本文關(guān)鍵詞：Tyr-Ala二肽的抗氧化作用及相關(guān)活性研究　出處：《吉林大學》2017年博士論文　論文類型：學位論文

  更多相關(guān)文章： 抗氧化 YA INS-1細胞 線蟲 二型糖尿病

【摘要】：活性肽因具有顯著的生理活性,如抗氧化作用、抗血栓、抗高血壓、降膽固醇、抗癌等,并且來源廣泛,已成為當今科學研究的熱點。其中,食源性活性肽因其制備工藝簡單且安全性高,已經(jīng)受到越來越多學者的關(guān)注。本文研究的小分子活性肽Tyr-Ala(YA)為兩種氨基酸——酪氨酸和丙氨酸組成的二肽,是在玉米蛋白酶解液中提取發(fā)現(xiàn)的一種單體肽。我們對其體內(nèi)外的抗氧化活性進行了研究,同時建立了線蟲模型和二型糖尿病動物模型,進一步探討了其抗氧化作用的機制以及抗衰老、治療糖尿病等相關(guān)活性研究。第一部分在對YA的抗氧化活性的體外研究中,我們采用DPPH自由基清除法、ABTS自由基清除法和超氧陰離子清除法等抗氧化性實驗進行綜合評估YA的體外抗氧化活性。研究發(fā)現(xiàn),YA具有清除DPPH自由基、ABTS自由基和超氧陰離子的能力。由此我們進一步進行了細胞實驗,發(fā)現(xiàn)YA本身對細胞并無毒性,在細胞受到嚴重的氧化損傷的情況下,具有保護細胞的功能。然后,我們使用DCFH-DA探針,檢測YA對胰島細胞ROS水平的影響。經(jīng)過氧化氫處理過的細胞的ROS水平明顯升高,在YA作用后ROS水平明顯下降。我們又利用Western Blot技術(shù),對細胞內(nèi)的PI3K-Akt通路與YA之間的關(guān)系進行研究。PI3K-Akt是影響體內(nèi)抗氧化能力的一個重要通路,結(jié)果顯示在高糖刺激下PI3K和磷酸化的Akt表達均有所下降,在YA干預(yù)后,PI3K和磷酸化的Akt表達均明顯上升。綜上所述,YA對氧化損傷的細胞具有保護作用,能夠清除自由基,并且可以調(diào)節(jié)PI3K-Akt這個通路。PI3K-Akt通路也是影響胰島素分泌的一個重要通路,因此我們設(shè)想,YA是否有促進胰島素分泌的作用,我們利用胰島素Elisa試劑盒對胰島素含量進行檢測。結(jié)果顯示,在高糖的刺激下,INS-1細胞分泌胰島素水平明顯降低,而YA可以促進高糖刺激下的INS-1細胞胰島素的分泌。第二部分,我們對YA在線蟲體內(nèi)的抗氧化活性進行了研究,建立了氧化應(yīng)激的線蟲模型,并對YA的抗氧化機制進行了探究。研究結(jié)果表明,YA在線蟲自然生長和在氧化應(yīng)激條件下,均可使線蟲的壽命延長,清除體內(nèi)過多的ROS,并且通過對CF1553線蟲的SOD-3蛋白表達的測定得出,YA是通過對SOD-3蛋白表達的升高起到清除線蟲體內(nèi)自由基的功效,YA還可以調(diào)節(jié)一些與抗氧化相關(guān)基因daf-2和daf-16的表達。第三部分,我們進行了YA在二型糖尿病小鼠體內(nèi)的研究。我們應(yīng)用高脂飲食配合STZ注射建立了實驗型二型糖尿病小鼠模型,檢測小鼠的體重、空腹血糖、胰島素水平;并進行小鼠的耐糖量測試;利用試劑盒測定小鼠血清中Hb A1c、TC、TG、SOD、MDA、GSH含量;稱量小鼠的肝臟、胰腺、脾和腎臟的重量,并利用HE染色法觀察胰腺和腎臟的病理形態(tài)學改變。實驗結(jié)果表明,模型組的小鼠的體重變化異常,空腹血糖值升高,胰島素分泌水平下降,而YA經(jīng)過尾靜脈注射4周的二型糖尿病小鼠,一般形態(tài)學狀態(tài)均好轉(zhuǎn),促進了胰島素的分泌。通過對小鼠血清中Hb A1c水平的檢測可見,YA有效的降低了Hb A1c的水平。Hb A1c反映了葡萄糖的水平,其升高的原因主要是由于二型糖尿病的血糖持續(xù)升高和微血管的損害。Hb A1c水平越高,則導(dǎo)致組織缺氧越嚴重,使蛋白質(zhì)結(jié)構(gòu)功能改變,尤其使發(fā)生在腎臟微血管豐富的部位,引起腎組織細胞缺血缺氧,導(dǎo)致腎組織損傷。二型糖尿病也常伴血脂異常,它是胰島素抵抗的始發(fā)因素也成為了動脈粥樣硬化、高血壓、高血脂癥等疾病的關(guān)鍵因素。所以改善二型糖尿病小鼠的內(nèi)脂代謝具有重要意義,并且對糖尿病的惡化或者產(chǎn)生并發(fā)癥起到緩解作用。YA有效的降低了TC、TG水平,可見YA具有良好的降血脂的作用。YA的抗氧化作用在二型糖尿病小鼠身上也得到了很好的體現(xiàn),有效升高了SOD水平,降低了MDA含量,促進了GSH的產(chǎn)生。二型糖尿病的β細胞的損傷主要就是通過線粒體氧化使葡萄糖代謝升高和FFA增加,從而升高線粒體膜電位,增加了過氧化物的產(chǎn)生。隨著過氧化物的產(chǎn)生增加,導(dǎo)致更多的細胞暴露在ROS下并且激活了解偶聯(lián)蛋白。所以YA的抗氧化性,可以抑制β細胞的氧化損傷,從而有治療二型糖尿病的作用。最后,通過對小鼠臟器的研究,發(fā)現(xiàn)YA對二型糖尿病小鼠的臟器有一定的修復(fù)作用。胰腺內(nèi)胰島細胞形態(tài)和活性經(jīng)過YA治療后有所改善,腎臟皮質(zhì)損傷得到恢復(fù),腎小球細胞得到治療。綜上所述,我們對YA的抗氧化活性及其機制進行了體內(nèi)外的研究,并且對二型糖尿病的影響等方面進行了探討,發(fā)現(xiàn)YA在體內(nèi)外都發(fā)揮著很好的抗氧化作用及相關(guān)活性功能。在未來的研究中,我們可以進一步對YA的其它相關(guān)活性和作用機制進行探索,對二肽YA在未來的應(yīng)用中起到指導(dǎo)性意義。
[Abstract]:Bioactive peptides, because of their significant physiological activities, such as antioxidation, antithrombotic, antihypertensive, cholesterol lowering, anticancer and so on, have become the focus of scientific research nowadays. Among them, food borne peptides have been paid more attention by more and more scholars because of their simple preparation and high safety. The small molecule active peptide Tyr-Ala (YA) is a two peptide composed of two kinds of amino acids tyrosine and alanine. It is a monomeric peptide extracted from corn protein hydrolysate. We studied the antioxidant activity in vivo and in vitro, and established the nematode model and type two diabetes animal model. We further explored the mechanism of its antioxidant activity, and related activities related to anti-aging and diabetes treatment. In the first part, in vitro study of the antioxidant activity of YA, we used DPPH free radical scavenging, ABTS radical scavenging and superoxide anion scavenging to evaluate the in vitro antioxidant activity of YA. It has been found that YA has the ability to scavenge DPPH radicals, ABTS radicals and superoxide anion. Therefore, we further carried out cell experiments, and found that YA itself is nontoxic to cells, and it has the function of protecting cells under severe oxidative damage. Then, we used the DCFH-DA probe to detect the effect of YA on the level of ROS in islet cells. The ROS level of the cells treated by hydrogen peroxide increased significantly, and the level of ROS decreased significantly after the action of YA. We also use Western Blot technology to study the relationship between the intracellular PI3K-Akt pathway and the YA. PI3K-Akt is an important pathway that affects the antioxidant capacity in vivo. The results showed that the expression of PI3K and phosphorylated Akt decreased under high glucose stimulation, and the expression of PI3K and phosphorylated Akt increased significantly after YA intervention. In summary, YA has a protective effect on oxidative damaged cells, which can scavenge free radicals and regulate the PI3K-Akt pathway. PI3K-Akt pathway is also an important pathway to affect insulin secretion. Therefore, we envisage whether YA can promote insulin secretion. We use insulin Elisa kit to detect the content of insulin. The results showed that the level of insulin secretion in INS-1 cells decreased significantly under the stimulation of high glucose, and YA could promote the secretion of insulin in INS-1 cells under high glucose stimulation. In the second part, we studied the antioxidant activity of YA worm, established the oxidative stress nematode model, and explored the antioxidant mechanism of YA. The results show that the YA online from natural growth and in the condition of oxidative stress, can prolong the life span in Caenorhabditis elegans, eliminate the excessive ROS, and the expression of CF1553 SOD-3 protein in nematode YA is obtained, through on the expression of SOD-3 protein increased to nematode free radical scavenging effect, expression YA can also regulate some antioxidant related genes DAF-2 and daf-16. In the third part, we conducted a study of YA in type two diabetic mice. We used high fat diet combined with STZ injection to establish experimental type two diabetes mice model, detection of mice body weight, fasting blood glucose, insulin levels and glucose tolerance in mice; TESTING; Determination of serum Hb A1c, TC, TG, SOD, MDA, GSH content by using the reagent kit; weighing the liver, the pancreas, spleen and kidney weight, and use HE staining to observe the pathological changes of pancreas and kidney. The experimental results showed that the body weight of mice in the model group was abnormal, the fasting blood glucose level increased and the insulin secretion level decreased, while the YA type two diabetic mice after 4 weeks of intravenous injection were all improved, which promoted the secretion of insulin. By detecting the level of Hb A1c in the mice serum, YA effectively reduced the level of Hb A1c. Hb A1c reflects the level of glucose, the cause of which is mainly due to the continuous rise in blood sugar in type two diabetes and the damage of microvessels. The higher the level of Hb A1c, the more severe the hypoxia is, which makes the structure and function of protein change, especially in the rich part of renal microvessels, causing the ischemia and anoxia of renal tissue, leading to renal tissue injury. Type two diabetes is also often associated with dyslipidemia. It is the initial factor of insulin resistance and also a key factor of atherosclerosis, hypertension, hyperlipidemia and other diseases. Therefore, it is of great significance to improve the internal lipid metabolism in type two diabetic mice, and to alleviate the deterioration of diabetes or to produce complications. YA effectively reduces the level of TC and TG, and it can be seen that YA has a good effect on lowering blood lipid. The antioxidant effect of YA was also well reflected in type two diabetic mice, which effectively raised the level of SOD, reduced the content of MDA, and promoted the production of GSH. Beta cell injury in type two diabetes is mainly caused by mitochondrial oxidation, which increases glucose metabolism and FFA, thereby increasing mitochondrial membrane potential and increasing peroxide production. As the production of peroxide increases, more cells are exposed to ROS and activate understanding of the coupling proteins. Therefore, the antioxidant activity of YA can inhibit the oxidative damage of beta cells, and thus have the effect on the treatment of type two diabetes. Finally, through the study of mouse organs, it is found that YA has a certain repair effect on the organs of type two diabetic mice. The morphology and activity of pancreatic islet cells in the pancreas were improved after YA treatment, and the renal cortical damage was restored, and the glomerular cells were treated. To sum up, we studied the antioxidant activity and mechanism of YA, and discussed the influence of type two diabetes. We found that YA played a very good role in antioxidant and related active functions both in vivo and in vitro. In future research, we can enter
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R96

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