基于代谢组学研究蜂胶和葡萄籽有效成分对糖尿病的作用机制

发布时间：2018-03-01 12:39

  本文关键词： 糖尿病 代谢组学 咖啡酸苯乙酯 原花青素 氧化应激　出处：《陕西科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：糖尿病(DM)是由于胰岛素缺乏或功能缺失引起的多功能慢性代谢紊乱疾病,主要症状以高血糖为特征,会引发多种严重的并发症,已经成为严重威胁人类的健康疾病之一。代谢组学是一门全面、系统研究生物体受到遗传修饰和外界刺激下随时间变化规律产生的所有代谢物变化应答的新兴学科。作为崭新的方法学,代谢组学具有高通量、高灵敏度及强特异性等优势,通过研究疾病以及服药后机体内源性代谢物的变化,来全面探索机体代谢途径,进而阐述疾病的发病机理和药物的作用机制,从而为DM的诊断开辟了新的途径。本研究拟采用高脂高糖喂养联合链脲佐菌素(STZ)诱导,构建糖尿病大鼠模型,蜂胶有效成分咖啡酸苯乙酯(CAPE)及葡萄籽有效成分原花青素(PC)作为保护药物,通过生理生化指标及组织形态学进行模型验证。利用代谢组学分析技术超高效液相-飞行时间质谱(UPLC-Q-TOF/MS),对正常对照组、糖尿病模型组及不同药物保护组的血清进行了代谢物谱的分析,采用多元统计分析及模式识别寻找出诱发糖尿病的潜在生物标记物。同时,探讨了糖尿病大鼠的发病机制及保护药物的保护机制,为糖尿病的临床诊断及药物的开发提供数据支持和理论依据。(1)DM大鼠模型的建立及模型验证SD雄性大鼠喂养高脂高糖饲料六周后,小剂量(30mg/kg)注射STZ诱导糖尿病大鼠模型。经生理生化指标及各主要脏器形态学分析模型构建成功。生理指标结果表明符合糖尿病“三多一少”的典型特征,生化指标结果检测表明血清甘油三酯(TG)、总胆固醇(TC)及低密度脂蛋白(LDL-C)较对照组相比,糖尿病模型组显著升高(P0.01,P0.001,P0.001),高密度脂蛋白(HDL-C)较对照组,DM模型组明显下降(P0.001),说明高脂高糖饲料联合STZ构建糖尿病大鼠造成了损伤,给予CAPE及PC保护后,其损伤得到了明显的改善,说明CAPE及PC对糖尿病具有一定的保护作用。(2)糖尿病模型大鼠给药前后血清代谢物谱的UPLC-Q-TOF/MS分析采用UPLC-Q-TOF/MS对对照组、DM模型组及CAPE和PC保护组血清进行检测,通过主成分分析法对对照组、DM模型组及保护组血清代谢物谱进行分类并寻找与DM发生有关的生物标记物。结果表示,各组大鼠血清样品实现完全分离,同时DM模型组大鼠不对称二甲基精氨酸、精胺、LPA(18:1)、PI(18:1/16:0)和LysoPC(18:2))较对照组明显增加,在给予CAPE及PC干预后,结果显示体内上述代谢物的含量有所下降。不对称二甲基精氨酸及磷脂类代谢物的变化均有可能与氧化应激有关,同时溶血性磷脂变化也与元素、生化指标的改变有关,而给予保护药保护后代谢物发生了相应的变化,体现了CAPE与PC对糖尿病均具有保护作用。(3)糖尿病大鼠发病机理的研究对各组大鼠组织氧化指标(蛋白羰基化(PCO)、丙二醛(MDA))、炎症因子(一氧化氮(NO))及抗氧化体系(超氧化物歧化酶(SOD)、谷胱甘肽(GSH)、过氧化氢酶(CAT))进行检测,结果表示DM模型组各组织氧化指标MDA、NO、PCO含量显著高于对照组(P0.05),抗氧化酶SOD、GSH、CAT活性明显低于对照组(P0.05),给予CAPE和PC治疗后氧化指标的上升得到抑制,抗氧化酶活性出现上升。说明糖尿病能够增加各脏器的氧化应激,诱导氧化损伤,而保护药能够减少氧化损伤,起到保护作用。同时对其各组织、血清、尿液中的元素进行含量检测,结果显示DM模型组各组织与血清中Zn~(2+)、Mg~(2+)、Ca~(2+)、Fe~(2+)的含量显著减少,Cu~(2+)的含量显著增加,经CAPE与PC保护后大鼠体内Zn~(2+)、Mg~(2+)、Ca~(2+)、Fe~(2+)元素的含量相对的上升,Cu~(2+)含量相对下降;同时糖尿病组大鼠尿液中Zn~(2+)、Cu~(2+)、Mg~(2+)、Ca~(2+)、Fe~(2+)的含量显著增加,经保护药保护后的大鼠尿液元素的含量有相对的下降。结果显示糖尿病的发生,一方面可能与其体内损伤ROS有关,另一方面与其体内元素变化有关。CAPE及PC对糖尿病的保护作用一方面与结构特征有关,另一方面可作为多种信号通路的激动剂,因此本实验为进一步阐明糖尿病的作用机制以及临床开发新物提供科学依据。
[Abstract]:Diabetes mellitus (DM) is due to insulin deficiency or loss of function caused by multi functional chronic metabolic disorders, the main symptoms are characterized by high blood glucose, can cause some serious complications, health human disease has become one of the serious threat. Metabolomics is an emerging discipline comprehensive, all metabolites change response system of organism by genetic modification and external stimulation with time under the change regularity. As a new methodology, metabonomics has high throughput, high sensitivity and strong specificity and other advantages, through the study of disease and medication after endogenous metabolite changes, to fully explore the metabolic pathway, and then elaborates the mechanism of pathogenesis and drug disease, which opens up a new way for the diagnosis of DM. The purpose of this study was feeding the high-fat diet combined with streptozotocin (STZ) induced by construction of diabetes The rat model of propolis effective component of caffeic acid phenethyl ester (CAPE) and the effective composition of grape seed procyanidins (PC) as a protective drug, verified by morphological and physiological biochemical index. Using metabonomics analysis technology of ultra high performance liquid chromatography time of flight mass spectrometry (UPLC-Q-TOF/MS), normal control group, the serum of diabetes the model group and different drug protection group analyzed the metabolite profile, using multivariate statistical analysis and pattern recognition to find potential biomarkers to induce diabetes. At the same time, to explore the pathogenesis of diabetic rats and protect the drug protection mechanism, to provide data support and theoretical basis for the development of clinical diagnosis and drug diabetes.. (1) DM rat model and model validation of male SD rats fed with high fat diet for six weeks, small dose (30mg/kg) model of diabetic rats induced by injection of STZ. The physiological and biochemical indexes and major organs morphology analysis model was constructed successfully. The results show that in line with the typical characteristics of physiological indexes of diabetes "three little", biochemical detection results showed that the serum triglyceride (TG), total cholesterol (TC) and low density lipoprotein (LDL-C) compared with the control group, diabetic model group increased significantly (P0.01. P0.001, P0.001), high density lipoprotein (HDL-C) compared with the control group, DM model group decreased significantly (P0.001), high fat and high glucose diet combined with STZ to construct diabetic rats caused damage, giving CAPE and PC protection after the injury has been significantly improved, indicating that CAPE and PC have some protective effect for diabetes. (2) diabetic rats before and after administration of serum metabolite profiles of UPLC-Q-TOF/MS were analyzed by UPLC-Q-TOF/MS in the control group, DM model group and CAPE and PC group serum were detected by the method of principal component analysis In control group, DM model group and protective group serum metabolite profile classification and search for biomarkers associated with the occurrence of DM. Results showed that the serum samples of rats to achieve complete separation, while DM model rats two asymmetric dimethylarginine, spermine, LPA (18:1), PI (18:1/16:0) and LysoPC (18:2)) significantly increased compared with the control group, CAPE and PC in the given intervention, results showed that the content in vivo metabolites decreased. Two asymmetric dimethylarginine and phospholipid metabolites were associated with oxidative stress, and hemolytic changes of phospholipid and elements, biochemical changes, and protective medicine to protect offspring metabolism changed, embodies the CAPE and PC have protective effect on diabetes. (3) the study of pathogenesis of diabetic rats on oxidative index tissue of rats (protein carbonyl (PCO), malondialdehyde (M DA)),鐐庣棁鍥犲瓙(涓，

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