發(fā)布時間：2018-03-01 12:39

  本文關(guān)鍵詞： 糖尿病 代謝組學(xué) 咖啡酸苯乙酯 原花青素 氧化應(yīng)激　出處：《陜西科技大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：糖尿病(DM)是由于胰島素缺乏或功能缺失引起的多功能慢性代謝紊亂疾病,主要癥狀以高血糖為特征,會引發(fā)多種嚴(yán)重的并發(fā)癥,已經(jīng)成為嚴(yán)重威脅人類的健康疾病之一。代謝組學(xué)是一門全面、系統(tǒng)研究生物體受到遺傳修飾和外界刺激下隨時間變化規(guī)律產(chǎn)生的所有代謝物變化應(yīng)答的新興學(xué)科。作為嶄新的方法學(xué),代謝組學(xué)具有高通量、高靈敏度及強特異性等優(yōu)勢,通過研究疾病以及服藥后機體內(nèi)源性代謝物的變化,來全面探索機體代謝途徑,進而闡述疾病的發(fā)病機理和藥物的作用機制,從而為DM的診斷開辟了新的途徑。本研究擬采用高脂高糖喂養(yǎng)聯(lián)合鏈脲佐菌素(STZ)誘導(dǎo),構(gòu)建糖尿病大鼠模型,蜂膠有效成分咖啡酸苯乙酯(CAPE)及葡萄籽有效成分原花青素(PC)作為保護藥物,通過生理生化指標(biāo)及組織形態(tài)學(xué)進行模型驗證。利用代謝組學(xué)分析技術(shù)超高效液相-飛行時間質(zhì)譜(UPLC-Q-TOF/MS),對正常對照組、糖尿病模型組及不同藥物保護組的血清進行了代謝物譜的分析,采用多元統(tǒng)計分析及模式識別尋找出誘發(fā)糖尿病的潛在生物標(biāo)記物。同時,探討了糖尿病大鼠的發(fā)病機制及保護藥物的保護機制,為糖尿病的臨床診斷及藥物的開發(fā)提供數(shù)據(jù)支持和理論依據(jù)。(1)DM大鼠模型的建立及模型驗證SD雄性大鼠喂養(yǎng)高脂高糖飼料六周后,小劑量(30mg/kg)注射STZ誘導(dǎo)糖尿病大鼠模型。經(jīng)生理生化指標(biāo)及各主要臟器形態(tài)學(xué)分析模型構(gòu)建成功。生理指標(biāo)結(jié)果表明符合糖尿病“三多一少”的典型特征,生化指標(biāo)結(jié)果檢測表明血清甘油三酯(TG)、總膽固醇(TC)及低密度脂蛋白(LDL-C)較對照組相比,糖尿病模型組顯著升高(P0.01,P0.001,P0.001),高密度脂蛋白(HDL-C)較對照組,DM模型組明顯下降(P0.001),說明高脂高糖飼料聯(lián)合STZ構(gòu)建糖尿病大鼠造成了損傷,給予CAPE及PC保護后,其損傷得到了明顯的改善,說明CAPE及PC對糖尿病具有一定的保護作用。(2)糖尿病模型大鼠給藥前后血清代謝物譜的UPLC-Q-TOF/MS分析采用UPLC-Q-TOF/MS對對照組、DM模型組及CAPE和PC保護組血清進行檢測,通過主成分分析法對對照組、DM模型組及保護組血清代謝物譜進行分類并尋找與DM發(fā)生有關(guān)的生物標(biāo)記物。結(jié)果表示,各組大鼠血清樣品實現(xiàn)完全分離,同時DM模型組大鼠不對稱二甲基精氨酸、精胺、LPA(18:1)、PI(18:1/16:0)和LysoPC(18:2))較對照組明顯增加,在給予CAPE及PC干預(yù)后,結(jié)果顯示體內(nèi)上述代謝物的含量有所下降。不對稱二甲基精氨酸及磷脂類代謝物的變化均有可能與氧化應(yīng)激有關(guān),同時溶血性磷脂變化也與元素、生化指標(biāo)的改變有關(guān),而給予保護藥保護后代謝物發(fā)生了相應(yīng)的變化,體現(xiàn)了CAPE與PC對糖尿病均具有保護作用。(3)糖尿病大鼠發(fā)病機理的研究對各組大鼠組織氧化指標(biāo)(蛋白羰基化(PCO)、丙二醛(MDA))、炎癥因子(一氧化氮(NO))及抗氧化體系(超氧化物歧化酶(SOD)、谷胱甘肽(GSH)、過氧化氫酶(CAT))進行檢測,結(jié)果表示DM模型組各組織氧化指標(biāo)MDA、NO、PCO含量顯著高于對照組(P0.05),抗氧化酶SOD、GSH、CAT活性明顯低于對照組(P0.05),給予CAPE和PC治療后氧化指標(biāo)的上升得到抑制,抗氧化酶活性出現(xiàn)上升。說明糖尿病能夠增加各臟器的氧化應(yīng)激,誘導(dǎo)氧化損傷,而保護藥能夠減少氧化損傷,起到保護作用。同時對其各組織、血清、尿液中的元素進行含量檢測,結(jié)果顯示DM模型組各組織與血清中Zn~(2+)、Mg~(2+)、Ca~(2+)、Fe~(2+)的含量顯著減少,Cu~(2+)的含量顯著增加,經(jīng)CAPE與PC保護后大鼠體內(nèi)Zn~(2+)、Mg~(2+)、Ca~(2+)、Fe~(2+)元素的含量相對的上升,Cu~(2+)含量相對下降;同時糖尿病組大鼠尿液中Zn~(2+)、Cu~(2+)、Mg~(2+)、Ca~(2+)、Fe~(2+)的含量顯著增加,經(jīng)保護藥保護后的大鼠尿液元素的含量有相對的下降。結(jié)果顯示糖尿病的發(fā)生,一方面可能與其體內(nèi)損傷ROS有關(guān),另一方面與其體內(nèi)元素變化有關(guān)。CAPE及PC對糖尿病的保護作用一方面與結(jié)構(gòu)特征有關(guān),另一方面可作為多種信號通路的激動劑,因此本實驗為進一步闡明糖尿病的作用機制以及臨床開發(fā)新物提供科學(xué)依據(jù)。
[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，

本文編號：1551987