泡桐花提取物的雌激素活性筛选及其对OVA致过敏性哮喘小鼠的保护作用

Screening of Estrogenic Activity of Paulownia fortunei Flower Extract and Its Protective Effect on OVA-Induced Allergic Asthma in Mice

目的:探讨泡桐花提取物雌激素活性及其对卵清白蛋白(OVA)致过敏性哮喘小鼠的保护作用。方法:体外采用MCF-7细胞模型筛选出泡桐花提取物具有雌激素样活性,采用雌激素受体拮抗剂(ICI182,780)、雌激素受体α拮抗剂(MPP)、雌激素受体β拮抗剂(THC)与雌激素膜受体GPR30拮抗剂(G15)干预雌激素活性有效剂量下的MCF-7细胞增殖,探讨其雌激素受体作用途径;采用小鼠子宫增重试验,计算性未成熟小鼠的子宫指数,以ELISA法检测血清雌二醇(E2)、促卵泡生成素(FSH)与黄体生成素(LH)含量;采用Westren blot法测定小鼠子宫中雌激素受体α(ERα)、雌激素受体β(ERβ)和雌激素膜受体(GPR30)表达水平,进一步体内验证泡桐花提取物雌激素活性;OVA致过敏性哮喘小鼠模型,分为正常对照组、模型对照组、地塞米松0.5 mg/kg组、泡桐花提取物155、310、620 mg/kg组,每组8只,同时设置雌激素受体拮抗剂ICI182,780组,雾化激发前30 min共同作用小鼠,连续7 d,末次给药24 h后,小鼠脱颈处死,计算免疫器官脾脏和胸腺指数,ELISA法检测小鼠血清以及肺泡灌洗液(BALF)中的细胞因子含量。结果:泡桐花提取物在1、10μg/m L时能够显著促进MCF-7细胞增殖(P<0.05或P<0.01),且ICI182,780、MPP能够阻断其促增殖作用;泡桐花提取物在310 mg/kg时,能明显增加小鼠子宫系数(P<0.05),明显降低FSH、LH活力,明显增加小鼠子宫中ERα蛋白的表达(P<0.05);在哮喘模型中,与模型对照组相比,能够降低哮喘小鼠BALF中Eotaxin及IL-4的含量(P<0.05或P<0.01),升高IL-10的含量(P<0.01),可以改善脾脏和胸腺代偿性增大(P<0.05);当泡桐花提取物与ICI182,780共同作用于哮喘小鼠时,发现ICI182,780能够阻断泡桐花提取物对BALF中Eotaxin和IL-10的影响,以及阻断其改善免疫器官代偿性增大的效果。结论:泡桐花提取物具有植物雌激素活性,其作用途径为通过ERα介导,可以通过激活雌激素受体途径显著改善OVA诱导的小鼠过敏性哮喘。

Objective:To investigate the estrogenic activity of the extract of Paulownia fortunei flowers and its protective effect on ovalbumin(OVA)-induced allergic asthma in mice.Methods:The P.fortunei flower extract with estrogenic activity was screened using the MCF-7cell model in vitro.The proliferative effect of the extract was assessed in MCF-7cells at effective doses for estrogenic activity with the intervention of estrogen receptor antagonist(ICI182,780),estrogen receptorαantagonist(MPP),estrogen receptorβantagonist(THC),and GPR30 antagonist(G15).The estrogen receptor pathways were investigated.A mouse uterotrophic assay was performed to calculate the uterine index of prepubertal mice.Serum levels of estradiol(E,),follicle-stimulating hormone(FSH),and luteinizing hormone(LH)were measured using enzyme-linked immunosorbent assay(ELISA).Western blot analysis was used to determine the expression levels of estrogen receptorα(ERα),estrogen receptorβ(ERβ),and CPR30 in the mouse uterus,further validating the estrogenic activity of the P.fortunei flower extract.An OVA-induced allergic asthma model was established in mice,and the model mice were divided into a normal control group,a model control group,a dexamethasone group,and P.fortunei flower extract groups at different doses(155,310,and 620 mg/kg),with eight mice in each group.An estrogen receptor antagonist(ICI182,780)group was also established.All mice were treated for 7 consecutive days 30 min before aerosol stimulation.After the last administration,the mice were sacrificed 24 hours later by cervical dislocation.The spleen and thymus indices were calculated,and the levels of cytokines in the serum and bronchoalveolar lavage fluid(BALF)were detected using ELISA.Results:P.fortunei flower extract significantly promoted the proliferation of MCF-7cells at 1 and 10μg/mL(P<0.05 or P<0.01),and this proliferative effect was blocked by ICI182,780 and MPP.P.fortunei flower extract at 310 mg/kg significantly increased the uterine index in mice(P<0.05)and significantly increased the expression of ERαprotein in the mouse uterus(P<0.05).In the asthma model,compared with the model control group,the P.fortunei flower extract reduced the levels of Eotaxin and IL-4 in the BALF of asthmatic mice(P<0.05 or P<0.01),increased the level of IL-10(P<0.01),and improved compensatory enlargement of the spleen and thymus(P<0.05).When P.fortunei flower extract and ICI182,780 were co-administered to asthmatic mice,ICI182,780 blocked the effects of the P.fortunei flower extract on Eotaxin and IL-10 in the BALF and inhibited the improvement of compensatory enlargement of immune organs.Conclusion:P.fortunei flower extract exhibits plant estrogenic activity,which is mediated through ERα,and it significantly improves OVA-induced allergic asthma in miceby activating estrogen receptor pathways.