法呢基硫代水杨酸对哮喘小鼠气道炎症，气道重塑和Th细胞分化的影响及其机制的初步研究
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摘要
背景：支气管哮喘是一种由多种细胞及细胞因子参与的慢性气道炎症性疾病,其病因复杂,发病机制至今尚未完全清楚,涉及到免疫学、神经内分泌学、遗传、环境等多种因素。气道炎症,气道高反应性,气道重塑是其三个基本特征。目前有关Ras通路与哮喘的关系的研究逐渐成为热点。Ras作为细胞生长分化的主要调节者,在细胞外刺激产生的信号转导通路中处于中枢地位。已有研究显示Ras介导的各下游信号传导通路在支气管哮喘发生发展的各个环节中均发挥重要作用。研究Ras信号转导通路有助于寻求新的药物作用靶点。
Ras信号传导通路主要由以下几条途径组成：1.丝裂原活化蛋白激酶信号通路(Mitogen-activated protein kinase, MAPK)。Ras-MAPK通路是目前最为明确的信号通路。细胞外信号调节蛋白激酶(Extracellular signal regulated kinase ERK)是MAPK信号通路下游家族成员之一,参与调控细胞有丝分裂,与细胞的增殖分化迁移密切相关。2.磷脂酰肌醇-3-激酶-蛋白激酶B信号通路(Phosphatidylinositol-3-kinase (PI3K)-protein kinase B (AKT))。在调节细胞代谢、凋亡、增殖和分化方面与Ras-MAPK途径同等重要。在肿瘤发生过程中起重要的辅助作用,介导气道炎性疾病嗜酸细胞的分化、降解、聚集及粘附分子的表达。3. GalGEF信号通路。使细胞免于凋亡,并加速细胞周期进程和细胞生长。4.其他。如Rac和Rho信号通路、Ras鸟嘌呤核苷酸交换因子(RasGRP)信号通路、NF-kappB信号通路、转化生长因子-β(TGF-β)信号通路等。
Ras传导通路与很多疾病(包括支气管哮喘、肿瘤等)的发生发展均有密切的关系。已经证实：基于ras信号通路为靶标的抗肿瘤治疗方法,在治疗由ras突变所导致的肿瘤中具有重要的价值。目前针对Ras信号途径的药物研究主要有以下几类：(1)法尼基化转移酶抑制剂(Farnesyltransferase inhibitors, FTIs)。法呢基转移酶(Farnesyltransferase, FTIase)是Ras蛋白加工过程中最重要的催化酶之一。Ras蛋白翻译后的修饰过程主要包括三个步骤：Ras蛋白羧基末端半胱氨酸(Cys)残基的法呢酯化,经法呢酯化的Cys残基的甲基化,以及清除羧基末端的氨基酸残基。其中羧基端的法尼基化是ras蛋白定位于细胞膜内侧进而发挥效能的关键修饰步骤。因此,抑制ras蛋白翻译后羧基端的法尼基化是抑制ras信号通路的一种重要治疗手段。(2)抗Ras的反义寡核苷酸。根据Ras蛋白和mRNA序列设计反义RNA,通过碱基互补配对的方式,抑制、破坏目的基因,以下调Ras的表达。(3)抑制ras的下游靶点。通过反义核苷酸技术合成的小分子化合物能特异抑制Ras及下游靶点RafmRNA的表达,下调ERK活性。(4)抑制Ras的上游靶点。Ras蛋白上游信号通路的异常活化亦可激活Ras。小分子酪氨酸激酶抑制剂和针对细胞外受体区域的抗体,通过阻断生长因子受体酪氨酸激酶的活性,从而抑制Ras的活化。(5)恢复Ras蛋白GTP酶的活性。Ras突变导致GTP酶失活,已研制的GTP类似物可代替GAP,更有效的水解Ras蛋白。(6)其他。包括以Ras通路为靶标的基因治疗、免疫治疗,以及以Ras外周成分为靶标的或以与Ras信号通路明显相关的其他信号通路为靶标的治疗方法等。目前,基于ras信号通路为靶标的治疗策略,在人类肿瘤的治疗中显示了广阔的前景。
但Ras抑制剂对支气管哮喘的干预作用及其机制尚罕见报道。反式法呢基硫代水杨酸(Trans-farnesyl thiosalicylic acid, FTS)是一种新型的Ras抑制剂,FTS对结合活化GTP的Ras具有明显的选择性,在动物模型中无明显毒性或不良副作用。
目的：本研究建立小鼠哮喘模型,着重研究FTS对哮喘小鼠气道炎症,气道重塑和Th细胞分化的影响,进一步探讨Ras通路与支气管哮喘发生发展的关系,以及FTS作为Ras抑制剂,对支气管哮喘的干预机制,为哮喘的治疗提供新的思路。
方法：6-8周级BALB/c小鼠60只,随机法分成六组,每组10只。正常对照组、哮喘模型组、地塞米松治疗组、5FTS干预组(5mg/kg)、10FTS干预组10mg/kg)、15FTS干预组(15mg/kg)。
1.制作哮喘小鼠模型：于实验第1d、第14d致敏,即卵白蛋白(OVA)40μg由1mg氢氧化铝乳化,总体积200μ1,腹腔注射。第21-27d,将小鼠置于密闭容器内,以超声雾化器雾化,3%OVA盐水气溶胶吸入激发,每天1次,每次30min。
2.药物干预：地塞米松治疗组,于每次激发前1小时腹腔注射2mg/kg地塞米松；FTS干预组,于每次激发前1小时分别腹腔注射5mg/kg、10mg/kg、15mg/kg FTS。
3.收集实验标本：支气管肺泡灌洗液,血清,肺组织、脾组织。
4.检测实验指标
4.1小鼠左肺组织HE染色,观察组织形态学变化。
4.2检测小鼠支气管肺泡灌洗液(BALF)细胞总数、嗜酸粒细胞数。
4.3酶联免疫吸附法检测小鼠血清中IL-5浓度。
4.4免疫印迹法(Western Blot)检测小鼠右肺组织pan-ras, ERK蛋白表达水平,脾组织GATA-3蛋白表达水平。
4.5实时荧光定量聚合酶链反应(Real-time Quantitative PCR, QPCR):检测小鼠右肺组织MMP-2、MMP-9mRNA表达,脾组织Foxp3, GATA-3mRNA表达。
结果：
1)肺组织形态学：正常对照组小鼠可见支气管壁光滑,上皮细胞完整,肺泡及支气管周围未见炎性细胞浸润,肺泡黏膜无肿胀。哮喘模型组小鼠,肺泡及支气管周围见可见明显炎性细胞浸润,支气管上皮水肿,气道周围小血管充血明显。地塞米松及FTS干预组,可见支气管及肺泡周围少量炎性细胞浸润,支气管及肺泡结构基本正常,炎症及充血不同程度的减轻。
2)小鼠支气管肺泡灌洗液炎症细胞计数、血清IL-5浓度在各组间均有显著性差异(BALF细胞总数F=52.27,嗜酸细胞数F=67.61,血清IL-5浓度F=23.57,P值均为0.00)。哮喘组小鼠支气管肺泡灌洗液中细胞总数、嗜酸细胞数及血清IL-5浓度均较正常组增高(P<0.05),地塞米松及FTS干预组各指标均低于未干预组(P<0.05),且随着FTS浓度的增加,各指标下降更明显。1OFTS及15FTS组与地塞米松组相比,有更显著地疗效。但1OFTS组与15FTS组间差异无显著性。
3)小鼠肺pan-ras蛋白的表达均值在各组间有显著性差异(F=153.55,P=0.00)。哮喘组小鼠肺组织中pan-Ras的蛋白表达均较正常对照组增高(P<0.05),地塞米松及FTS干预组较未干预组表达减少(P<0.05),与地塞米松治疗组相比,FTS的下调作用更为明显,但无浓度依赖性。
4)小鼠脾GATA-3蛋白及GATA-3mRNA的表达均值在各组间均有显著性差异(GATA-3蛋白F=106.44, P=0.00; GATA-3mRNA F=6.02, P=0.00)。哮喘组脾组织GATA-3mRNA和GATA-3蛋白的表达较正常对照组均有增加(P<0.05),地塞米松及FTS干预组较未干预组表达减少(P<0.05),减少程度与FTS浓度无关。对GATA-3mRNA的表达,FTS各组疗效与地塞米松相当。对GATA-3蛋白表达,地塞米松组和FTS各相比差异显著(P<0.05),提示FTS对脾GATA-3蛋白表达的抑制作用大于地塞米松组。
5)小鼠脾Foxp3mRNA的表达均值在各组间有显著性差异(F=2.85,P=0.029)。哮喘组小鼠脾组织中Foxp3mRNA表达较正常对照组减少(P<0.05)。FTS干预后,表达水平较未干预组增高(P<0.05),且与浓度无关。地塞米松干预后虽有升高趋势,但与未干预组相比无统计学意义(P>0.05)。
6)小鼠肺ERK1/2蛋白的表达均值在各组间有显著性差异(F=92.34,P=0.00)。哮喘组小鼠肺组织ERKl/2的蛋白表达较正常对照组增高(P<0.05),干预后,表达水平较未干预组减少(P<0.05),减少程度与FTS浓度无关。地塞米松组与FTS组各相比差异显著(P<0.05),提示FTS的作用强于地塞米松。
7)小鼠肺MMP-2、MMP-9mRNA的表达均值在各组间有显著性差异(MMP-2F=4.51, P=0.003; MMP-9F=4.68, P=0.002)。哮喘小鼠肺组织MMP-2、MMP-9表达较正常对照组增高(P<0.05),干预后,表达水平较未干预组减少(P<0.05),减少程度与FTS剂量无关。地塞米松组和FTS各组相比疗效无显著差异(P>0.05)。
结论：本文从免疫机制、神经内分泌机制等角度探讨了Ras通路与支气管哮喘发生发展的关系。通过抑制Ras信号通路可以减轻支气管哮喘小鼠的气道炎症及气道重塑,为哮喘的治疗开拓了新的思路。本研究中,FTS作为Ras抑制剂,与糖皮质激素一样,能明显改善哮喘模型小鼠气道炎症细胞浸润、减少细胞因子IL-5的分泌,下调肺组织pan-ras、ERK1/2蛋白及MMP-9、MMP-2mRNA表达水平,下调脾组织GATA-3mRNA、GATA-3蛋白表达水平,上调脾组织Foxp3mRNA表达水平。减轻哮喘小鼠气道炎症、气道重塑,促进TH1/TH2细胞平衡的恢复,增加Treg的数量及其抑制功能。其机制可能是通过抑制不同的Ras下游级联反应通路,从而调节复杂的细胞内信号传导过程,最终发挥治疗哮喘的作用。但哮喘的发病机制极为复杂,Ras信号转导途径亦是一个复杂的相互交联的网络,且与其他的信号系统相互联系。本研究亦发现FTS与地塞米松的治疗效应并不完全一致,可能二者通过影响不同的信号通路机制起效。总之,在治疗支气管哮喘方面,FTS的作用机制仍未完全清晰,尚需进一步更深入的研究。
Background:Asthma is a disease of chronic airway inflammation associated with many cells and cytokines. Up to now, it not very clear to the complex nosogenesis. It involve the immunology, neurology, heredity, endocrinology, environment and so on. The inflammation, hyperresponsiveness and remodling of airway are the basic propty of asthma. At present, researches about the relationship between Ras pathway and asthma has become a hot spot. As the main moderator of cell growth and differentiation, Ras lies the central position in signal transduction pathways of the extracellular stimulations. It has been showed that the signal transduction Mediated by Ras play an important role in the occurrence and development of bronchial asthma. Research on Ras signal transduction pathways contribute to seek the new drug targets.
The Ras signal transduction pathway consists mainly of the following ways:1. Mitogen-activated protein kinase (MAPK), The Ras-MAPK pathway is the most clear signal pathway at present. Extracellular signal regulated kinase(ERK) Is one of the family members of MAPK. Participate in the regulation of cell mitosis, Closely associated with the proliferation, differentiation and migration of cells.2. Phosphatidylinositol-3-kinase and protein kinase B signaling pathway.. Equally important as Ras-MAPK pathway in the regulation of cell metabolism, proliferation,differentiation and apoptosis. It plays an important helping role in the tumorigenesis, and mediate the differentiation, degradation, assemble and the expression of adhension molecules.3. GalGEF signal pathway. It adviod the cells from apoptosis and accelerate the growth of cell cycle progression.4. For example,Rac and Rho signal pathway, Ras guanine nucleotide exchange factor (RasGRP) signaling pathway, NF-kappB signal pathway, Transforming growth factor-β(TGF-β) signal pathway, and etc.
Ras signaling pathway have close relationship with the occurrence and development of many diseases (including cancer, bronchial asthma, etc). It has been improved that the method for tumor treatment based on the RAS signal pathway has important value in the treatment of tumors caused by Ras mutations. The current research of drug about the Ras signal pathway mainly in the following categories:1) Farnesyltransferase inhibitors, FTIs. Faniki Farnesyltransferase is one of the most important catalytic enzyme in the Ras protein processing. The Ras protein post-translational modificated processing consists of three steps:The farnesyl esterification of the C-terminal cysteine (Cys) residues of Ras protein. The methylation of the farnesyl esterificated Cys residues. And removing the amino acid residues from carboxyl terminus. The C-terminal farnesylation play a key modification steps of Ras protein in the location on the cell membrane and then developing the effectiveness. Therefore, inhibition of Ras protein post-translational farnesylation of carboxyl terminal is an important treatment for inhibiting Ras signal pathway.2) Antisense oligonucleotide against Ras. According to the Ras protein and mRNA sequence, we design ed the antisense RNA,. By the way of complementary base pairing, inhibited and damaged the aimed gene expression, then down regulated the expression of Ras.3) Inhibition of Ras downstream targets. By antisense oligodeoxynucleotide, the small molecule compounds can inhibit Ras expression and its downstream target of RafmRNA, down-regulate the activity of ERK.4) Inhibition of Ras upstream targets. Tthe Ras can be activated by the abnormal activation of the upstream signaling pathway of Ras protein.The small molecule tyrosine kinase inhibitors and the antibodies to the receptors of extracellular region, can inhibite Ras activation by blocking the activation of growth factor receptor's tyrosine kinase.5) Recovery of Ras protein GTP activity. Ras mutations lead to GTP inactivation, GTP analogues have been developed can be used instead of GAP, can hydrolyze the Ras protein more effectivly.6) et al. Include the gene therapy and immune therapy targeting the Ras pathway, as well as the therapy targeting Ras peripheral components or other signaling pathway which was significantly related to the Ras signal pathway, and etc. In short,the therapy strategy based on the Ras signaling pathway has showed a broad prospects in the treatment of human cancer.
But the intervention effect and mechanism of Ras inhibitors on the pathogenesis of asthma is seldom reported. Farnesyl thiosalicylic acid (trans-farnesylthiosalicylic, acid, FTS) is a kind of novel Ras inhibitors., FTS has obvious selectivity for the activation of GTP-bound Ras,. It has no toxic or adverse side effects In the animal models.
Objectives:This study established a murine model of asthma, focuses on the study of FTS on airway inflammation, airway remodeling and the differentiation of Th cell. To further explore the relationship between Ras pathway and bronchial asthma.and the intervention mechanism on bronchial asthma of FTS. To provide a new idea for the treatment of asthma
Methods:60BALB/c mice (6-8week), are randomly divided into six groups, each group has10mice. Control group(n=10), asthma group(n=10), dexamethasone treatment group(n=10),5FTS group,(n=10,5mg/kg),10FTS group,(n=10,10mg/kg),15FTS group,(n=10,15mg/kg).
1. Making a mouse asthma model:Sensitated the mice with ova/AL(OH)3intraperitoneal injection (40μgOVA each mouse) at1st day and Fourteenth day. Motivated the mice with3%OVA saline aerosol by Ultrasonic nebulizer1times a day from twenty-first day to twenty-seventh day,for30min each time.
2. medicine treatment:Dexamethasone group, intraperitoneal injection of2mg/kg dexamethasone before motivation each time. FTS group:intraperitoneal injection of FTS before motivation each time. The doses are5mg/kg,10mg/kg and15mg/kg for the three groups.
3. Collection of specimens:Bronchoalveolar lavage fluid, serum, lung tissue, spleen tissue.
4. Testing index:
a) Lung tissue in mice with HE staining, observe the histological changes.
b) Detection of mouse bronchoalveolar lavage (BALF) cell counts, eosinophil count s
c) Enzyme linked immunosorbent assay for the detection of IL-5concentration in serum of mice
d) Western Blot for the detection of the expression level of pan-ras, ERK protein in lung tissues, and GATA-3protein in spleen tissues of mice.
e) Real-time fluorescence quantitative polymerase chain reaction (Real-time Quantitative PCR, QPCR):to detect the expression of MMP-2, MMP-9mRNA in lung tissue of mice, and the expression of Foxp3, GATA-3mRNA in spleen tissues of mice
Results:
1. The Histomorphology of lung tissures:The lung tissures of the mice in the normal control group have smooth bronchial wall, intact alveolar epithelial cells, surrounded by no infiltration of inflammatory cells, and no alveolar mucosa swelling. And in the asthma model group, the lung tissures have obvious infiltration of inflammatory cells around the alveolus, edema of alveolar mucosa, hyperplasia, shedding, and mucus secretion of bronchial epithelial. Furthermore, there are contraction of bronchial, inflammatory cells infiltration of peribronchial, significantly congestion of the small vascular Airway around the airway, following a large number of inflammatory cells infiltration. In lung tissures of DEX and FTS intervention group, we can see the that the bronchiolar epithelial structures are basically normal, a few infiltration of inflammatory cells in the submucosal, alveolar structure basically normal. So,there is reduced inflammation to varying degrees.
2. Each index in the six groups of mice were a number of significant difference (BALF cell total F=52.27, eosinophil numbers F=67.61, serum IL-5concentration of F=23.57, P=0).The number of the BALF cell counts, eosinophil counts and serum IL-5concentrations of asthma mice were higher than the normal control group (P<0.05), They were lower in dexamethasone and FTS intervention group than in those of non-intervention group (P<0.05). and with the increase of FTS concentration, the all indexes fell more obviously. But the difference between the10mgFTS group and the15mgFTS group has no significant. Comparison of the dexamethasone group, the10mgFTS and15mgFTS group have the more significant effect.
3. There were significant differences among the groups in pan-ras protein expression in mouse lung (F=153.55, P=0.00)。 Expression of pan-Ras protein in lung tissues of asthmatic group were higher than control group (P<0.05). They were lower in dexamethasone and FTS intervention group than in those of non-intervention group (P<0.05). Compared with dexamethasone treatment group, the effction of down-regulation of FTS is more apparent, and there is no concentration dependence.
4. Among the groups, there were significant differences of the expression of GATA-3protein and GATA-3mRNA in mice spleen,(GATA-3protein F=106.44, P=0.00; GATA-3mRNA F=6.02, P=0.00). The expression of GATA-3mRNA and GATA-3protein in splenic tissues of asthmatic group were increased compared with normal control group (P<0.05), They were lower in dexamethasone and FTS intervention group than in those of non-intervention group (P<0.05). The effection were independent of the concentration of FTS.The effects of FTS and dexamethasone was the same on the expression of GATA-3mRNA. But on the expression of GATA-3protein, the effect of FTS is more stronger than that of dexamethasone group.
5. There were significant differences in mean of Foxp3mRNA expression among each group of mice spleen.(F=2.85, P=0.029). Compared with the normal control group, the expression of Foxp3mRNA were reduced in spleen tissue in asthmatic mice (P<0.05).After intervention of FTS, the expression levels were higher than in the no-intervention group (P<0.05), and is independent of concentration. Although the expression in dexamethasone group has increased, there were no statistical significance compared with non-intervention group (P>0.05).
6. There were significant differences among the groups in ERK1/2protein expression in mouse lung (F=92.34, P=0.00). Expression of ERK1/2protein in lung tissue of asthmatic mice are higher than in normal control group (P<0.05), After intervention of FTS and dexamethasone, the expression levels were lower than in the no-intervention group (P<0.05), and is independent of FTS concentration. Compared with dexamethasone,there was more significant reduce in FTS group (P<0.05).
7. There were significant differences in mean of MMP-2and MMP-9mRNA expression among each group of mice lung.(MMP-2F=4.51, P=0.003; MMP-9F=4.68, P=0.002). The expression of MMP-2and MMP-9mRNA in lung tissue of asthmatic group was increased compared with normal group (P<0.05). Compared with no intervention group, the expression level of the two indexes were decreased in FTS and dexamethasone groups (P<0.05). There was no FTS dose dependent. There was no ignificant difference in the effect of dexamethasone group and FTS groups (P>0.05).
Conclusions:This paper discusses the relationship between Ras pathway and bronchial asthma from the point of view of mechanism of immune and neuroendocrine. The alleviation of inflammation and airway remodeling in mice bronchial asthma by inhibiting Ras signal pathway, opened up a new way for the treatment of asthma. In this study, As Ras inhibitors, FTS as well as glucocorticoid, can significantly improve the inflammatory cells infiltration of airway model mice, reduce the secretion of cytokine IL-5, down-regulate the levels of ERK1/2protein, pan-ras protein, MMP-9and MMP-2mRNA in lung tissues, levels, lower the levels of GATA-3mRNA and GATA-3protein in spleen tissues, increased the levels of Foxp3mRNA in spleen tissues. Alleviating the inflammation, airway remodeling in asthmatic mice airway, promoting recovery of TH1/TH2cells balance, increasing the quantity of Treg and its inhibition function. The mechanism is probably through the inhibition of different Ras downstream cascade pathway, thereby regulating the complex process of intracellular signal transduction, ultimately play in asthma. But the pathogenesis of asthma is very complex, the Ras signal transduction pathway is also a complex interconnected network, and interaction with other signal system. The study found that the treatment effect of FTS and dexamethasone is not entirely consistent, may be through the different signal transduction pathways. In short, the action mechanism of FTS in the treatment of bronchial asthma is not perfectly clear. The further research must be required.
Ras信号传导通路主要由以下几条途径组成：1.丝裂原活化蛋白激酶信号通路(Mitogen-activated protein kinase, MAPK)。Ras-MAPK通路是目前最为明确的信号通路。细胞外信号调节蛋白激酶(Extracellular signal regulated kinase ERK)是MAPK信号通路下游家族成员之一,参与调控细胞有丝分裂,与细胞的增殖分化迁移密切相关。2.磷脂酰肌醇-3-激酶-蛋白激酶B信号通路(Phosphatidylinositol-3-kinase (PI3K)-protein kinase B (AKT))。在调节细胞代谢、凋亡、增殖和分化方面与Ras-MAPK途径同等重要。在肿瘤发生过程中起重要的辅助作用,介导气道炎性疾病嗜酸细胞的分化、降解、聚集及粘附分子的表达。3. GalGEF信号通路。使细胞免于凋亡,并加速细胞周期进程和细胞生长。4.其他。如Rac和Rho信号通路、Ras鸟嘌呤核苷酸交换因子(RasGRP)信号通路、NF-kappB信号通路、转化生长因子-β(TGF-β)信号通路等。
Ras传导通路与很多疾病(包括支气管哮喘、肿瘤等)的发生发展均有密切的关系。已经证实：基于ras信号通路为靶标的抗肿瘤治疗方法,在治疗由ras突变所导致的肿瘤中具有重要的价值。目前针对Ras信号途径的药物研究主要有以下几类：(1)法尼基化转移酶抑制剂(Farnesyltransferase inhibitors, FTIs)。法呢基转移酶(Farnesyltransferase, FTIase)是Ras蛋白加工过程中最重要的催化酶之一。Ras蛋白翻译后的修饰过程主要包括三个步骤：Ras蛋白羧基末端半胱氨酸(Cys)残基的法呢酯化,经法呢酯化的Cys残基的甲基化,以及清除羧基末端的氨基酸残基。其中羧基端的法尼基化是ras蛋白定位于细胞膜内侧进而发挥效能的关键修饰步骤。因此,抑制ras蛋白翻译后羧基端的法尼基化是抑制ras信号通路的一种重要治疗手段。(2)抗Ras的反义寡核苷酸。根据Ras蛋白和mRNA序列设计反义RNA,通过碱基互补配对的方式,抑制、破坏目的基因,以下调Ras的表达。(3)抑制ras的下游靶点。通过反义核苷酸技术合成的小分子化合物能特异抑制Ras及下游靶点RafmRNA的表达,下调ERK活性。(4)抑制Ras的上游靶点。Ras蛋白上游信号通路的异常活化亦可激活Ras。小分子酪氨酸激酶抑制剂和针对细胞外受体区域的抗体,通过阻断生长因子受体酪氨酸激酶的活性,从而抑制Ras的活化。(5)恢复Ras蛋白GTP酶的活性。Ras突变导致GTP酶失活,已研制的GTP类似物可代替GAP,更有效的水解Ras蛋白。(6)其他。包括以Ras通路为靶标的基因治疗、免疫治疗,以及以Ras外周成分为靶标的或以与Ras信号通路明显相关的其他信号通路为靶标的治疗方法等。目前,基于ras信号通路为靶标的治疗策略,在人类肿瘤的治疗中显示了广阔的前景。
但Ras抑制剂对支气管哮喘的干预作用及其机制尚罕见报道。反式法呢基硫代水杨酸(Trans-farnesyl thiosalicylic acid, FTS)是一种新型的Ras抑制剂,FTS对结合活化GTP的Ras具有明显的选择性,在动物模型中无明显毒性或不良副作用。
目的：本研究建立小鼠哮喘模型,着重研究FTS对哮喘小鼠气道炎症,气道重塑和Th细胞分化的影响,进一步探讨Ras通路与支气管哮喘发生发展的关系,以及FTS作为Ras抑制剂,对支气管哮喘的干预机制,为哮喘的治疗提供新的思路。
方法：6-8周级BALB/c小鼠60只,随机法分成六组,每组10只。正常对照组、哮喘模型组、地塞米松治疗组、5FTS干预组(5mg/kg)、10FTS干预组10mg/kg)、15FTS干预组(15mg/kg)。
1.制作哮喘小鼠模型：于实验第1d、第14d致敏,即卵白蛋白(OVA)40μg由1mg氢氧化铝乳化,总体积200μ1,腹腔注射。第21-27d,将小鼠置于密闭容器内,以超声雾化器雾化,3%OVA盐水气溶胶吸入激发,每天1次,每次30min。
2.药物干预：地塞米松治疗组,于每次激发前1小时腹腔注射2mg/kg地塞米松；FTS干预组,于每次激发前1小时分别腹腔注射5mg/kg、10mg/kg、15mg/kg FTS。
3.收集实验标本：支气管肺泡灌洗液,血清,肺组织、脾组织。
4.检测实验指标
4.1小鼠左肺组织HE染色,观察组织形态学变化。
4.2检测小鼠支气管肺泡灌洗液(BALF)细胞总数、嗜酸粒细胞数。
4.3酶联免疫吸附法检测小鼠血清中IL-5浓度。
4.4免疫印迹法(Western Blot)检测小鼠右肺组织pan-ras, ERK蛋白表达水平,脾组织GATA-3蛋白表达水平。
4.5实时荧光定量聚合酶链反应(Real-time Quantitative PCR, QPCR):检测小鼠右肺组织MMP-2、MMP-9mRNA表达,脾组织Foxp3, GATA-3mRNA表达。
结果：
1)肺组织形态学：正常对照组小鼠可见支气管壁光滑,上皮细胞完整,肺泡及支气管周围未见炎性细胞浸润,肺泡黏膜无肿胀。哮喘模型组小鼠,肺泡及支气管周围见可见明显炎性细胞浸润,支气管上皮水肿,气道周围小血管充血明显。地塞米松及FTS干预组,可见支气管及肺泡周围少量炎性细胞浸润,支气管及肺泡结构基本正常,炎症及充血不同程度的减轻。
2)小鼠支气管肺泡灌洗液炎症细胞计数、血清IL-5浓度在各组间均有显著性差异(BALF细胞总数F=52.27,嗜酸细胞数F=67.61,血清IL-5浓度F=23.57,P值均为0.00)。哮喘组小鼠支气管肺泡灌洗液中细胞总数、嗜酸细胞数及血清IL-5浓度均较正常组增高(P<0.05),地塞米松及FTS干预组各指标均低于未干预组(P<0.05),且随着FTS浓度的增加,各指标下降更明显。1OFTS及15FTS组与地塞米松组相比,有更显著地疗效。但1OFTS组与15FTS组间差异无显著性。
3)小鼠肺pan-ras蛋白的表达均值在各组间有显著性差异(F=153.55,P=0.00)。哮喘组小鼠肺组织中pan-Ras的蛋白表达均较正常对照组增高(P<0.05),地塞米松及FTS干预组较未干预组表达减少(P<0.05),与地塞米松治疗组相比,FTS的下调作用更为明显,但无浓度依赖性。
4)小鼠脾GATA-3蛋白及GATA-3mRNA的表达均值在各组间均有显著性差异(GATA-3蛋白F=106.44, P=0.00; GATA-3mRNA F=6.02, P=0.00)。哮喘组脾组织GATA-3mRNA和GATA-3蛋白的表达较正常对照组均有增加(P<0.05),地塞米松及FTS干预组较未干预组表达减少(P<0.05),减少程度与FTS浓度无关。对GATA-3mRNA的表达,FTS各组疗效与地塞米松相当。对GATA-3蛋白表达,地塞米松组和FTS各相比差异显著(P<0.05),提示FTS对脾GATA-3蛋白表达的抑制作用大于地塞米松组。
5)小鼠脾Foxp3mRNA的表达均值在各组间有显著性差异(F=2.85,P=0.029)。哮喘组小鼠脾组织中Foxp3mRNA表达较正常对照组减少(P<0.05)。FTS干预后,表达水平较未干预组增高(P<0.05),且与浓度无关。地塞米松干预后虽有升高趋势,但与未干预组相比无统计学意义(P>0.05)。
6)小鼠肺ERK1/2蛋白的表达均值在各组间有显著性差异(F=92.34,P=0.00)。哮喘组小鼠肺组织ERKl/2的蛋白表达较正常对照组增高(P<0.05),干预后,表达水平较未干预组减少(P<0.05),减少程度与FTS浓度无关。地塞米松组与FTS组各相比差异显著(P<0.05),提示FTS的作用强于地塞米松。
7)小鼠肺MMP-2、MMP-9mRNA的表达均值在各组间有显著性差异(MMP-2F=4.51, P=0.003; MMP-9F=4.68, P=0.002)。哮喘小鼠肺组织MMP-2、MMP-9表达较正常对照组增高(P<0.05),干预后,表达水平较未干预组减少(P<0.05),减少程度与FTS剂量无关。地塞米松组和FTS各组相比疗效无显著差异(P>0.05)。
结论：本文从免疫机制、神经内分泌机制等角度探讨了Ras通路与支气管哮喘发生发展的关系。通过抑制Ras信号通路可以减轻支气管哮喘小鼠的气道炎症及气道重塑,为哮喘的治疗开拓了新的思路。本研究中,FTS作为Ras抑制剂,与糖皮质激素一样,能明显改善哮喘模型小鼠气道炎症细胞浸润、减少细胞因子IL-5的分泌,下调肺组织pan-ras、ERK1/2蛋白及MMP-9、MMP-2mRNA表达水平,下调脾组织GATA-3mRNA、GATA-3蛋白表达水平,上调脾组织Foxp3mRNA表达水平。减轻哮喘小鼠气道炎症、气道重塑,促进TH1/TH2细胞平衡的恢复,增加Treg的数量及其抑制功能。其机制可能是通过抑制不同的Ras下游级联反应通路,从而调节复杂的细胞内信号传导过程,最终发挥治疗哮喘的作用。但哮喘的发病机制极为复杂,Ras信号转导途径亦是一个复杂的相互交联的网络,且与其他的信号系统相互联系。本研究亦发现FTS与地塞米松的治疗效应并不完全一致,可能二者通过影响不同的信号通路机制起效。总之,在治疗支气管哮喘方面,FTS的作用机制仍未完全清晰,尚需进一步更深入的研究。
Background:Asthma is a disease of chronic airway inflammation associated with many cells and cytokines. Up to now, it not very clear to the complex nosogenesis. It involve the immunology, neurology, heredity, endocrinology, environment and so on. The inflammation, hyperresponsiveness and remodling of airway are the basic propty of asthma. At present, researches about the relationship between Ras pathway and asthma has become a hot spot. As the main moderator of cell growth and differentiation, Ras lies the central position in signal transduction pathways of the extracellular stimulations. It has been showed that the signal transduction Mediated by Ras play an important role in the occurrence and development of bronchial asthma. Research on Ras signal transduction pathways contribute to seek the new drug targets.
The Ras signal transduction pathway consists mainly of the following ways:1. Mitogen-activated protein kinase (MAPK), The Ras-MAPK pathway is the most clear signal pathway at present. Extracellular signal regulated kinase(ERK) Is one of the family members of MAPK. Participate in the regulation of cell mitosis, Closely associated with the proliferation, differentiation and migration of cells.2. Phosphatidylinositol-3-kinase and protein kinase B signaling pathway.. Equally important as Ras-MAPK pathway in the regulation of cell metabolism, proliferation,differentiation and apoptosis. It plays an important helping role in the tumorigenesis, and mediate the differentiation, degradation, assemble and the expression of adhension molecules.3. GalGEF signal pathway. It adviod the cells from apoptosis and accelerate the growth of cell cycle progression.4. For example,Rac and Rho signal pathway, Ras guanine nucleotide exchange factor (RasGRP) signaling pathway, NF-kappB signal pathway, Transforming growth factor-β(TGF-β) signal pathway, and etc.
Ras signaling pathway have close relationship with the occurrence and development of many diseases (including cancer, bronchial asthma, etc). It has been improved that the method for tumor treatment based on the RAS signal pathway has important value in the treatment of tumors caused by Ras mutations. The current research of drug about the Ras signal pathway mainly in the following categories:1) Farnesyltransferase inhibitors, FTIs. Faniki Farnesyltransferase is one of the most important catalytic enzyme in the Ras protein processing. The Ras protein post-translational modificated processing consists of three steps:The farnesyl esterification of the C-terminal cysteine (Cys) residues of Ras protein. The methylation of the farnesyl esterificated Cys residues. And removing the amino acid residues from carboxyl terminus. The C-terminal farnesylation play a key modification steps of Ras protein in the location on the cell membrane and then developing the effectiveness. Therefore, inhibition of Ras protein post-translational farnesylation of carboxyl terminal is an important treatment for inhibiting Ras signal pathway.2) Antisense oligonucleotide against Ras. According to the Ras protein and mRNA sequence, we design ed the antisense RNA,. By the way of complementary base pairing, inhibited and damaged the aimed gene expression, then down regulated the expression of Ras.3) Inhibition of Ras downstream targets. By antisense oligodeoxynucleotide, the small molecule compounds can inhibit Ras expression and its downstream target of RafmRNA, down-regulate the activity of ERK.4) Inhibition of Ras upstream targets. Tthe Ras can be activated by the abnormal activation of the upstream signaling pathway of Ras protein.The small molecule tyrosine kinase inhibitors and the antibodies to the receptors of extracellular region, can inhibite Ras activation by blocking the activation of growth factor receptor's tyrosine kinase.5) Recovery of Ras protein GTP activity. Ras mutations lead to GTP inactivation, GTP analogues have been developed can be used instead of GAP, can hydrolyze the Ras protein more effectivly.6) et al. Include the gene therapy and immune therapy targeting the Ras pathway, as well as the therapy targeting Ras peripheral components or other signaling pathway which was significantly related to the Ras signal pathway, and etc. In short,the therapy strategy based on the Ras signaling pathway has showed a broad prospects in the treatment of human cancer.
But the intervention effect and mechanism of Ras inhibitors on the pathogenesis of asthma is seldom reported. Farnesyl thiosalicylic acid (trans-farnesylthiosalicylic, acid, FTS) is a kind of novel Ras inhibitors., FTS has obvious selectivity for the activation of GTP-bound Ras,. It has no toxic or adverse side effects In the animal models.
Objectives:This study established a murine model of asthma, focuses on the study of FTS on airway inflammation, airway remodeling and the differentiation of Th cell. To further explore the relationship between Ras pathway and bronchial asthma.and the intervention mechanism on bronchial asthma of FTS. To provide a new idea for the treatment of asthma
Methods:60BALB/c mice (6-8week), are randomly divided into six groups, each group has10mice. Control group(n=10), asthma group(n=10), dexamethasone treatment group(n=10),5FTS group,(n=10,5mg/kg),10FTS group,(n=10,10mg/kg),15FTS group,(n=10,15mg/kg).
1. Making a mouse asthma model:Sensitated the mice with ova/AL(OH)3intraperitoneal injection (40μgOVA each mouse) at1st day and Fourteenth day. Motivated the mice with3%OVA saline aerosol by Ultrasonic nebulizer1times a day from twenty-first day to twenty-seventh day,for30min each time.
2. medicine treatment:Dexamethasone group, intraperitoneal injection of2mg/kg dexamethasone before motivation each time. FTS group:intraperitoneal injection of FTS before motivation each time. The doses are5mg/kg,10mg/kg and15mg/kg for the three groups.
3. Collection of specimens:Bronchoalveolar lavage fluid, serum, lung tissue, spleen tissue.
4. Testing index:
a) Lung tissue in mice with HE staining, observe the histological changes.
b) Detection of mouse bronchoalveolar lavage (BALF) cell counts, eosinophil count s
c) Enzyme linked immunosorbent assay for the detection of IL-5concentration in serum of mice
d) Western Blot for the detection of the expression level of pan-ras, ERK protein in lung tissues, and GATA-3protein in spleen tissues of mice.
e) Real-time fluorescence quantitative polymerase chain reaction (Real-time Quantitative PCR, QPCR):to detect the expression of MMP-2, MMP-9mRNA in lung tissue of mice, and the expression of Foxp3, GATA-3mRNA in spleen tissues of mice
Results:
1. The Histomorphology of lung tissures:The lung tissures of the mice in the normal control group have smooth bronchial wall, intact alveolar epithelial cells, surrounded by no infiltration of inflammatory cells, and no alveolar mucosa swelling. And in the asthma model group, the lung tissures have obvious infiltration of inflammatory cells around the alveolus, edema of alveolar mucosa, hyperplasia, shedding, and mucus secretion of bronchial epithelial. Furthermore, there are contraction of bronchial, inflammatory cells infiltration of peribronchial, significantly congestion of the small vascular Airway around the airway, following a large number of inflammatory cells infiltration. In lung tissures of DEX and FTS intervention group, we can see the that the bronchiolar epithelial structures are basically normal, a few infiltration of inflammatory cells in the submucosal, alveolar structure basically normal. So,there is reduced inflammation to varying degrees.
2. Each index in the six groups of mice were a number of significant difference (BALF cell total F=52.27, eosinophil numbers F=67.61, serum IL-5concentration of F=23.57, P=0).The number of the BALF cell counts, eosinophil counts and serum IL-5concentrations of asthma mice were higher than the normal control group (P<0.05), They were lower in dexamethasone and FTS intervention group than in those of non-intervention group (P<0.05). and with the increase of FTS concentration, the all indexes fell more obviously. But the difference between the10mgFTS group and the15mgFTS group has no significant. Comparison of the dexamethasone group, the10mgFTS and15mgFTS group have the more significant effect.
3. There were significant differences among the groups in pan-ras protein expression in mouse lung (F=153.55, P=0.00)。 Expression of pan-Ras protein in lung tissues of asthmatic group were higher than control group (P<0.05). They were lower in dexamethasone and FTS intervention group than in those of non-intervention group (P<0.05). Compared with dexamethasone treatment group, the effction of down-regulation of FTS is more apparent, and there is no concentration dependence.
4. Among the groups, there were significant differences of the expression of GATA-3protein and GATA-3mRNA in mice spleen,(GATA-3protein F=106.44, P=0.00; GATA-3mRNA F=6.02, P=0.00). The expression of GATA-3mRNA and GATA-3protein in splenic tissues of asthmatic group were increased compared with normal control group (P<0.05), They were lower in dexamethasone and FTS intervention group than in those of non-intervention group (P<0.05). The effection were independent of the concentration of FTS.The effects of FTS and dexamethasone was the same on the expression of GATA-3mRNA. But on the expression of GATA-3protein, the effect of FTS is more stronger than that of dexamethasone group.
5. There were significant differences in mean of Foxp3mRNA expression among each group of mice spleen.(F=2.85, P=0.029). Compared with the normal control group, the expression of Foxp3mRNA were reduced in spleen tissue in asthmatic mice (P<0.05).After intervention of FTS, the expression levels were higher than in the no-intervention group (P<0.05), and is independent of concentration. Although the expression in dexamethasone group has increased, there were no statistical significance compared with non-intervention group (P>0.05).
6. There were significant differences among the groups in ERK1/2protein expression in mouse lung (F=92.34, P=0.00). Expression of ERK1/2protein in lung tissue of asthmatic mice are higher than in normal control group (P<0.05), After intervention of FTS and dexamethasone, the expression levels were lower than in the no-intervention group (P<0.05), and is independent of FTS concentration. Compared with dexamethasone,there was more significant reduce in FTS group (P<0.05).
7. There were significant differences in mean of MMP-2and MMP-9mRNA expression among each group of mice lung.(MMP-2F=4.51, P=0.003; MMP-9F=4.68, P=0.002). The expression of MMP-2and MMP-9mRNA in lung tissue of asthmatic group was increased compared with normal group (P<0.05). Compared with no intervention group, the expression level of the two indexes were decreased in FTS and dexamethasone groups (P<0.05). There was no FTS dose dependent. There was no ignificant difference in the effect of dexamethasone group and FTS groups (P>0.05).
Conclusions:This paper discusses the relationship between Ras pathway and bronchial asthma from the point of view of mechanism of immune and neuroendocrine. The alleviation of inflammation and airway remodeling in mice bronchial asthma by inhibiting Ras signal pathway, opened up a new way for the treatment of asthma. In this study, As Ras inhibitors, FTS as well as glucocorticoid, can significantly improve the inflammatory cells infiltration of airway model mice, reduce the secretion of cytokine IL-5, down-regulate the levels of ERK1/2protein, pan-ras protein, MMP-9and MMP-2mRNA in lung tissues, levels, lower the levels of GATA-3mRNA and GATA-3protein in spleen tissues, increased the levels of Foxp3mRNA in spleen tissues. Alleviating the inflammation, airway remodeling in asthmatic mice airway, promoting recovery of TH1/TH2cells balance, increasing the quantity of Treg and its inhibition function. The mechanism is probably through the inhibition of different Ras downstream cascade pathway, thereby regulating the complex process of intracellular signal transduction, ultimately play in asthma. But the pathogenesis of asthma is very complex, the Ras signal transduction pathway is also a complex interconnected network, and interaction with other signal system. The study found that the treatment effect of FTS and dexamethasone is not entirely consistent, may be through the different signal transduction pathways. In short, the action mechanism of FTS in the treatment of bronchial asthma is not perfectly clear. The further research must be required.
引文
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3.Fish JE. Peters SP. Airway remodeling and persistent airway obstruction in asthma [Review]. J Allegy Clin Immunol.1999:104(3 Pt):509-516.
4.Shibata Y, Kamata T, Kimura M, e tal. Ras activation in T cells determines the development of antigen-induced airway hyperresponsiveness and eosinophilic inflammation.2002 J Immunol. 169(4):2134-40.
5.Elomaa AP, Niskanen L, et al. Elevated levels of serum IL-5 are associated with an increased likehood of major depressive disordre.BMC Psychiatry.2012V12N:2.
6.Parsa A T, Holland E C. Cooperative translational control of gene exp ression by Ras and Akt in cancer. Trend Mol M ed,2004,10 (12):607-613.
7. Masakatsu Yamashita, Motoko Kimura, Masato Kubo, e tal. T cell antigen receptor-mediated activation of the Ras/mitogen-activated protein kinase pathway controls interleukin 4 receptor function and type-2 helper T cell differentiation. Proc. Natl. Acad. Sci. USA.1999; 96: 1024-1029.
8. Citro S, Ravasi S, Rovati GE, et al. Thromboxane prostanoid receptor signals through Gi protein to rapidly activate extracellular signal-regulated kinasein human airways[J]. Am J Respir CellMol Biol,2005,32(4):326-333
9.BAI JING, LIU XS, XU YJ, et al. Extracellular signal-regulated kinase activation in airway smooth muscle cell proliferation in chronic asthmatic rats[J]. Acta Physiologica Sinica,2007, 59(3):311-318.
10.白晶,刘先胜等.ERK对慢性哮喘大鼠气道平滑肌细胞凋亡的影响.细胞与分子免疫学杂志.2010,26(8)；738-741
11.Daniel M,Tessier and Laura EP.Activation of MAP kinases by hexavalent chromium, manganese and nickel in human lung epithelial cells[J]. Toxcol Lett,2006,167(2):114-121
12. Walker K, Olson M F. Targeting Ras and Rho GTPases as opportunities for cancer therapeutics. Curr Opin Genet Dev,2005,15(1):62-68.
13.Wong CK, Ip WK, et al. Biochemical assessment of intracellular signal transduction pathways in eosinophils:implications for pharmacotherapy. Crit Rev Clin Lab Sci.2004V4N 1:79-113
14.Springett GM, Kawasaki H, et al. Non-kinase second-messenger signaling:new pathways with new promise. Bioessays.2004V26N7:730-8
15.Tang Y, Katuri V, et al. Disruption of transforming growth factor-beta signaling in Elf beta-spetrin-deficient mice[J]. Science.2003,299(5606):574-577
16. Park J I,Lee MG, Cho K, et a 1. Transforming growth factor-betal activa tes inte rleukin-6 expression in prosta tecancer ce lls through the syne rgistic collaboration of the Smad-p38-N-2kappB, JNK, and Ra s singaling pathways[J]. Oncogene,2003,22 (28) 431424332.
17. Uozumi, N., K. Kume, T. Nagase, N. Nakatani, S. Ishii, F. Tashiro, Y. Komagata,K. Maki, K. Ikuta, Y. Ouchi, et al.1997. Role of cytosolic phospholipase A2 in allergic response and parturition. Nature 390:618.
18.Genot, E. and Cantrell, D. A., Ras regulation and function in lymphocytes.Curr. Opin. Immunol.2000.12:289-294.
19. Brode, S., Raine, T., Zaccone, P. and Cooke, A., Cyclophosphamide induced type-1 diabetes in the NOD mouse is associated with a reduction of CD4+CD25+Foxp3+ regulatory T cells. J. Immunol.2006.177:6603-6612.
20. Genot, E., Cleverley, S., Henning, S. and Cantrell, D., Multiple p21ras effector pathways regulate nuclear factor of activated T cells. EMBO J.1996.15:3923-3933
21. Adi Mor, Gad Keren, Yoel Kloog, e tal. N-Ras or K-Ras inhibition increases the number and enhances the function of Foxp3 regulatory T cells. Eur. J. Immunol.2008;38:1493-1502
22.Kay A B. Allergy and allergic diseases. First of two parts. N Engl J Med,2001; 344 (1) 30-37.
23. Smyth L J,Eustace A,et al.Increased airway T regulatory cell in asthmatic subjects. Chest. 2010V138N4:905-12
24. Dechene L. Th1/Th2 immune response. J Allergy Clin Immunol,2002; 110 (3):539-540.
25.吉宁飞等,地塞米松对哮喘大鼠转录因子GATA-3表达的影响。南京医科大学学报,2006： 26(9)：791
26. Turner, H., and D. A. Cantrell.1997. Distinct Ras effector pathways are involved in FcεR1 regulation of the transcriptional activity of Elk-1 and NFAT in mast cells. J. Exp. Med.185:43.
27.Prieschl, E. E., G. G. Pendl, N. E. Harrer, and T. Baumruker.1995. p21ras links FcεRI to NF-AT family member in mast cells. The AP3-like factor in this cell type is an NF-AT family member. J. Immunol.155:4963.
28.Zhu, X., N. M. Munoz, K. P. Kim, H. Sano, W. Cho, and A. R. Leff.1999. Cytosolic phospholipase A2 activation is essential for p1 and β2 integrin-dependent adhesion of human eosinophils. J. Immunol.163:3423.
29. Myou, S., X. Zhu, E. Boetticher, S. Myo, A. Meliton, A. Lambertino, N. M. Munoz, and A. R. Leff.2002. Blockade of focal clustering and active conformation in β2-integrin-mediated adhesion of eosinophils to intercellular adhesion molecule-1 caused by transduction of HIV TAT-dominant negative Ras. J. Immunol.169:2670.
30. David J. Hall, Jin Cui, Mary Ellen Bates,e tal. Transduction of a dominant-negative H-Ras into human eosinophils attenuates extracellular signal-regulated kinase activation and interleukin-5-mediated cell viability. Blood.2001; 98:2014-2021.
31. Myou, S., H. Sano, M. Fujimura, X. Zhu, K. Kurashima, T. Kita, S. Nakao,. Nonomura, T. Shioya, K. P. Kim, et al.2001. Blockade of eosinophil migration and airway hyperresponsiveness by cPLA2-inhibition. Nat. Immunol.2:145.
32. S. Myou, X. Zhu, S. Myo, e tal.Blockade of Airway Inflammation and Hyperresponsiveness by HIV-TAT-Dominant Negative Ras. J. Immunol.2003 171:4379-4384.[]
33. Langier S; Sade K et al. Regulary T cells in allergic asthma. Isr Med Assoc J.2012V14N3:180-3 [IF:1.018]
34. Fontenot JD, Rudensky AY1 A well adap ted regulatory contrivance:regulatory T cell development and the forkhead family transcrip tion factor Foxp31 Nat Immunol,2005,6 (4): 331-337.
35.Smyth LJ, Eustace A, el al. Increased airway T regulatory cells in asthmatic subjects. Chest. 2010V138N4:905-12
36. Lin W,Truong N,Grossma WJ, et al. Allergic dysregulation and hyper-immunoglobulinemia E in Foxp3 mutant mice. J Allergy Clin Immunol,2005,116 (5):1106-1115.
37. AkdisM, Blaser K, Akdis CA. T regulatory cells in allergy:novel concepts in the pathogenesis, p revent, and treatment of allergic diseases. J Allergy Clin Immunol,2005,116 (5):961-968.
38.李茜,沈华浩。哮喘小鼠CD4+CD25+调节性T细胞及Foxp3表达的变化[J]。中华急诊医学杂志,2008,17(2)：177
39.黄东辉,甘考。喘可治注射液诱导哮喘小鼠Foxp3+调节性T细胞的免疫机制探讨。时珍国医国药。2012,23(6)：1487
40.Nassenstein C, Kutschker J,Tumes D,et al.Neuro-immune interaction in allergic asthma:role of neurotrophins[J].Biochem Soc Trans.2006,34(pt4):591-593
41.Feng JT, Hu CP. Dysfunction of releasing adrenline in asthma by nerve growth factor[J]. Med Hypotheses,2005,65(6):1043-1046
42.汤渝玲,胡成平,冯俊涛等。神经生长因子调控哮喘神经源性炎症Ras-MAPK信号转导通路。中南大学学报(医学版),2006；31：319-325.
43.宋立强,戚好文,郎兵,刘飞。Ras-MAPK信号转导系统在哮喘气道重塑中的作用。第四军医大学学报,2002；23：1158-1160。
44. Ammit AJ, Kane SA, Panettieri RA,et al. Activation of K-p21ras and N-p21 ras, but not H-p21 ras, is necessary for mitogen-induced human airway smooth-muscle proliferation. Am J Respir Cell Mol Biol,1999; 21:719-727.
45. Lin CC, Shyr MH, Chien CS, et al. Thrombin-stimulated cell proliferation mediated through activation of Ras/Raf/MEK/MAPKpathway in canine cultured tracheal smooth muscle. Cell Signal,2002; 14:265-275
46. Jacob George, Jessica Sack, Iris Barshack,e tal. Inhibition of Intimal Thickening in the Rat Carotid Artery Injury Model by a Nontoxic Ras Inhibitor. Arterioscler. Thromb. Vase. Biol.2004; 24:363-368.
47.Araujo BB, Dolhnikoff M, Silva LF. Extracellular matrix components and regulators in the airway smooth muscle in asthma[J]. Eur Respir J,2008,32(1):61-69
48.李锋,白云凯。基质金属蛋白酶-9与支气管哮喘[J].2004,24(5)：343-345
49. Doinita Serban, Jie Leng, David Cheresh. H-Ras Regulates Angiogenesis and Vascular Permeability by Activation of Distinct Downstream Effectors. Circ Res.2008;102:1350-1358.2,
50. Yamashita M, Shinnakasu R, Asou H, et al. Ras-ERK MAPK cascade regulates GATA3 stability and Th2 differentiation through ubiquitin-proteasome pathway. J Biol Chem,2005, 280(33):29409-29419.
51.刘晓湘,曹德寿。哮喘豚鼠下呼吸道MMP-2的表达及NGF的调节作用。解剖科学进展。2005,11(4)：307-310
52. Sepp-Lorenzino L, Ma Z, Rands E, et al. A peptidominmetic inhibitor of farnesyl:protein transferase blocks the anchorage-dependent and independent growth of human tumor cell line[J]. Cancer Res,1995,55(22):5302.
53. Crooke S T. Potential roles of antisense technology in cancer chemotherapy[J]. Oncogene, 2000,19(56):6651-6659
54. Brummelkamp T R, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference[J]. Cancer Cell,2002,2(3):243-247.
55. Gysin S, Lee S H, Dean N M, et al. Pharmacologic inhibition of RAF— MEK— ERK signaling elicits pancreatic cancer cell cycle arrest through induced expression of p27Kipl[J]. Cancer Res,2005,65(11):4870-4880.
2.Schaich M, Illmer T. Mdrl gene expression and mutations in Ras proto-oncogene in acute myeloid leukemia. Leuk Lymphoma,2002,43:1345-1354.
3.Fish JE. Peters SP. Airway remodeling and persistent airway obstruction in asthma [Review]. J Allegy Clin Immunol.1999:104(3 Pt):509-516.
4.Shibata Y, Kamata T, Kimura M, e tal. Ras activation in T cells determines the development of antigen-induced airway hyperresponsiveness and eosinophilic inflammation.2002 J Immunol. 169(4):2134-40.
5.Elomaa AP, Niskanen L, et al. Elevated levels of serum IL-5 are associated with an increased likehood of major depressive disordre.BMC Psychiatry.2012V12N:2.
6.Parsa A T, Holland E C. Cooperative translational control of gene exp ression by Ras and Akt in cancer. Trend Mol M ed,2004,10 (12):607-613.
7. Masakatsu Yamashita, Motoko Kimura, Masato Kubo, e tal. T cell antigen receptor-mediated activation of the Ras/mitogen-activated protein kinase pathway controls interleukin 4 receptor function and type-2 helper T cell differentiation. Proc. Natl. Acad. Sci. USA.1999; 96: 1024-1029.
8. Citro S, Ravasi S, Rovati GE, et al. Thromboxane prostanoid receptor signals through Gi protein to rapidly activate extracellular signal-regulated kinasein human airways[J]. Am J Respir CellMol Biol,2005,32(4):326-333
9.BAI JING, LIU XS, XU YJ, et al. Extracellular signal-regulated kinase activation in airway smooth muscle cell proliferation in chronic asthmatic rats[J]. Acta Physiologica Sinica,2007, 59(3):311-318.
10.白晶,刘先胜等.ERK对慢性哮喘大鼠气道平滑肌细胞凋亡的影响.细胞与分子免疫学杂志.2010,26(8)；738-741
11.Daniel M,Tessier and Laura EP.Activation of MAP kinases by hexavalent chromium, manganese and nickel in human lung epithelial cells[J]. Toxcol Lett,2006,167(2):114-121
12. Walker K, Olson M F. Targeting Ras and Rho GTPases as opportunities for cancer therapeutics. Curr Opin Genet Dev,2005,15(1):62-68.
13.Wong CK, Ip WK, et al. Biochemical assessment of intracellular signal transduction pathways in eosinophils:implications for pharmacotherapy. Crit Rev Clin Lab Sci.2004V4N 1:79-113
14.Springett GM, Kawasaki H, et al. Non-kinase second-messenger signaling:new pathways with new promise. Bioessays.2004V26N7:730-8
15.Tang Y, Katuri V, et al. Disruption of transforming growth factor-beta signaling in Elf beta-spetrin-deficient mice[J]. Science.2003,299(5606):574-577
16. Park J I,Lee MG, Cho K, et a 1. Transforming growth factor-betal activa tes inte rleukin-6 expression in prosta tecancer ce lls through the syne rgistic collaboration of the Smad-p38-N-2kappB, JNK, and Ra s singaling pathways[J]. Oncogene,2003,22 (28) 431424332.
17. Uozumi, N., K. Kume, T. Nagase, N. Nakatani, S. Ishii, F. Tashiro, Y. Komagata,K. Maki, K. Ikuta, Y. Ouchi, et al.1997. Role of cytosolic phospholipase A2 in allergic response and parturition. Nature 390:618.
18.Genot, E. and Cantrell, D. A., Ras regulation and function in lymphocytes.Curr. Opin. Immunol.2000.12:289-294.
19. Brode, S., Raine, T., Zaccone, P. and Cooke, A., Cyclophosphamide induced type-1 diabetes in the NOD mouse is associated with a reduction of CD4+CD25+Foxp3+ regulatory T cells. J. Immunol.2006.177:6603-6612.
20. Genot, E., Cleverley, S., Henning, S. and Cantrell, D., Multiple p21ras effector pathways regulate nuclear factor of activated T cells. EMBO J.1996.15:3923-3933
21. Adi Mor, Gad Keren, Yoel Kloog, e tal. N-Ras or K-Ras inhibition increases the number and enhances the function of Foxp3 regulatory T cells. Eur. J. Immunol.2008;38:1493-1502
22.Kay A B. Allergy and allergic diseases. First of two parts. N Engl J Med,2001; 344 (1) 30-37.
23. Smyth L J,Eustace A,et al.Increased airway T regulatory cell in asthmatic subjects. Chest. 2010V138N4:905-12
24. Dechene L. Th1/Th2 immune response. J Allergy Clin Immunol,2002; 110 (3):539-540.
25.吉宁飞等,地塞米松对哮喘大鼠转录因子GATA-3表达的影响。南京医科大学学报,2006： 26(9)：791
26. Turner, H., and D. A. Cantrell.1997. Distinct Ras effector pathways are involved in FcεR1 regulation of the transcriptional activity of Elk-1 and NFAT in mast cells. J. Exp. Med.185:43.
27.Prieschl, E. E., G. G. Pendl, N. E. Harrer, and T. Baumruker.1995. p21ras links FcεRI to NF-AT family member in mast cells. The AP3-like factor in this cell type is an NF-AT family member. J. Immunol.155:4963.
28.Zhu, X., N. M. Munoz, K. P. Kim, H. Sano, W. Cho, and A. R. Leff.1999. Cytosolic phospholipase A2 activation is essential for p1 and β2 integrin-dependent adhesion of human eosinophils. J. Immunol.163:3423.
29. Myou, S., X. Zhu, E. Boetticher, S. Myo, A. Meliton, A. Lambertino, N. M. Munoz, and A. R. Leff.2002. Blockade of focal clustering and active conformation in β2-integrin-mediated adhesion of eosinophils to intercellular adhesion molecule-1 caused by transduction of HIV TAT-dominant negative Ras. J. Immunol.169:2670.
30. David J. Hall, Jin Cui, Mary Ellen Bates,e tal. Transduction of a dominant-negative H-Ras into human eosinophils attenuates extracellular signal-regulated kinase activation and interleukin-5-mediated cell viability. Blood.2001; 98:2014-2021.
31. Myou, S., H. Sano, M. Fujimura, X. Zhu, K. Kurashima, T. Kita, S. Nakao,. Nonomura, T. Shioya, K. P. Kim, et al.2001. Blockade of eosinophil migration and airway hyperresponsiveness by cPLA2-inhibition. Nat. Immunol.2:145.
32. S. Myou, X. Zhu, S. Myo, e tal.Blockade of Airway Inflammation and Hyperresponsiveness by HIV-TAT-Dominant Negative Ras. J. Immunol.2003 171:4379-4384.[]
33. Langier S; Sade K et al. Regulary T cells in allergic asthma. Isr Med Assoc J.2012V14N3:180-3 [IF:1.018]
34. Fontenot JD, Rudensky AY1 A well adap ted regulatory contrivance:regulatory T cell development and the forkhead family transcrip tion factor Foxp31 Nat Immunol,2005,6 (4): 331-337.
35.Smyth LJ, Eustace A, el al. Increased airway T regulatory cells in asthmatic subjects. Chest. 2010V138N4:905-12
36. Lin W,Truong N,Grossma WJ, et al. Allergic dysregulation and hyper-immunoglobulinemia E in Foxp3 mutant mice. J Allergy Clin Immunol,2005,116 (5):1106-1115.
37. AkdisM, Blaser K, Akdis CA. T regulatory cells in allergy:novel concepts in the pathogenesis, p revent, and treatment of allergic diseases. J Allergy Clin Immunol,2005,116 (5):961-968.
38.李茜,沈华浩。哮喘小鼠CD4+CD25+调节性T细胞及Foxp3表达的变化[J]。中华急诊医学杂志,2008,17(2)：177
39.黄东辉,甘考。喘可治注射液诱导哮喘小鼠Foxp3+调节性T细胞的免疫机制探讨。时珍国医国药。2012,23(6)：1487
40.Nassenstein C, Kutschker J,Tumes D,et al.Neuro-immune interaction in allergic asthma:role of neurotrophins[J].Biochem Soc Trans.2006,34(pt4):591-593
41.Feng JT, Hu CP. Dysfunction of releasing adrenline in asthma by nerve growth factor[J]. Med Hypotheses,2005,65(6):1043-1046
42.汤渝玲,胡成平,冯俊涛等。神经生长因子调控哮喘神经源性炎症Ras-MAPK信号转导通路。中南大学学报(医学版),2006；31：319-325.
43.宋立强,戚好文,郎兵,刘飞。Ras-MAPK信号转导系统在哮喘气道重塑中的作用。第四军医大学学报,2002；23：1158-1160。
44. Ammit AJ, Kane SA, Panettieri RA,et al. Activation of K-p21ras and N-p21 ras, but not H-p21 ras, is necessary for mitogen-induced human airway smooth-muscle proliferation. Am J Respir Cell Mol Biol,1999; 21:719-727.
45. Lin CC, Shyr MH, Chien CS, et al. Thrombin-stimulated cell proliferation mediated through activation of Ras/Raf/MEK/MAPKpathway in canine cultured tracheal smooth muscle. Cell Signal,2002; 14:265-275
46. Jacob George, Jessica Sack, Iris Barshack,e tal. Inhibition of Intimal Thickening in the Rat Carotid Artery Injury Model by a Nontoxic Ras Inhibitor. Arterioscler. Thromb. Vase. Biol.2004; 24:363-368.
47.Araujo BB, Dolhnikoff M, Silva LF. Extracellular matrix components and regulators in the airway smooth muscle in asthma[J]. Eur Respir J,2008,32(1):61-69
48.李锋,白云凯。基质金属蛋白酶-9与支气管哮喘[J].2004,24(5)：343-345
49. Doinita Serban, Jie Leng, David Cheresh. H-Ras Regulates Angiogenesis and Vascular Permeability by Activation of Distinct Downstream Effectors. Circ Res.2008;102:1350-1358.2,
50. Yamashita M, Shinnakasu R, Asou H, et al. Ras-ERK MAPK cascade regulates GATA3 stability and Th2 differentiation through ubiquitin-proteasome pathway. J Biol Chem,2005, 280(33):29409-29419.
51.刘晓湘,曹德寿。哮喘豚鼠下呼吸道MMP-2的表达及NGF的调节作用。解剖科学进展。2005,11(4)：307-310
52. Sepp-Lorenzino L, Ma Z, Rands E, et al. A peptidominmetic inhibitor of farnesyl:protein transferase blocks the anchorage-dependent and independent growth of human tumor cell line[J]. Cancer Res,1995,55(22):5302.
53. Crooke S T. Potential roles of antisense technology in cancer chemotherapy[J]. Oncogene, 2000,19(56):6651-6659
54. Brummelkamp T R, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference[J]. Cancer Cell,2002,2(3):243-247.
55. Gysin S, Lee S H, Dean N M, et al. Pharmacologic inhibition of RAF— MEK— ERK signaling elicits pancreatic cancer cell cycle arrest through induced expression of p27Kipl[J]. Cancer Res,2005,65(11):4870-4880.
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