黄芪多糖APS-Ⅰ、APS-Ⅱ制备及其对溃疡性结肠炎小鼠抗炎活性  

Preparation of Astragalus polysaccharides APS-Ⅰ and APS-Ⅱ and their anti-inflammatory activity in mice with ulcerative colitis

作　　者：文雨薇 李科[1,2,3,4] 吕弯弯 冯仕红 秦雪梅 杜昱光[4] 李震宇 WEN Yuwei;LI Ke;LYU Wanwan;FENG Shihong;QIN Xuemei;DU Yuguang;LI Zhenyu(Modern Research Center of Traditional Chinese Medicine,Shanxi University,Taiyuan 030006,China;Key Laboratory of Chemical Biology and Molecular Engineering,Ministry of Education,Shanxi University,Taiyuan 030006,China;Shanxi Provincial Key Laboratory of Research and Utilization of Functional Substances in Traditional Chinese Medicine,Taiyuan 030006,China;Institute of Process Engineering,Chinese Academy of Sciences,Beijing 100190,China)

机构地区：[1]山西大学中医药现代研究中心,山西太原030006 [2]山西大学化学生物学与分子工程教育部重点实验室,山西太原030006 [3]地产中药功效物质研究与利用山西省重点实验室,山西太原030006 [4]中国科学院过程工程研究所,北京100190

出　　处：《中草药》2024年第14期4759-4770,共12页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金资助项目(81872962);国家博士后科学基金资助项目(2019M650851);国家重点研发计划项目(2019YFC1710800);山西省重点研发计划重点项目(201603D311101);山西省优秀人才科技创新项目(201605D211030,201705D211020);山西省科技创新人才团队专项基金。

摘　　要：目的从黄芪多糖(Astragalus polysaccharides,APS)中分离制备出2种多糖APS-Ⅰ(相对分子质量>2×10~6)、APS-Ⅱ(相对分子质量1×10~4),明确APS、APS-Ⅰ、APS-Ⅱ对葡聚糖硫酸钠(dextran sulphate sodium,DSS)诱导的小鼠溃疡性结肠炎的疗效。方法对APS-Ⅰ、APS-Ⅱ进行分离制备,并进行相对分子质量、糖醛酸含量、单糖组成以及糖苷键连接方式测定。建立BALB/c小鼠溃疡性结肠炎模型,分别给予APS、APS-Ⅰ、APS-Ⅱ,记录小鼠疾病活动指数(disease activity index,DAI)评分、结肠长度、脾脏指数、肝脏指数;采用苏木素-伊红(HE)染色观察结肠组织病理变化;测定结肠组织髓过氧化物酶(myeloperoxidase,MPO)活性和Th1相关细胞因子肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、γ干扰素(interferon-γ,IFN-γ)以及Th2相关细胞因子白细胞介素-4(interleukin-4,IL-4)、IL-10水平;采用qRT-PCR检测结肠组织TNF-α、IFN-γ、IL-4、IL-10 mRNA表达;免疫组化法测定结肠组织T细胞特异性转录因子(transcription factor T-bet,T-bet)、GATA结合蛋白3(GATA binding protein 3,GATA-3)蛋白表达,比较APS、APS-Ⅰ、APS-Ⅱ疗效。结果APS中共有2个组分APS-Ⅰ、APS-Ⅱ,其相对分子质量分别为>2×10~6、1×10~4,糖醛酸质量分数分别为26.25%、1.62%。单糖结果显示,APS-Ⅰ和APS-Ⅱ均由甘露糖、鼠李糖、半乳糖醛酸、葡萄糖、半乳糖、阿拉伯糖组成,APS-Ⅰ中甘露糖、阿拉伯糖和半乳糖占比均大于APS-Ⅱ。糖苷键结果显示,APS-Ⅰ以1,4连接的葡萄糖和1,6连接的半乳糖为主,APS-Ⅱ以1,4连接的葡萄糖为主。与模型组比较,APS-Ⅰ可显著改善结肠长度降低、体质量减轻、DAI评分增加、脾脏和肝脏指数增加等临床症状(P<0.05、0.01、0.001),减轻结肠组织病理损伤,降低结肠组织中MPO活性及TNF-α、IFN-γ水平(P<0.01、0.001),升高结肠组织中IL-4、IL-10水平(P<0.05、0.01、0.001),下调结肠组织TNF-α、IFN-γmRNA表达和T-bet蛋白�Objective To isolate Astragalus polysaccharides-Ⅰ(APS-Ⅰ)(relative molecular mass > 2 × 10~6) and APS-Ⅱ(relative molecular mass 1 × 10~4) from APS,and further clarify the efficacy of APS,APS-Ⅰ and APS-Ⅱ on dextran sulphate sodium(DSS)-induced ulcerative colitis in mice.Methods APS-Ⅰ and APS-Ⅱ were isolated and prepared,relative molecular mass,uronic acid content,monosaccharide composition and glycosidic linkage were determined.The BALB/c mice model of ulcerative colitis was established,APS,APS-Ⅰ and APS-Ⅱ were given.The disease activity index(DAI),colon length,spleen index and liver index of mice were measured;Hematoxylin-eosin(HE) staining was used to observe pathological changes in colon tissue;The activity of myeloperoxidase(MPO) and levels of Th1 related cytokines tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ),as well as Th2related cytokines interleukin-4(IL-4) and IL-10 in colon tissue were measured;qRT-PCR was used to detect the mRNA expressions of TNF-α,IFN-γ,IL-4 and IL-10 in colon tissue;Immunohistochemical method was used to measure the expressions of T cell specific transcription factor T-bet(T-bet) and GATA binding protein 3(GATA-3) protein in colon tissue.The efficacy of APS,APS-Ⅰ and APSII was compared.Results APS was composed of two components APS-Ⅰ and APS-Ⅱ,with relative molecular weights > 2 × 10~6and 1 × 10~4,respectively;The mass fractions of uronic acid were 26.25% and 1.62%,respectively.The monosaccharide results showed that APS-Ⅰ and APS-Ⅱ were both composed of mannose,rhamnose,galacturonic acid,glucose,galactose and arabinose.The proportion of mannose,arabinose and galactose in APS-Ⅰ was higher than that in APS-Ⅱ.The glycosidic bond results showed that APS-Ⅰ was mainly composed of 1,4-linked glucose and 1,6-linked galactose,while APS-Ⅱ was mainly composed of 1,4-linked glucose.Compared with model group,APS-Ⅰ could significantly improve clinical symptoms such as decreased colon length,decreased body weight,increased DAI score,increased sple

关 键 词：黄芪多糖 黄芪多糖-Ⅰ 黄芪多糖-Ⅱ 甘露糖 鼠李糖 半乳糖醛酸 葡萄糖 半乳糖 阿拉伯糖 溃疡性结肠炎 Th1/Th2免疫平衡 

分 类 号：R285.5[医药卫生—中药学]