S-Equol调控Txnip表达对高糖培养INS-1细胞胰岛素分泌的影响及机制研究

发布时间：2018-05-15 08:51

本文选题：S型雌马酚 + 胰岛素分泌　；参考：《第三军医大学》2015年硕士论文

【摘要】：糖尿病(diabetic mellitus,DM)是最常见的慢性代谢性疾病之一,严重危害着人类身体健康,给个人及家庭带来极重的精神和经济负担。并且DM发病机制极为复杂,至今仍未完全阐明。目前认为胰岛素抵抗和胰岛素分泌功能缺陷是DM发病的两大主要因素,已成为糖尿病防治的重要靶点。然而,临床上针对其使用的药物大都存在诸多不良反应。因此,不断寻找和开辟糖尿病防治的新途径已成为当今医学研究的焦点,尤其是经过膳食途径改善糖尿病及其并发症具有非常重要的意义。雌马酚(equol,Eq)是由大豆生长过程中形成的次生代谢物大豆异黄酮(soy isoflavones,SI)经微生物代谢形成的重要产物,包括S、R两种异构体,而在动物体内均为S型[1]。大量研究表明,SI干预可增加胰岛素分泌,促进葡萄糖摄取和利用,从而有效控制血糖浓度,改善糖尿病症状。而进一步研究发现,SI的生物学活性主要由Eq来实现[2,3]。Eq具有比SI更高的生物活性和生物利用度,能够有效的发挥雌激素样、抗炎和抗氧化等作用,不仅在骨质疏松症、更年期综合症以及心血管疾病等方面表现出较好的防治效果,而且对乳腺癌及前列腺癌也具有很好的预防作用[4]。已有研究表明,Eq可显著增加葡萄糖耐量[5],促进葡萄糖摄取和利用,提高胰岛素敏感性[6],但其具体的作用机制有待进一步研究。最新研究发现,硫氧还蛋白相互作用蛋白(thioredoxin-interacting protein,Txnip)在调控胰岛素分泌中发挥着关键作用。糖尿病发病过程中,胰岛素敏感性改变、高血糖、糖皮质激素变化和β细胞凋亡均可诱导Txnip表达,抑制葡萄糖摄取与利用。Txnip的表达受多条细胞外葡萄糖调控路径影响,其中碳水化合物反应元件结合蛋白(carbohydrate response element binding protein,ChREBP)和Max样蛋白(Max-like protein X,MLx)是调节Txnip表达的最主要转录因子。高糖可促进ChREBP去磷酸化,形成活化的ChREBP。当活化ChREBP进入细胞核,与异质二聚体伴侣MLX,Txnip募集的辅助剂和组蛋白乙酰化P300结合,导致组蛋白H4乙酰化,从而进行染色质修饰,同时促使RNA聚合酶Ⅱ(PolⅡ)转移到启动子区,致使Txnip基因开始转录[7]。那么,S-Eq是否能够通过ChREBP-Txnip信号通路调节胰岛细胞分泌功能,从而实现其抗DM的作用,相关研究未见报道。本课题采用体外培养大鼠胰岛素瘤(INS-1)细胞株,利用高糖刺激建立体外胰岛细胞损伤模型,采用CCK-8法检测细胞活力,ELISA法测定葡萄糖刺激胰岛素分泌(glucose-stimulated insulin secretion,GSIS)功能,TUNEL法联合Annexin V-FITC/PI流式细胞术检测细胞凋亡,Realtime PCR检测前胰岛素原(preproinsulin,PPI)、葡萄糖转运蛋白2(glucose transporter 2,Glut2)、线粒体阴离子载体解偶联蛋白2(uncoupling protein2,UCP2)mRNA表达,Western blot检测Glut2及UCP2蛋白表达,以探讨S-Eq对INS-1细胞胰岛素分泌功能的影响。并在此基础上,采用PKA及PP2A试剂盒分别检测PKA及PP2A活性,同时通过转染si RNA和Western blot检测ChREBP、Txnip蛋白表达,结合Chip及双荧光素酶报告基因等技术深入研究ChREBP-Txnip信号通路在S-Eq调控胰岛细胞分泌功能中的作用。本实验的主要实验结果和结论如下:(1)S-Eq可显著增加高糖培养条件下INS-1细胞活力,减少细胞凋亡(P0.05)。(2)S-Eq可显著上调高糖培养条件下INS-1细胞PPI mRNA表达水平,改善高糖培养条件下INS-1细胞GSIS功能。同时可显著增加高糖处理后细胞Glut2的转录表达而降低UCP2的转录表达水平(P0.05)。(3)S-Eq可显著降低高糖培养INS-1细胞内PP2A活性,升高PKA活性,抑制INS-1细胞内ChREBP蛋白表达(P0.05)。(4)S-Eq可显著减少ChREBP在ChoRE顺式作用元件上募集,下调ChREBP(wt)转染后INS-1细胞内Txnip启动子区转录活性,进而抑制Txnip表达。而在转染ChREBP(dm)质粒后,各组INS-1细胞内Txnip启动子区转录活性均显著降低。进一步采用siRNA沉默ChREBP基因表达后,ChREBP和Txnip表达均显著降低,同时发现S-Eq对高糖诱导Txnip的表达有显著抑制作用(P0.05)。全文结论:S-Eq可能通过调节ChREBP活性进而抑制Txnip信号通路,从而减少高糖培养条件下INS-1细胞凋亡、增强Glut2表达和降低UCP2的转录表达,进而改善INS-1细胞功能,增加胰岛素分泌。
[Abstract]:Diabetic mellitus (DM) is one of the most common chronic metabolic diseases. It seriously endangers the health of human beings and brings serious mental and economic burden to individuals and families. And the pathogenesis of DM is very complex and is still not fully elucidated. At present, insulin resistance and insulin secretion deficiency are the two major causes of DM. The main factors have become an important target for the prevention and control of diabetes. However, most of the drugs used in clinical use have many adverse reactions. Therefore, it has become the focus of modern medical research to find and open up new ways to prevent and cure diabetes. Especially, it is very important to improve diabetes and its complications through dietary ways. Equol (Eq) is an important product formed by the metabolism of soy isoflavones (SI), the secondary metabolite of Soybean (soy isoflavones, SI), formed in the process of soybean growth, including S, R two isomers, and in the animal body, a large amount of S type [1]. studies show that SI intervention can increase insulin secretion, promote glucose uptake and utilization, so as to promote the uptake and utilization of glucose. Further studies have found that the biological activity of SI is mainly based on Eq to achieve higher bioactivity and bioavailability of [2,3].Eq than SI, and can effectively play the role of estrogen like, anti-inflammatory and antioxidant activities, not only in osteoporosis, menopause syndrome, and cardiovascular disease, and so on. [4]. has shown good preventive effects and has a good preventive effect on breast and prostate cancer. Studies have shown that Eq can significantly increase glucose tolerance [5], promote glucose uptake and utilization, improve insulin sensitivity [6], but its specific mechanism remains to be further studied. The latest research has found that sulphur and oxygen are still eggs. Thioredoxin-interacting protein (Txnip) plays a key role in the regulation of insulin secretion. In the course of diabetes, insulin sensitivity changes, hyperglycemia, glucocorticoid changes and beta cell apoptosis can induce Txnip expression, and the inhibition of glucose uptake and use of.Txnip is affected by multiple extracellular Portuguese. The effects of glucose regulation pathway, including carbohydrate response element binding protein (ChREBP) and Max like protein (Max-like protein X, MLx), are the most important transcription factors regulating Txnip expression. Hetero two polymer chaperone MLX, Txnip raised auxiliary and histone acetylation P300 binding, causing histone H4 acetylation, making chromatin modification, and promoting the transfer of RNA polymerase II (Pol II) to the promoter region, causing the Txnip gene to begin to transcribe [7]., then whether S-Eq can regulate islet cell scores through the ChREBP-Txnip signaling pathway. The role of secreting function to achieve its anti DM effect was not reported. This topic used in vitro culture of rat insulinoma (INS-1) cell lines, using high glucose to build a three-dimensional external islet cell damage model, using the CCK-8 method to detect cell viability, ELISA assay of glucose stimulated insulin secretion (glucose-stimulated insulin secretion, GS). IS) function, TUNEL method combined with Annexin V-FITC/PI flow cytometry to detect cell apoptosis. Realtime PCR was used to detect proinsulin (preproinsulin, PPI), glucose transporter 2 (glucose transporter 2, Glut2), mitochondrial anion carrier uncoupling protein 2 In order to investigate the effect of S-Eq on the insulin secretion of INS-1 cells, the activity of PKA and PP2A was detected by PKA and PP2A kits respectively. Meanwhile, the expression of Txnip protein was detected by the transfection of Si RNA and Western blot, and the expression of Txnip protein, combined with the double luciferase reporter gene and other techniques, was deeply studied. The main experimental results and conclusions of this experiment are as follows: (1) S-Eq can significantly increase the activity of INS-1 cells and decrease apoptosis (P0.05). (2) S-Eq can significantly increase the level of PPI mRNA in INS-1 cells under high glucose culture, and improve the GSIS function of INS-1 cells under high glucose conditions. At the same time, the transcriptional expression of Glut2 was significantly increased after high glucose treatment, and the transcriptional expression level of UCP2 (P0.05) was reduced. (3) S-Eq significantly reduced the PP2A activity in the INS-1 cells of high glucose, increased the PKA activity and inhibited the ChREBP protein expression in INS-1 cells (P0.05). (4) S-Eq can significantly reduce the recruitment of ChREBP in the cis acting element. The transcriptional activity of Txnip promoter in INS-1 cells after transfection of BP (WT), and then the expression of Txnip was inhibited. The transcriptional activity of Txnip promoter in INS-1 cells was significantly reduced after transfection of ChREBP (DM) plasmid. The ChREBP and expression were significantly reduced after the siRNA silent ChREBP gene was expressed. The expression of P has significant inhibitory effect (P0.05). Conclusion: the full text conclusion: S-Eq may inhibit the Txnip signaling pathway by regulating the activity of ChREBP, thus reducing the apoptosis of INS-1 cells under high glucose conditions, enhancing the expression of Glut2 and reducing the transcriptional expression of UCP2, and then improving the function of INS-1 cells and increasing the secretion of insulin.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.1

【参考文献】