Effect Of Farnesylthiosalicylic Acid (FTS) On The Airway Inflammation, Airway Remodeling And TH Differentiation In Asthmatic Mouse Model

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 asthmaMethods: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 sc) Enzyme linked immunosorbent assay for the detection of IL-5concentration in serum of miced) 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 miceResults: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.