Tyr-Ala二肽的抗氧化作用及相关活性研究

发布时间：2017-12-27 01:27

本文关键词：Tyr-Ala二肽的抗氧化作用及相关活性研究　出处：《吉林大学》2017年博士论文　论文类型：学位论文

更多相关文章： 抗氧化 YA INS-1细胞 线虫 二型糖尿病

【摘要】：活性肽因具有显著的生理活性,如抗氧化作用、抗血栓、抗高血压、降胆固醇、抗癌等,并且来源广泛,已成为当今科学研究的热点。其中,食源性活性肽因其制备工艺简单且安全性高,已经受到越来越多学者的关注。本文研究的小分子活性肽Tyr-Ala(YA)为两种氨基酸——酪氨酸和丙氨酸组成的二肽,是在玉米蛋白酶解液中提取发现的一种单体肽。我们对其体内外的抗氧化活性进行了研究,同时建立了线虫模型和二型糖尿病动物模型,进一步探讨了其抗氧化作用的机制以及抗衰老、治疗糖尿病等相关活性研究。第一部分在对YA的抗氧化活性的体外研究中,我们采用DPPH自由基清除法、ABTS自由基清除法和超氧阴离子清除法等抗氧化性实验进行综合评估YA的体外抗氧化活性。研究发现,YA具有清除DPPH自由基、ABTS自由基和超氧阴离子的能力。由此我们进一步进行了细胞实验,发现YA本身对细胞并无毒性,在细胞受到严重的氧化损伤的情况下,具有保护细胞的功能。然后,我们使用DCFH-DA探针,检测YA对胰岛细胞ROS水平的影响。经过氧化氢处理过的细胞的ROS水平明显升高,在YA作用后ROS水平明显下降。我们又利用Western Blot技术,对细胞内的PI3K-Akt通路与YA之间的关系进行研究。PI3K-Akt是影响体内抗氧化能力的一个重要通路,结果显示在高糖刺激下PI3K和磷酸化的Akt表达均有所下降,在YA干预后,PI3K和磷酸化的Akt表达均明显上升。综上所述,YA对氧化损伤的细胞具有保护作用,能够清除自由基,并且可以调节PI3K-Akt这个通路。PI3K-Akt通路也是影响胰岛素分泌的一个重要通路,因此我们设想,YA是否有促进胰岛素分泌的作用,我们利用胰岛素Elisa试剂盒对胰岛素含量进行检测。结果显示,在高糖的刺激下,INS-1细胞分泌胰岛素水平明显降低,而YA可以促进高糖刺激下的INS-1细胞胰岛素的分泌。第二部分,我们对YA在线虫体内的抗氧化活性进行了研究,建立了氧化应激的线虫模型,并对YA的抗氧化机制进行了探究。研究结果表明,YA在线虫自然生长和在氧化应激条件下,均可使线虫的寿命延长,清除体内过多的ROS,并且通过对CF1553线虫的SOD-3蛋白表达的测定得出,YA是通过对SOD-3蛋白表达的升高起到清除线虫体内自由基的功效,YA还可以调节一些与抗氧化相关基因daf-2和daf-16的表达。第三部分,我们进行了YA在二型糖尿病小鼠体内的研究。我们应用高脂饮食配合STZ注射建立了实验型二型糖尿病小鼠模型,检测小鼠的体重、空腹血糖、胰岛素水平;并进行小鼠的耐糖量测试;利用试剂盒测定小鼠血清中Hb A1c、TC、TG、SOD、MDA、GSH含量;称量小鼠的肝脏、胰腺、脾和肾脏的重量,并利用HE染色法观察胰腺和肾脏的病理形态学改变。实验结果表明,模型组的小鼠的体重变化异常,空腹血糖值升高,胰岛素分泌水平下降,而YA经过尾静脉注射4周的二型糖尿病小鼠,一般形态学状态均好转,促进了胰岛素的分泌。通过对小鼠血清中Hb A1c水平的检测可见,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续升高和微血管的损害。Hb A1c水平越高,则导致组织缺氧越严重,使蛋白质结构功能改变,尤其使发生在肾脏微血管丰富的部位,引起肾组织细胞缺血缺氧,导致肾组织损伤。二型糖尿病也常伴血脂异常,它是胰岛素抵抗的始发因素也成为了动脉粥样硬化、高血压、高血脂症等疾病的关键因素。所以改善二型糖尿病小鼠的内脂代谢具有重要意义,并且对糖尿病的恶化或者产生并发症起到缓解作用。YA有效的降低了TC、TG水平,可见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体现,有效升高了SOD水平,降低了MDA含量,促进了GSH的产生。二型糖尿病的β细胞的损伤主要就是通过线粒体氧化使葡萄糖代谢升高和FFA增加,从而升高线粒体膜电位,增加了过氧化物的产生。随着过氧化物的产生增加,导致更多的细胞暴露在ROS下并且激活了解偶联蛋白。所以YA的抗氧化性,可以抑制β细胞的氧化损伤,从而有治疗二型糖尿病的作用。最后,通过对小鼠脏器的研究,发现YA对二型糖尿病小鼠的脏器有一定的修复作用。胰腺内胰岛细胞形态和活性经过YA治疗后有所改善,肾脏皮质损伤得到恢复,肾小球细胞得到治疗。综上所述,我们对YA的抗氧化活性及其机制进行了体内外的研究,并且对二型糖尿病的影响等方面进行了探讨,发现YA在体内外都发挥着很好的抗氧化作用及相关活性功能。在未来的研究中,我们可以进一步对YA的其它相关活性和作用机制进行探索,对二肽YA在未来的应用中起到指导性意义。
[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
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R96

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