| Background: Bronchial asthma is characterized by infiltration ofeosinophil, mast cell and T lymphocyte associated with chronic airwayinflammation. Eosinophil can act as one of prominent effector cells inthe initiation and perpetuation of airway inflammation by producingvarious phalogic mediator. Recently, growing evidence show that chemotaxisof eosinophil play an important role in initiation, development andaggravation of asthma. Then CCR3 the receptor express on surface ofeosinophil and its main lignans such as eotaxin, RANTES and MCP-3,4 astherapeutic targets draw more and more attention.It is now widely accepted inhaled corticosteroids remain the mostimportant anti-inflammatory treatment for asthma. However, they arerather non-specific in their actions and their use raises concerns overside effects and compliance issues, particularly in children andadolescents. Moreover, a significant sub-group of asthmatic patientsresponds poorly or not at all to high-dose inhaled or systemic steroidtreatment. Therefore much effort is being to develop novel more specificand safer therapy for asthma. According to previous studies from ourlaboratory, we observed Epimedium can reduce the dose and abatement theside-effect of steroids. Otherwise it can decrease the production of TNF-αwhich can affect the production of asthma-relevant chemokine, anddecrease the activity of nuclear transcription factor kappa-B(NF-κB)which regulate the transcription and synthesis of many asthma-relevantchemokines and cytokines. But whether it can affect the chemoataxis migration of eosinophil by regulating its recptor CCR3 and its mainligands, then abatement the asthmatic symptom is still unkown.Objectives: The effects of Epimedium on regulating asthmatic ratseosinophils' chemotaxis:directly affect the expression of CCR3 and it' schief ligands, indirecty affect eosinophil' s chemotaxis migration byregulating the concentration of some cytokines. And whether this regulation exists dose-effect relationship.Materials and methods: Animals'Brown Norway rats, male, 3～6months old, illumination time wasfixed, water and food were taken unconventionally.Drug preparation: Epimedium was obtained from Korean epimedium herbraised in Liaoling province of China. Batch NO-060517.Methods: 72 BN rats were randomly divided into 6 groups. Group A isnormal control group. Group B is allergic asthma model group, which wassensitised by ovalbumin(OVA) at first day and challegend by OVA atfifteenth day and lasted 14 days. Group C is treated with Dexamethasone.Group D,E,F were treated with low, medium, high dose of Epimedium. GroupC,D,E,F were sensitized and challegened by OVA as same as Group B. Thedifference between C and B is at fifteenth day the group C is peritonealinjected with Dexamethasone 0.5mg/kg once a day and for 14 days. The groupD,E,F were intragastric administrated with respective dose of Epimediumat fifteenth day once a day and for 14 days. All the rats were anesthetized4 hours later after the last challegen by OVA, then samples were taken. Theeosinophils were isolated from bronchoalveolar lavage fluid(BALF) ,thentheir CCR3mRNA were tested with real-time PCR. The lung tissue'schemokine such as eotaxin(CCL11),RANTES(CCL5),MCP-3(CCLT)mRNA weremeasured also with real-time PCR. Meanwhile we used western blot to testthe lung tissue's expression of eotaxin and RANTES protein. Besides theconcentration of TNF-α,IL-6, IL-4, IL-5 and IFN-γin serum and BALF weremeasured by Enzyme linked immunosorbent assay(ELISA) in all groups.Results:1. The expression of CCR3mNNA in normal, allergic asthma model, differenttreatment groups were 0.0081±0.0047,0.0343±0.0107,0.0126±0.0055,0.0264±0.0163, 0.0205±0.0110, 0.0203±0.0125. When compared withthat in normal group, the expression CCR3mRNA in asthma model groupupregulated significantly(P＜0.01). When compared with asthma modelgroup, all treatment group except that low dose group downregulated atsignificant level(P＜0.05).AS compared with Dexamethasone group, the expr-ession of CCR3mRNA of medium, high dose group increased but have nostatistical significance (P＞0.05).2. The expression of eotaxin, PANTES, MCP-3 mRNA in lung tissue.The expression of eotaxin mRNA in normal, allergic asthma model, different treatment groups were 0.0099±0.0091,0.2477±0.0063,0.0175±0.0068,0.2363±0.0026,0.1950±0.0075,0.0295±0.0024. When compared with that in normalgroup, the expression of eotaxin mRNA in asthma model group raisedsignificantly(P＜0.01).But as compared with asthma model group, only theDexamethasone group and high dose group decreased significantly(P＜0.01).Contrast to Dexamethasone group and high dose group, the low and mediumdose group both augmented P＜0.01).There are no obvious diference betweenDexamethasone and high dose group(P＞0.05).The expression of RANTES mRNA in normal, allergic asthma model, differenttreatment groups were 0.0192±0.0133, 0.7707±0.0225, 0.0210±0.0079,0.0591±0.0278, 0.0638±0.0345, 0.0259±0.0216. When compared with thatin normal group, the expression of RANTES mRNA in asthma model groupincreased remarkably(P＜0.01).As compared withe asthma model group ,alltreatment group decreased markedly(P＜0.01).There are no significantdiference between respective treatment group. (P＞0.05).The expression of MCP-3 mRNA in normal, allergic asthma model, differenttreatment groups were 0.0113±0.0051,0.1307±0.0240,0.0339±0.0200,0.0789±0.0337,0.0533±0.0325,0.0565±0.0302. When compared with that in normalgroup, the expression of MCP-3 mRNA in asthma model group increased mar-Kedly(P＜0. 05).Contrast to asthma model group, the Dexamethasone group,medium and high dose group's expression of MCP-3 mRNA downregulated si-gnificantly(P＜0.05). There are no diference between treatment groups(P＞0.05).3. The expression of eotaxin and RANTES protein in lung tissueThe expression of eotaxin protein in normal, allergic asthma model,different treatment groups were 0.1155±0.1261,0.6896±0.4581,0.2371±0.1659,0.6837±0.5089,0.5732±0.3220, 0.3374±0.2684. When compared tothat in normal group, the asthma model and low dose groups increasedremarkably(P＜0.01).As compared with that in asthma model group ,theexpression of eotaxin protein in the Dexamethasone group decreasedsignificantly(P＜0.05).Although that in low, medium and high dose groupdroped in comparison with asthma model group, there are no significantdiference(P＞0.05).The expression of RANTES mRNA in normal, allergic asthma model, different treatment groups were 0.3945±0.3470,0.7161±0.5167,0.5709±0.3509,0.7155±0.4240,0.5949±0.2579,0.5959±0.2311. we don't see significant diference betwe-en all 6 groups(P＞0.05).4.Changes of cytokinesThere were significant diference in serum TNF-α,IL-6,IL-4,IL-5 and IFN-γamong 6groups but no such remarkable diference in BALF. Meanwhile all these cytokines'concentration in BALF were higher than in serum.In comparison with normal group,TNF-α,IL-6,IL-4,IL-5 in asthma model group increased obviously both in serum andBALF(P＜0.05). But IFN-γchanged differently from other cytokines, itdecreased markably when compared with normal group. As compared withasthma model group, all treatment groups' concentrtion of the serumTNF-αand IL-6dropped significantly (P＜0.05). But there are no such great diference seenin that of IL-4 and IL-5. Actually we only see the Dexamethasone group ,the mediumand high dose group's concentration of IL-4 and IL-5 decreased significantly in comp-arison with the asthma model group. At the same time we still observe the opposite c-hange in IFN-γ. In this study we observed definite dose-effect relationship among low,medium and high dose groups. The medium and high dose groups' therapeutic effectdid better than the low dose group.Conclusions:From our study we do observe the Epimedium can affect the chemotaxisof the eosinophil, ro show concretely it can downregulate the level ofCCR3mRNA, and make the expression of eotaxin, RANTES and MCP-3 mRNA inlung tissue decrease, probably further influence the expression of eotaxinprotein in lung tissue. Besides it also can reduce the production ofcytokines, which can promote the expression of CCR3 and have thesynergistic effect with the chemokine above. As for in this study we findno diference between the Dexamethasone group and the medium , high dosegroup, we consider the reason is the dose of Dexamethasone we used isminidose. So it's not difficult to explain there are no inequalitybetween the Dexamethasone and Epimedium group. Markable diference exsistsamong the low, medium and high dose group. We observe the medium and highdose groups' therapeutic effect are better than the low group, however itneed a further study in vitro to prove it. |
PDF Full Text Request