 cells in part by stimulating phosphorylation of Src homology 2β(SH2-Bβ) on serine/threonine kinase (Akt/PKB). SH2-Bβis a member of a widely expressed, highly conserved family of adaptor proteins and can bind to Janus kinase 2 (JAK2) and the activated receptor tyrosine kinases for insulin, insulin-like growth factor I, NGF, platelet-derived growth factor, and fibroblast growth factor. Moreover, SH2-Bβis critical for both NGF-dependent neurite outgrowth and neurite maintenance.Additionally, SH2-Bβenhances and prolongs of NGF-induced Akt phosphorylation and of Akt enzymatic activity in PC 12 cells. Li has also characterized SH2-Bβexpression in alveolar macrophages in the bronchoalveolar lavage fluid (BALF) of asthmatic guinea pigs and its role in NGF-TrkA-mediated asthma. Together, these studies suggest that SH2-Bβplays a fundamental role in receptor tyrosine kinase-mediated cellular functions. Together, these studies suggest that SH2-Bβplays a fundamental role in receptor tyrosine kinase-mediated cellular functions. Based on the above information, we hypothesized that upregulation of Akt phosphorylation by SH2-Bβmay contribute to the pathogenesis of NGF-mediated asthma.
By the study, using the mouse model of asthma, we observed expression of Akt at asthmatic lung tissue, observed the relationship of between NGF and SH2-B(3 with airway hyperresponsiveness, observed lung tissue pathological changes and changes in the number of inflammatory cells after blocking NGF and SH2-Bβ, observed effects of NGF and SH2-B(3 on the activity of Akt in the pathogenesis of asthma using several methods. These findings may pave the way for exploring the NGF-mediated asthma pathogenesis and the development of new therapeutic approaches to allergic airway diseases.
Methods
1、Animals
Fifty-five normal female BALB/c mice (6 to 8 weeks old) weighing 22±2.5 g were obtained from the Laboratory Animal Research Center in Beijing, China, they were randomly divided into four groups:control group, ovalbumin group, R555E group, and anti-NGF group.
2、OVA-treated asthma mice model and Drugs
On days (d) 0,7,14, and 21, all mice except the control group were actively sensitized with an intra-abdominal injection of 1 mg OVA (Sigma) and 100 mg aluminum hydroxide in 0.5 ml phosphate buffered saline (PBS), for the control group only with 0.5 ml PBS. The control group mice were then exposed to aerosolized PBS for 30 min per day from d 22 to d 30. Meanwhile, the OVA-sensitized mice were exposed to 10 mg/ml aerosolized OVA (1 g OVA in 100 ml sterile PBS in a nebulizer) for 30 min per day from d 22 to d 33. The anti-NGF group and R555E group mice were anesthetized and respectively administered an intravenous injection of 50μl polyclonal rabbit anti-mouse NGF antibody (Sigma, St Louis, MO, USA) diluted 1:10 in PBS and 50μl R555E. Intravenous treatment was administered 3h before allergen aerosol challenge on d 31,32, and 33.
3、Determination of Airway responsiveness
Airway responsiveness to muscarinic acetylcholine(mAch) was measured in AniRes2005 lung function system. Inspiratory resistance (IR) and expiratory resistance(ER) are detected.
4、Preparing the histological slices
The animals were perfused transcardially with 4% paraformaldehyde in PBS(0.1M, pH 7.4). The lungs were removed and postfixed. The lung slices at same positions of four groups.
5、Detecting the expressions of Akt at lungs by using immunohisto-chemical technique.
6、Total BALF cell counts were performed using hemacytometer. A differential cell count was carried out by Wright-Giemsa staining on the basis of morphologic criteria.
7、Determinating p-Akt of expression in lungs of 4 groups by Immunofluorescence
8、The expressions of p-Akt in lungs were detected by Western blotting.
9、The expressions of Akt mRNA in lungs were detected by Western blotting.
10、Hematoxylin-Eosin(HE) staining was employed to detect the histological changes in different groups.
The frozen lungs slices from different groups at 24 h reperfusion were stained with HE solution, and the histopathologic changes of hippocampuses were observed under the microscope.
11、Statistics
All values are expressed as the mean±SD. One-way analysis of variance (ANOVA) was used to compare group differences in Akt levels, while two-way ANOVA was used to assess differences in airway resistance.
1、Respiratory frequencys have no significant differences in each group
2、Pathological changes in the bronchi and lung tissue
Inflammatory cells, mucous edema, and epithelial lesions were significantly more prevalent in asthmatic mice than in those treated with anti-NGF or R555E. Many inflammatory cells, including mononuclear cells, macrophages, lymphocytes, and eosinophils, were observed in asthmatic pulmonary alveoli and the surrounding bronchi, in contrast with control mice. However, these finds reduced anti-NGF-and R555E-treated mice.
3、BALF cell counting and differential counting
The total number of cells in the BALF was significantly higher in OVA-sensitized mice than in control mice. The numbers of macrophages, eosinophils, and lymphocytes were also significantly increased in OVA-sensitized mice but reduced in anti-NGF-and R555E-treated mice.
4、Airway resistance measurement
The airway response to MCH was increased in the OVA group compared with the control group (P<0.01). Treatment of OVA-sensitized mice with intravenous anti-NGF and R555E prior to OVA challenge prevented this increase in airway reactivity.
5、Immunohistochemistry observed expression of Akt
Akt levels significantly increased in OVA group, but reduced in NGF group.
6、Immunofluorescent analysis of the effects of NGF and SH2-Bβon Akt activation
Immunofluorescent staining showed that p-Akt levels significantly increased in macrophages, eosinophils, and lymphocytes and bronchial tissue of the OVA group's lung tissue (P< 0.01), as compared with the control group. Meanwhile, the overexpression of p-Akt at the above sites was reduced in both anti-NGF-and R555E-treated groups.
7、Western blot analysis revealed p-Akt protein expression
In the lung tissue of the control group, p-Akt was expressed at low levels, but these levels increased markedly in the OVA group. However, anti-NGF and R555E greatly decreased the p-Akt upregulation caused by OVA.
8、Effects of anti-NGF and R555E on Akt mRNA
Akt mRNA expression was significantly higher in the OVA group than in the control group, but that anti-NGF and R555E treatment significantly decreased Akt mRNA expression in these OVA-sensitized mice.
Conclusions
1、The airway response was increased in the OVA group, NGF participates in airway hyperresponsiveness and inflammatory response of in the lungs of asthmatic mice. Blocking NGF significantly reduced airway hyperresponsiveness and inflammatory response in the lungs of asthmatic mice
2、Akt may be involved in pathogenesis of asthma, NGF regulates activity of AKT may be one of the mechanisms of NGF involved in asthma.
3、SH2-Bβinvolved in airway hyper-responsiveness and inflammatory response of lung of asthmatic mice, and blocking SH2-Bβcould alleviate airway hyperresponsiveness and inflammatory response in the lungs of asthmatic mice.
4、NGF mediated SH2-Bβ/Akt signaling pathway may be one of the pathways NGF-mediated asthma.
支气管哮喘(bronchial asthma,简称哮喘),是由多种炎性细胞特别是巨噬细胞,肥大细胞、嗜酸性粒细胞和T淋巴细胞等参与的慢性气道炎症。国内外支气管哮喘患病率,死亡率逐渐上升,全世界支气管哮喘者约1亿人,成为严重威胁人们健康的主要慢性疾病。我国的哮喘发病率为1%,儿童达3%。神经生长因子(Nerve growth factor, NGF)在炎症反应中起重要作用,被认为是连接神经系统和免疫系统的纽带,许多研究都证明NGF参与哮喘发病,它通过提高气道高反应和诱导气道炎症参与哮喘发病,阻断NGF后可有效减轻哮喘病理变化和降低多种炎性因子的表达。但是NGF参与哮喘发病的机制尚不完全清楚。
NGF促进神经细胞分化、发育和营养等生物学作用主要是通过结合其高亲合力特异性受体TrkA (tyrosine kinase receptor A)实现的。此外,神经生长因子介导SH2-Bβ(Src homology 2β)-Akt/PKB (protein kinase B,逆转录病毒Akt8癌基因V-akt编码的蛋白产物,故也称Akt)是调节嗜铬细胞瘤(PC12)细胞的信号途径之一。SH2-Bβ是广泛表达的高度保守的调节蛋白家族成员之一,能结合非酪氨酸激酶2(Janus kinase 2, Jas2)、胰岛素受体酪氨酸激酶和胰岛素样生长因子、NGF、血小板衍生因子、和成纤维细胞因子。SH2-Bp对NGF依赖的轴突生长和轴突存活非常关键。SH2-Bp加强NGF在PC12细胞中Akt的磷酸化和延长Akt酶活性(NGF激活TrkA使其与SH2-B结合,促使SH2-B发生酪氨酸磷酸化之后作用于丝氨酸／苏氨酸激酶Akt／PKB,Akt进一步激活分叉头转录因子(Forkhead transcription factors),促使PC12细胞分化增殖。研究表明SH2-Bβ在哮喘豚鼠肺组织巨噬细胞中过表达,同时还证明它参与NGF与其受体TrkA介导的哮喘发病。这些研究表明SH2-Bp在NGF介导的细胞功能中发挥基础性作用。基于以上信息,我们推测SH2-Bβ调节Akt磷酸化可能参与NGF介导的哮喘发病。
本课题利用哮喘小鼠模型,以肺组织为研究对象,观察Akt在哮喘肺组织及炎性细胞中的表达情况,观察NGF和SH2-Bβ与气道高反应的关系,观察阻断NGF和SH2-Bβ肺组织病理变化和炎性细胞数量的变化,应用多种方法观察NGF和SH2-Bβ在哮喘发病机制中对Akt及其活性的影响。为探讨NGF介导的哮喘的可能的发病机制和寻找过敏性气道疾病新的治疗靶点提供理论依据。
材料和方法
一、主要试剂
卵清蛋白(OVA, sigma,美国)；乙酰甲胆碱(mAch, sigma,美国)；RPMI1640培养基(GIBCO);定点突变试剂盒(Stratagene,美国)；兔抗鼠NGF抗体(Sigma,美国)；定点突变试剂盒(Stratagene,美国)；鼠多克隆p-Akt抗体(CellSignaling,美国),辣根过氧化酶免疫组化试剂盒(SABC,博士德,中国);ECL (enhanced chemi luminescence)试剂盒(Sigma Chemical,美国);Trizol裂解液(Santa Cruz,美国)；RNA PCR Kit (AMV) Ver.3.0 (Takara Biotechnology,中国大连);RNAout (Takara Biotechnology,中国)。
二、主要仪器
电子天平(BS110S, Startorius,德国)；恒温冷冻切片机(Leice,德国)；-80℃深低温冰箱(Toshiba,日本);低温冷冻超速离心机(3K18, Hitachi, Japan);超声粉碎机(Dr. Hielscher,德国)；通用电泳仪(Bio-Rad,美国)；普通光学显微镜(COIC, XSZ-H);超纯水系统(Millipore A10):MILLIPOREMILLI-B(美国)；超声雾化器(JWC-201D型,北京超声仪器厂)；AniRes2005动物肺功能分析系统(北京贝兰博科技有限公司)；MetaMorph显微图像分析系统(UIC/OLYMPUS); GDS8000凝胶图像处理系统；PCR仪(Biometra,德国)；Motic Images Advanced 3.2 (Motic, Xiamen)。
三、实验方法
1、动物及分组
雌性BALB／c小鼠85只,6-8周龄,体重20.0-22.0 g,清洁级,购自中国科学院实验动物繁育中心。随机分为4组：正常对照组,OVA组(哮喘组),R555E组,NGF阻断组。
2、哮喘模型复制及给药方法
1 mg OVA (Sigma, St Louis, MO, USA)和100mg氢氧化铝溶于0.5 ml PBS中,分别于0,7,14和21天腹腔注射给OVA组,R555E组和anti-NGF组小鼠致敏,从22天到30天雾化吸入10 mg／ml OVA PBS激发哮喘,每天一次,每次30分钟。OVA组继续雾化激发三天,而R555E组和anti-NGF组分别在激发前3小时尾静脉注射以1：10稀释于PBS中的50μl多克隆抗体兔抗鼠R555E和NGF,连续三天。正常组以等量的PBS腹腔注射和雾化吸入。
3、气道反应性测定
采用梯度浓度的mAch激发气道高反应,AniRes2005动物肺功能分析系统进行分析。测定小鼠气道阻力。
4、组织切片的准备
以上各组动物用多聚甲醛固定,取肺,相同位置切片。
5、免疫组织化学方法测定肺组织Akt表达。
6、肺泡灌洗液(BAIF)细胞计数及分类计数。用细胞计数板计数细胞总数,涂片进行Wright Giemsa染色作细胞分类计数。
7、免疫荧光测定p-Akt表达情况。
8、Western blot测定p-Akt蛋白水平,图像分析及数据处理。
9、RT-PCR检测Akt mRNA表达,图像分析及数据处理。
10、苏木素伊红(HE)染色观察肺组织病理变化。常规包埋,冰冻切片,进行苏木素伊红(HE)染色,观察肺组织病理变化。
11、统计学处理
所有数据采用SPSS14.0软件分析,实验数据以x±s表示。P<0.05为差异有显著性。
1、呼吸频率计数
呼吸频率各组无明显差异(P>0.05)。
2、支气管和肺组织病理变化
炎症细胞,粘膜水肿和上皮损伤anti-NGF组和R555E组小鼠没有未加干预的哮喘鼠明显(P<0.01)。在哮喘肺组织肺泡和支气管周围发现许多炎性细胞包括单核细胞,巨噬细胞,淋巴细胞和嗜酸性粒细胞。而正常鼠肺组织没有上述变化。
3、BALF细胞计数和分类
炎症细胞,粘膜水肿和上皮损伤anti-NGF组和R555E组小鼠没有未加干预的哮喘鼠明显(P<0.01)。OVA组小鼠的BALF的总细胞数明显高于正常鼠。巨噬细胞,嗜酸性粒细胞和淋巴细胞数与NGF组和R555E组比较OVA组明显升高。在NGF组和R555E组小鼠肺组织上述变化与OVA组比较明显减轻。
4、气道阻力测定
哮喘鼠对MCH的气道反应性比正常鼠增强(P<0.01)。静脉注射NGF抗体和R555E的OVA致敏鼠气道反应性明显低于OVA哮喘鼠。
5、免疫组织化学染色
哮喘组Akt／PKB在各种炎性细胞表达,包括嗜酸性粒细胞、嗜中性粒细胞、巨噬细胞、淋巴细胞、浆细胞等,并且这些炎性细胞非常多,肺泡内见大量的巨噬细胞,淋巴细胞和浆细胞等。正常组和NGF阻断组炎性细胞表达Akt／PKB很少,并且炎性细胞非常少。
6、免疫荧光分析NGF和SH2-Bβ对Akt活性影响
肺组织免疫荧光染色显示在OVA组p-Akt水平与正常组比较明显高表达。而上述部位过表达的p-Akt水平在NGF组和R555E组又呈降低趋势。
7、免疫印迹法分析p-Akt蛋白表达
在正常鼠的肺组织p-Akt蛋白表达处于低水平,而在OVA组则明显升高。NGF组和R555E组由OVA上调的p-Akt水平反而降低。
8、Anti-NGF和R555E对Akt mRNA的影响
OVA组Akt mRNA水平明显高于正常组,而NGF组和R555E组致敏鼠的Akt mRNA水平与OVA组比较明显降低。
结论
1、哮喘小鼠气道高反应明显增高,NGF参与哮喘小鼠的气道高反应和肺部炎症反应,阻断NGF明显减轻哮喘小鼠的气道高反应和肺部炎症反应。
2、SH2-Bβ参与哮喘气道高反应和肺部炎症反应,阻断SH2-Bβ减轻哮喘小鼠气道高反应和肺部炎症反应。
3、Akt信号分子在哮喘时被激活,NGF调节Akt活性可能是NGF参与哮喘发病的机制之一。
4、NGF介导的SH2-Bβ/Akt/PKB信号通路可能是NGF参与哮喘发病的通路之一
Bronchial asthma (bronchial asthma, referred to as asthma), is a chronic airway inflammation which many inflammatory cells, especially macrophages, mast cells, eosinophils and T lymphocytes take part in. The death rate and prevalence of bronchial asthma increase gradually at home and abroad. Bronchial asthma invalid some 100 million people around the world, becoming a serious threat to people's health a major chronic diseases. China's asthma incidence is 1%, children up to 3%.
Nerve growth factor (NGF) has been specifically implicated in asthma pathogenesis due to its ability to promote sensory neuron hyperreactivity and induce airway inflammation. Blocking NGF can effectively relieve asthmatic pathological changes and reduce a variety of cytokines. While the mechanism of NGF involved in the pathogenesis of asthma not fully understood. In addition, NGF regulates PC 12 cells in part by stimulating phosphorylation of Src homology 2β(SH2-Bβ) on serine/threonine kinase (Akt/PKB). SH2-Bβis a member of a widely expressed, highly conserved family of adaptor proteins and can bind to Janus kinase 2 (JAK2) and the activated receptor tyrosine kinases for insulin, insulin-like growth factor I, NGF, platelet-derived growth factor, and fibroblast growth factor. Moreover, SH2-Bβis critical for both NGF-dependent neurite outgrowth and neurite maintenance.Additionally, SH2-Bβenhances and prolongs of NGF-induced Akt phosphorylation and of Akt enzymatic activity in PC 12 cells. Li has also characterized SH2-Bβexpression in alveolar macrophages in the bronchoalveolar lavage fluid (BALF) of asthmatic guinea pigs and its role in NGF-TrkA-mediated asthma. Together, these studies suggest that SH2-Bβplays a fundamental role in receptor tyrosine kinase-mediated cellular functions. Together, these studies suggest that SH2-Bβplays a fundamental role in receptor tyrosine kinase-mediated cellular functions. Based on the above information, we hypothesized that upregulation of Akt phosphorylation by SH2-Bβmay contribute to the pathogenesis of NGF-mediated asthma.
By the study, using the mouse model of asthma, we observed expression of Akt at asthmatic lung tissue, observed the relationship of between NGF and SH2-B(3 with airway hyperresponsiveness, observed lung tissue pathological changes and changes in the number of inflammatory cells after blocking NGF and SH2-Bβ, observed effects of NGF and SH2-B(3 on the activity of Akt in the pathogenesis of asthma using several methods. These findings may pave the way for exploring the NGF-mediated asthma pathogenesis and the development of new therapeutic approaches to allergic airway diseases.
Methods
1、Animals
Fifty-five normal female BALB/c mice (6 to 8 weeks old) weighing 22±2.5 g were obtained from the Laboratory Animal Research Center in Beijing, China, they were randomly divided into four groups:control group, ovalbumin group, R555E group, and anti-NGF group.
2、OVA-treated asthma mice model and Drugs
On days (d) 0,7,14, and 21, all mice except the control group were actively sensitized with an intra-abdominal injection of 1 mg OVA (Sigma) and 100 mg aluminum hydroxide in 0.5 ml phosphate buffered saline (PBS), for the control group only with 0.5 ml PBS. The control group mice were then exposed to aerosolized PBS for 30 min per day from d 22 to d 30. Meanwhile, the OVA-sensitized mice were exposed to 10 mg/ml aerosolized OVA (1 g OVA in 100 ml sterile PBS in a nebulizer) for 30 min per day from d 22 to d 33. The anti-NGF group and R555E group mice were anesthetized and respectively administered an intravenous injection of 50μl polyclonal rabbit anti-mouse NGF antibody (Sigma, St Louis, MO, USA) diluted 1:10 in PBS and 50μl R555E. Intravenous treatment was administered 3h before allergen aerosol challenge on d 31,32, and 33.
3、Determination of Airway responsiveness
Airway responsiveness to muscarinic acetylcholine(mAch) was measured in AniRes2005 lung function system. Inspiratory resistance (IR) and expiratory resistance(ER) are detected.
4、Preparing the histological slices
The animals were perfused transcardially with 4% paraformaldehyde in PBS(0.1M, pH 7.4). The lungs were removed and postfixed. The lung slices at same positions of four groups.
5、Detecting the expressions of Akt at lungs by using immunohisto-chemical technique.
6、Total BALF cell counts were performed using hemacytometer. A differential cell count was carried out by Wright-Giemsa staining on the basis of morphologic criteria.
7、Determinating p-Akt of expression in lungs of 4 groups by Immunofluorescence
8、The expressions of p-Akt in lungs were detected by Western blotting.
9、The expressions of Akt mRNA in lungs were detected by Western blotting.
10、Hematoxylin-Eosin(HE) staining was employed to detect the histological changes in different groups.
The frozen lungs slices from different groups at 24 h reperfusion were stained with HE solution, and the histopathologic changes of hippocampuses were observed under the microscope.
11、Statistics
All values are expressed as the mean±SD. One-way analysis of variance (ANOVA) was used to compare group differences in Akt levels, while two-way ANOVA was used to assess differences in airway resistance.
1、Respiratory frequencys have no significant differences in each group
2、Pathological changes in the bronchi and lung tissue
Inflammatory cells, mucous edema, and epithelial lesions were significantly more prevalent in asthmatic mice than in those treated with anti-NGF or R555E. Many inflammatory cells, including mononuclear cells, macrophages, lymphocytes, and eosinophils, were observed in asthmatic pulmonary alveoli and the surrounding bronchi, in contrast with control mice. However, these finds reduced anti-NGF-and R555E-treated mice.
3、BALF cell counting and differential counting
The total number of cells in the BALF was significantly higher in OVA-sensitized mice than in control mice. The numbers of macrophages, eosinophils, and lymphocytes were also significantly increased in OVA-sensitized mice but reduced in anti-NGF-and R555E-treated mice.
4、Airway resistance measurement
The airway response to MCH was increased in the OVA group compared with the control group (P<0.01). Treatment of OVA-sensitized mice with intravenous anti-NGF and R555E prior to OVA challenge prevented this increase in airway reactivity.
5、Immunohistochemistry observed expression of Akt
Akt levels significantly increased in OVA group, but reduced in NGF group.
6、Immunofluorescent analysis of the effects of NGF and SH2-Bβon Akt activation
Immunofluorescent staining showed that p-Akt levels significantly increased in macrophages, eosinophils, and lymphocytes and bronchial tissue of the OVA group's lung tissue (P< 0.01), as compared with the control group. Meanwhile, the overexpression of p-Akt at the above sites was reduced in both anti-NGF-and R555E-treated groups.
7、Western blot analysis revealed p-Akt protein expression
In the lung tissue of the control group, p-Akt was expressed at low levels, but these levels increased markedly in the OVA group. However, anti-NGF and R555E greatly decreased the p-Akt upregulation caused by OVA.
8、Effects of anti-NGF and R555E on Akt mRNA
Akt mRNA expression was significantly higher in the OVA group than in the control group, but that anti-NGF and R555E treatment significantly decreased Akt mRNA expression in these OVA-sensitized mice.
Conclusions
1、The airway response was increased in the OVA group, NGF participates in airway hyperresponsiveness and inflammatory response of in the lungs of asthmatic mice. Blocking NGF significantly reduced airway hyperresponsiveness and inflammatory response in the lungs of asthmatic mice
2、Akt may be involved in pathogenesis of asthma, NGF regulates activity of AKT may be one of the mechanisms of NGF involved in asthma.
3、SH2-Bβinvolved in airway hyper-responsiveness and inflammatory response of lung of asthmatic mice, and blocking SH2-Bβcould alleviate airway hyperresponsiveness and inflammatory response in the lungs of asthmatic mice.
4、NGF mediated SH2-Bβ/Akt signaling pathway may be one of the pathways NGF-mediated asthma.
引文
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25 李东培,才丽平,方秀斌.哮喘豚鼠下呼吸道及内脏传人部位NGF表达的研究[J].解剖学报,2002,33(4)：350-4.
26 刘晓湘,曹德寿,方秀斌等.NG寸哮喘豚鼠下呼吸道及内脏感觉传人部位TNF-mRNA表达的上调作用.解剖学报,2004,35(3)：225-229.
27 刘晓湘,方秀斌.神经生长因子对实验性哮喘豚鼠降钙素基因相关肤的调节作用[J].中 国临床康复,2004,8(24)：5020-5021.
28 刘晓湘,曹德寿,方秀斌等.NGF对哮喘豚鼠下呼吸道及内脏感觉传人部位IL-1 B的影响[J].神经解剖学杂志,2005,21(21)：597-602.
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31 才丽平,方秀斌,高杰,等.实验性哮喘豚鼠的内脏感觉传人部位sP免疫反应的研究[J].神经解剖学杂志,2001,17(1)：68-70.
32 曹德寿,刘晓湘,方秀斌,哮喘豚鼠初级传人神经元ERK的表达及NGF的调节作用[J],解剖科学进展,2004,10(1)：23-26.
33 曹德寿,刘晓湘,方秀斌等,实验性支气管哮喘豚鼠肺和内脏感觉传人系统蛋白激酶c的表达及神经生长因子的调节作用[J].中华结核和呼吸杂志,2005,28(8)：525—529.
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23 Zhu JQ, Feng JT, Hu CP, et al. Experimental study on the mechanisms of regulating airway neurogenic inflammation in asthma by never growth factor and leukemia inhibitory factor [J]. Zhonghua Jie He He Hu Xi Za Zhi,2006,29(6):376-80.
24 方秀斌,刘晓湘,张鸿.实验性哮喘时神经生长因子对神经激肽A的上调作用[J].中国病理生理杂志,2004,20(1)：80-2.
25 李东培,才丽平,方秀斌.哮喘豚鼠下呼吸道及内脏传人部位NGF表达的研究[J].解剖学报,2002,33(4)：350-4.
26 刘晓湘,曹德寿,方秀斌等.NG寸哮喘豚鼠下呼吸道及内脏感觉传人部位TNF-mRNA表达的上调作用.解剖学报,2004,35(3)：225-229.
27 刘晓湘,方秀斌.神经生长因子对实验性哮喘豚鼠降钙素基因相关肤的调节作用[J].中 国临床康复,2004,8(24)：5020-5021.
28 刘晓湘,曹德寿,方秀斌等.NGF对哮喘豚鼠下呼吸道及内脏感觉传人部位IL-1 B的影响[J].神经解剖学杂志,2005,21(21)：597-602.
29 刘晓湘,方秀斌,张鸿等.实验性哮喘时神经生长因子对P物质的上调作用[J].中国病理生理杂志,2005,21(10)：1978-1980.
30 方秀斌,刘晓湘,张鸿.实验性哮喘时神经生长因子对神经激肽A的上调作用中国病理生理杂志,2004,20(1)：80-82.
31 才丽平,方秀斌,高杰,等.实验性哮喘豚鼠的内脏感觉传人部位sP免疫反应的研究[J].神经解剖学杂志,2001,17(1)：68-70.
32 曹德寿,刘晓湘,方秀斌,哮喘豚鼠初级传人神经元ERK的表达及NGF的调节作用[J],解剖科学进展,2004,10(1)：23-26.
33 曹德寿,刘晓湘,方秀斌等,实验性支气管哮喘豚鼠肺和内脏感觉传人系统蛋白激酶c的表达及神经生长因子的调节作用[J].中华结核和呼吸杂志,2005,28(8)：525—529.
34 王巧玲,齐金萍,方秀斌,TrkA对哮喘小鼠下呼吸道气道阻力及IL-1 B表达的上调作用[J],解剖科学进展,2006,12(2)：97-99.
35 Jinping Q, Deshou C, Xiaoxiang L, et al, The expression of SH2-BB in the lung and visceral sensory aferent system of the asthmatic mice[J]. Acta Anatomica Sinica,2006,36:350-354.
36 齐金萍,刘晓湘,方秀斌等.哮喘小鼠肺内和初级传人神经元SH2-Bp表达的调节作用[J],解剖科学进展,2006,12(1)：23-26.
37 黄小艳,吴宜林,刘凤英,等.转化生长因子TGF2 B 1在卵巢上皮性肿瘤中的表达及义[J].湖南医科大学学报,2002,27(1)：26-8.
38 Citterio F, Pozzetto U, Romagnoli J, et al. Plasma levels of transforming growth factor-B 1 in renal transplant recipients receiving diferent immunosuppressive regimens [J]. Transplant Proc,2004,36(3):698-9.
39 Nef LM, Borgatti P, Capitani S, et al .The nuclear phosphoinositide 3-kinase/AKT pathway:a new second messenger system[J]. Biochim Biophy Acta,2002,1584(2-3):73-80.
40 Matsuzaki H, Yamamoto T, Kikkawa U. Distinct Activation mechanisms of protein kinase B by growth-factor stimulationand heat-shock treatment[J]. Bio-chemistry, 2004,43(14):4284-4293.
41 Madelon B, Ed van de Graaf,Jan-Willem J L, et al. In vivo priming of FcR functioning on eosinophils of allergic asthmatics[J]-Leukocyte Biology,2000,68:655-61.
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