不同脂肪酸对INS-1细胞TXNIP表达的影响及其机制

发布时间：2018-06-28 15:59

本文选题：游离脂肪酸 + 软脂酸　；参考：《山西医科大学》2017年硕士论文

【摘要】：研究背景:糖尿病的患病率逐年上升,目前已成为严重威胁人类健康的疾病之一,胰岛β细胞凋亡增加是糖尿病发生、发展的关键因素[1]。近年来有研究表明,2型糖尿病患者体内长期高水平的游离脂肪酸(free fatty acids,FFAs),特别是饱和脂肪酸可以引起胰岛素抵抗、胰岛β细胞功能紊乱,以及β细胞凋亡的增加,进而加重糖尿病[2-4]。硫氧还蛋白相互作用蛋白(Thioredoxin interacting protein,TXNIP)又名VDUP1(Vitamin D(3)up-regulated protein 1)和TBP2(Thioredoxin-binding protein-2),属于α抑制蛋白家族,是目前发现的唯一内源性的硫氧还蛋白(Thioredoxin,Trx)结合抑制蛋白,在糖尿病各组织中其表达均显著升高,已有大量研究证明高糖可引起TXNIP表达明显升高,并介导β细胞功能损伤和凋亡[5-7]。但是,作为糖尿病主要损伤因素之一的游离脂肪酸,其对TXNIP表达的影响,以及不同饱和程度的游离脂肪酸对TXNIP表达的可能差别目前尚不清楚。因此本课题旨在研究不同饱和程度的脂肪酸对INS-1胰岛细胞TXNIP表达的影响,并分析其可能机制。目的:1.使用不同饱和程度的游离脂肪酸孵育INS-1细胞,观察不同类型脂肪酸对INS-1胰岛细胞凋亡及TXNIP表达的影响。2.分析脂肪酸调节TXNIP表达的可能机制。方法:1.实验分组:将正常培养的INS-1细胞分为四组:分别是正常对照组(含0.55%fatty acid free-BSA的RPMI1640培养基)、饱和脂肪酸-软脂酸组(0.5 mmol/L软脂酸+0.55%fatty acid free-BSA)、单不饱和脂肪酸-棕榈油酸组(0.5 mmol/L棕榈油酸+0.55%fatty acid free-BSA)、多不饱和脂肪酸-DHA 组(0.5 mmol/L DHA+0.55%fatty acid free-BSA),均于培养24 h后收集细胞进行指标测定。2.采用Real-time PCR方法测定FFAs处理后INS-1细胞TXNIP mRNA表达量。3.采用 Western blot方法检测FFAs处理后 INS-1 细胞 TXNIP、Cleaved caspase-3、Caspase-3、ChREBP、FOXO1、p-NF-KB 蛋白表达。4.采用免疫荧光技术检测FFAs处理后INS-1细胞中ChREBP蛋白表达。5.使用Annexin V-FITC/PI染色流式细胞术检测INS-1细胞凋亡情况。结果:1.不同饱和程度的FFAs对INS-1细胞TXNIP表达的影响不同。与对照组相比,饱和脂肪酸软脂酸组TXNIP mRNA和蛋白表达均显著上调,而不饱和脂肪酸棕榈油酸组和DHA组INS-1细胞TXNIP表达均没有明显的改变(见图1)。如图2所示,软脂酸引起的TXNIP的蛋白表达从18 h开始增加,并于24 h达到最高。36 h时,软脂酸诱导的TXNIP表达降低到原蛋白水平。而棕榈油酸和DHA在12 h、18 h、24 h、36 h不同的时间点对TXNIP表达与对照组相比均无统计学差异。2.FFAs由于其饱和程度的不同对INS-1细胞凋亡也具有不同的影响。如图3所示,软脂酸组Cleaved caspase-3/caspase-3的比值与对照组相比明显增加。棕榈油酸组和DHA组Cleaved caspase-3/caspase-3比值与对照组相比无显著性差异。流式细胞术结果显示,软脂酸组INS-1细胞的凋亡指数明显增加,而棕榈油酸组和DHA组对INS-1细胞凋亡的影响与对照组相比无统计学差异(见图4)。3.软脂酸对转录因子ChREBP和FOXO1蛋白表达的影响。使用Western blot和免疫荧光方法研究发现,与对照组相比软脂酸组ChREBP蛋白表达显著上调,转录因子FOXO1蛋白表达降低。(见图5-6)4.软脂酸调节INS-1细胞TXNIP蛋白表达与NF-κB磷酸化相关。与对照组相比,软脂酸组p-NF-κB p65蛋白表达量明显增加(见图7)。加入NF-κB抑制剂PDTC后,与不加抑制剂组相比,软脂酸孵育后引起的TXNIP mRNA和蛋白水平的上调受到明显的抑制(见图8)。同样,使用一种多肽类的NF-κB抑制剂SN50也可降低软脂酸诱导的TXNIP表达上调(见图9)。同时发现,PDTC能够显著降低软脂酸诱导的ChREBP表达上调(见图10),而对软脂酸下调FOXO1的表达没有影响(见图11)。5.p38 MAPK参与软脂酸诱导的TXNIP表达上调。与不加抑制剂组相比,使用p38 MAPK抑制剂SB203580可显著抑制软脂酸处理后引起的INS-1细胞TXNIP mRNA和蛋白水平的增加(见图12)。同时,SB203580也可阻断了软脂酸诱导的p-NF-κB p65蛋白表达上调(见图13),以及软脂酸对转录因子ChREBP和FOXO1蛋白表达的调节作用(见图14,15)。结论:1.饱和脂肪酸软脂酸可能通过诱导TXNIP表达上调,进而促进了 INS-1胰岛细胞凋亡。而不饱和脂肪酸棕榈油酸和DHA对TXNIP表达以及INS-1细胞凋亡均无显著作用。2.软脂酸上调TXNIP表达的机制与p38 MAPK/NF-κB/ChREBP信号通路有关,FOXO1可能也参与了 TXNIP的调节。
[Abstract]:Background: the prevalence of diabetes is increasing year by year, and now it has become one of the serious threats to human health. The increase of islet beta cell apoptosis is the occurrence of diabetes. The key factor in the development of diabetes [1]. in recent years has shown that the long-term high level of free fatty acids (FFAs), especially saturated fat, in patients with type 2 diabetes mellitus Fatty acids can cause insulin resistance, islet beta cell dysfunction, and the increase of beta cell apoptosis, and further aggravate the diabetes [2-4]. thioredoxin interaction protein (Thioredoxin interacting protein, TXNIP), also known as VDUP1 (Vitamin D (3) up-regulated protein 1) and TBP2 (Thioredoxin-binding), belonging to the alpha suppressor family. The only endogenous thioredoxin (Thioredoxin, Trx) binding inhibitor is found at present, and its expression is significantly increased in all diabetic tissues. A large number of studies have shown that high glucose can cause a significant increase in the expression of TXNIP and mediate the function damage and apoptosis of beta cells, but it is one of the major damage factors of diabetes. The effects of fatty acids on the expression of TXNIP and the possible difference in the expression of TXNIP with different saturation degrees of free fatty acids are still unclear. Therefore, the purpose of this study is to investigate the effect of fatty acids on the expression of TXNIP in INS-1 islet cells at different saturation levels and to analyze its possible mechanism. Objective: 1. the use of free fat with different saturation degrees is used. INS-1 cells were incubated with fatty acids to observe the effects of different types of fatty acids on the apoptosis of INS-1 islet cells and the expression of TXNIP..2. analysis of the possible mechanism of TXNIP expression by fatty acids. Methods: 1. experimental groups: the normal cultured INS-1 cells were divided into four groups: the normal control group (RPMI1640 culture containing 0.55%fatty acid free-BSA), saturated fat, respectively. Fatty acid and palmitic acid group (0.5 mmol/L +0.55%fatty acid free-BSA), monounsaturated fatty acid palmitic acid group (0.5 mmol/L palmitic oleic acid +0.55%fatty acid free-BSA), polyunsaturated fatty acid -DHA group (0.5 mmol/L DHA+0.55%fatty acid) The expression of TXNIP mRNA in INS-1 cells after FFAs treatment was measured by Western blot method to detect INS-1 cell TXNIP, Cleaved caspase-3. Cell apoptosis was used to detect the apoptosis of INS-1 cells. Results: 1. the effects of FFAs on the expression of TXNIP in INS-1 cells were different. Compared with the control group, the expression of TXNIP mRNA and protein in the saturated fatty acid palmitic acid group were significantly up, while the TXNIP expression of the unsaturated fatty acid palmitoleic acid group and the DHA group had no significant changes (see Figure 1). As shown in Figure 2, the protein expression of TXNIP caused by palmitic acid increased from 18 h, and the TXNIP expression induced by palmitic acid decreased to the original protein level when 24 h reached the highest.36 H. While palmitic acid and DHA were at 12 h, 18 h, 24 h, 36 h, there was no significant difference between the expression of TXNIP and the control group because of its saturation degree The difference in the apoptosis of INS-1 cells was also different. As shown in Figure 3, the ratio of Cleaved caspase-3/caspase-3 in the palmitic acid group was significantly increased compared with the control group. The ratio of Cleaved caspase-3/caspase-3 in the palmitic and DHA groups was not significantly different from that of the control group. The flow cytometry results showed that the INS-1 cell in the palmitic acid group was withered. The death index increased significantly, while the effect of palmitic acid group and DHA group on INS-1 cell apoptosis was not significantly different from that of the control group (see Figure 4) the effect of.3. palmitic acid on the expression of ChREBP and FOXO1 protein. The use of Western blot and immunofluorescence methods found that the expression of ChREBP protein in the palmitic acid group was significantly up-regulated compared with the control group. The expression of transcription factor FOXO1 protein decreased. (see Figure 5-6) 4. palmitic acid regulated INS-1 cell TXNIP protein expression was associated with NF- kappa B phosphorylation. Compared with the control group, the expression of p-NF- kappa B p65 protein in the palmitic acid group increased significantly (see Figure 7). After adding NF- kappa B inhibitor PDTC, the TXNIP and egg caused by the incubation of palmitic acid compared with the non inhibitor group. The upregulation of white level was significantly inhibited (see Figure 8). Similarly, the use of a peptide NF- kappa B inhibitor SN50 could also reduce the up-regulated TXNIP expression induced by palmitic acid (see Figure 9). At the same time, it was found that PDTC could significantly reduce the up-regulated ChREBP expression induced by palmitic acid (see Figure 10), but no effect on the expression of palmitic acid down FOXO1 (see Figure 11).5.p 38 MAPK was involved in the up-regulated TXNIP expression induced by palmitic acid. Compared with the non inhibitor group, the use of p38 MAPK inhibitor SB203580 significantly inhibited the increase of TXNIP mRNA and protein levels in INS-1 cells induced by palmitic acid (see Figure 12). At the same time, SB203580 also blocked the up regulation of p-NF- kappa B p65 protein expression induced by palmitic acid (see Figure 13). And the regulation of palmitic acid on the expression of transcription factor ChREBP and FOXO1 protein (see Figure 14,15). Conclusion: 1. saturated fatty acid palmitic acid may increase the expression of TXNIP by inducing TXNIP expression, and thus promote the apoptosis of INS-1 islet cells, while unsaturated fatty acid palmitoleic acid and DHA have no significant effect on TXNIP expression and INS-1 cell apoptosis. The mechanism of upregulation of TXNIP expression is related to p38 MAPK/NF- kappa B/ChREBP signaling pathway. FOXO1 may also play a role in TXNIP regulation.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1

【参考文献】