【作者】 吴迪；

【导师】 杨正涛；

【作者基本信息】 吉林大学 ， 临床兽医学， 2021， 硕士

【摘要】 过敏性哮喘是一种常见的慢性呼吸系统疾病,其特征在于出现各种不同的气喘、气短、胸闷或咳嗽症状,严重者甚至可以导致窒息。根据流行病调查学显示,在不同国家中,哮喘约占人口中的1-18%,约有3.34亿人,严重影响全球人健康,除此之外,哮喘在动物中也较为常见。尽管目前的一些药物如喘定片虽然治疗过敏性哮喘有效,但是会带来诸多副作用如头晕,心律失常以及引起胃肠道反应。目前,寻求安全、有效的治疗药物依然是防治过敏性哮喘重要途径,中药作为一种安全、无毒、副作用低的治疗药物进入了人们的视野,其中具有多种药理学功能的黄酮类化合物山姜素是重要代表之一。多种文献证明山姜素具有重要的生物学特性包括抗炎、抗氧化及抗肿瘤作用,同时在临床上也被证明具体降血压、降血脂、降血糖、止吐、镇痛等功能。但山姜素能否治疗过敏性哮喘尚未见报道。本文旨在初步为验证山姜素对过敏性哮喘保护效果,并且探究其作用机制,为过敏性哮喘提供有效药物治疗的研究基础。本实验利用OVA(卵清蛋白)诱导小鼠建立过敏性哮喘模型,在第1和第14天采用OVA滴鼻激发,在第21、22、23天利用OVA腹腔注射并采取不同浓度的山姜素(25、50、100 mg/kg)对该进行预处理。第24天处死小鼠后,首先取小鼠肺脏,并对其病理组织学HE和PAS染色进行观察;其次检测肺泡灌洗液(BALF)中的中性粒细胞、巨噬细胞和嗜酸性粒细胞的数量;接下来利用酶联免疫吸附(ELISA)测定测定肺泡灌洗液中IL-5和IL-13以血清中的Ig E和IL-4等;最后蛋白免疫印记(Western Blotting)检测肺组织的NF-κB/PI3K/AKT和HO-1信号通路。实验结果显示,当山姜素处理浓度在25、50和100 mg/kg水平时,与OVA模型组相比,小鼠肺脏的HE和PAS染色切片显示小鼠肺部组织损伤程度减轻,如减少炎性细胞浸润和粘液分泌;血清中Ig E和IL-4,BALF中嗜酸性粒细胞、中性粒细胞、淋巴细胞细胞数目减少,IL-5和IL-13的含量明显降低,NF-κB,PI3K/AKT等蛋白磷酸化水平随着山姜素处理浓度的升高而降低。为了更进一步验证山姜素在体外的作用机制,我们采用LPS刺激小鼠巨噬细胞RAW 264.7构建体外模型用于接下来的研究。首先采取细胞毒性实验(CCK-8)筛选出山姜素浓度为15、30、60μg/m L,ELISA法检测细胞上清液炎症细胞因子TNF-α、IL-1β、IL-6,利用Western blot检测NF-κB和PAKT/PI3K信号通路中蛋白的磷酸化水平探讨山姜素作用机制。结果显示,与生理盐水处理的空白对照组相比,山姜素预处理细胞后,TNF-α、IL-1β、IL-6的水平降低,并且山姜素可以有效抑制RAW264.7中NF-κB/PI3K/AKT信号通路来发挥抗炎作用。本论文利用OVA建立小鼠过敏性哮喘体内模型和LPS刺激RAW264.7细胞体外模型,运用病理组织切片、ELISA和Western Blot等检测方法探究山姜素对在体内和体外抗炎抗过敏效果以及作用机制,证实了山姜素25、50和100 mg/kg水平上对过敏性哮喘具有保护作用,为山姜素在治疗过敏性哮喘疾病以及后续相关原料药物研发提供理论及数据支持。更多还原

【Abstract】 Allergic asthma is a common chronic respiratory disease characterized by various symptoms of wheezing,shortness of breath,chest tightness or coughing.In severe cases,it can even lead to suffocation.According to epidemiological surveys,asthma accounts for about 1-18% of the population,with about 334 million people,which seriously affects the health of people around the world.In addition,asthma is also more common in animals.Although some current drugs are effective in treating allergic asthma,they also cause many side effects such as dizziness,arrhythmia and gastrointestinal reactions.At present,seeking safe and effective therapeutic drugs is still an important way to prevent allergic asthma.Traditional Chinese medicine has entered people’s field of vision as a safe,non-toxic,and low-side-effect therapeutic drug.Among them,flavonoids with multiple pharmacological functions are included.Ginger Su is one of the important representatives.A variety of documents have proved that alpinein has important biological properties including anti-inflammatory,anti-oxidant and anti-tumor effects.It has also been clinically proven to have specific functions such as lowering blood pressure,lowering blood lipids,lowering blood sugar,antiemetic,and analgesia.However,the effect of alpinein on allergic asthma has not been reported yet.This article aims to initially verify the protective effect of alpinein on allergic asthma,explore its mechanism of action,and provide a research basis for effective drug treatment for allergic asthma.In this experiment,OVA(ovalbumin)was used to establish an allergic asthma model.OVA was used for nasal stimulation on the 1st and 14 th days,and OVA was injected intraperitoneally on the 21 st,22nd,and 23 rd days and different concentrations of mountain ginger were taken.The allergic asthma model was pretreated with alpinein(25,50,100 mg/kg).After the mice were sacrificed on the24 th day,the lungs were used for histopathological HE and PAS staining;secondly,the neutrophils,macrophages and eosinophils in the alveolar lavage fluid(BALF)were counted;then enzyme-linked immunosorbent assay(ELISA)was used to measure IL-5 and IL-13 in alveolar lavage fluid and serum Ig E and IL-4;Finally,lung tissue NF-κB/PI3K/AKT and HO-1 signaling pathway were detected by Western Blotting.The experimental results showed that alpinetin significantly ameliorated OVA-induced pathologic changes of lungs,such as decreasing massive inflammatory cell infiltration and mucus hypersecretion,and reduced the number of eosinophils,neutrophils,and lymphocytes in BALF.Alpinetin also decreased the OVA-induced levels of IL-4,IL-5,IL-13 and Ig E.Furthermore,alpinetin inhibited OVA-induced phosphorylation of p65,IκB,PI3 K and AKT,and the activity of HO-1 in vivo.In order to further verify the mechanism of alpinetin in vitro,LPS was used to stimulate mouse macrophages RAW 264.7 to bulid an in vitro inflammatory response model.Firstly,the cytotoxicity test(CCK-8)was used to screen out the concentrations of alpinein were 15,30,and 60 μg/m L.The ELISA was used to detect the inflammatory cytokines TNF-α,IL-1β,and IL-6 in the cell supernatant,and Western blot was used to detect the phosphorylation level of proteins in the NF-κB and PAKT/PI3 K signaling pathways to explore the mechanism of alpinetin.The results found that alpinetin could effectively reduce the production of TNF-α IL-6,and IL-1β,and inhibited the activities of PI3K/AKT/NF-κB signaling pathways in LPS-induced RAW264.7 cells,which also confirm that alpinetin alleviates OVA-induced allergic asthma is related to the inhibition of the activities of the PI3K/AKT/NF-κB signaling pathway.In this article,OVA was used to establish an in vivo model of allergic asthma in mice and an in vitro model of LPS stimulated RAW264.7 cells,pathological tissue sections,ELISA and Western Blot to explore the anti-inflammatory and anti-allergic effects and effects of alpinetin in vivo and in vitro.In conclusion,above results indicate that alpinetin exhibites a potent anti-inflammatory activity in allergic asthma through modulating PI3K/AKT/NF-κB and HO-1 signaling pathways,which would be used as a promising therapy agent for allergic asthma.更多还原