Neu-P11通过抑制氧化应激改善脂肪细胞胰岛素抵抗

发布时间：2018-03-13 08:14

本文选题：Neu-P11　切入点：褪黑素　出处：《南华大学》2015年硕士论文　论文类型：学位论文

【摘要】：研究背景褪黑素(melatonin,Mel)是脑松果腺合成和分泌的一种吲哚类神经内分泌活性物质,具有调节昼夜节律、改善睡眠等功能。Neu-P11则是一种新型褪黑素非选择性受体激动剂,我们前期的研究表明Neu-P11可调节高糖高脂高胆固醇诱导的肥胖大鼠糖脂代谢并改善其胰岛素抵抗(insulin resistance,IR)状态,体外细胞试验也证实Neu-P11能够抑制甘油三酯聚积改善3T3-L1脂肪细胞IR。有研究报道,细胞内活性氧(reactive oxygen species,ROS)水平过高造成的氧化应激和线粒体功能障碍是导致胰岛素抵抗的主要因素。基于Neu-P11、氧化应激以及IR之间的联系,本课题在前期实验的基础上提出假想:Neu-P11是否通过抑制氧化应激而改善高糖高胰岛素(high glucose and high insulin,HGI)诱导的3T3-L1脂肪细胞IR。方法(1)3T3-L1脂肪细胞的诱导及分化:经典“鸡尾酒”法(0.5mmol/L IBMX,1μmol/L DEX,10μg/ml INS)诱导前脂肪细胞分化为成熟3T3-L1脂肪细胞,油红O染色鉴定脂肪细胞形态及3T3-L1前脂肪细胞的分化程度。(2)IR模型的制备:高糖(25 mmol/L)高胰岛素(1μmol/L INS)孵育脂肪细胞24 h,葡萄糖氧化酶法检测糖消耗量验证胰岛素抵抗细胞模型构建是否成功。(3)荧光探针法、罗丹明123及葡萄糖氧化酶法分别检测Neu-P11或Mel干预前后各组脂肪细胞中ROS含量、线粒体膜电位(mitochondrial transmembrane potential,MMP)变化及培养基内葡萄糖含量。(4)Western Blot检测细胞c-jun氨基末端激酶(Jun N-terminal kinase,JNK)、核转录蛋白p65(Nuclear factor kappa B p65,NF-κBp65)、蛋白激酶B(Protein kinase B,Akt)的表达变化及Neu-P11或Mel干预对上述蛋白表达的影响。结果3T3-L1前脂肪细胞经“鸡尾酒”法诱导10 d后通过油红O染色可见90%以上细胞呈现明显的“戒环样”结构:细胞呈圆形,内含大量脂滴,且呈环形排列,即表明已分化为典型的成熟脂肪细胞。实验结果显示:高糖高胰岛素联合作用3T3-L1脂肪细胞24 h能明显降低细胞的葡萄糖消耗量,成功诱导其发生胰岛素抵抗。另外,实验结果还表明:与Control组相比,IR组细胞ROS含量明显增高,MMP下降,细胞内JNK/NF-κBp65蛋白表达增加,胰岛素信号蛋白Akt表达下降。而经Neu-P11处理可以明显逆转高糖高胰岛素引起的生理效应,差异均具有统计学意义,且与Mel组作用相似。结论Neu-P11可以改善HGI诱导的3T3-L1脂肪细胞IR状态,其机制可能与Neu-P11清除ROS而改善线粒体功能、抑制JNK/NF-κBp65蛋白的活性有关。
[Abstract]:Background melatonin melatonin (melatonin) is an indole neuroendocrine active substance synthesized and secreted by the pineal gland of the brain. Neu-P11 is a novel melatonin nonselective receptor agonist with the functions of regulating circadian rhythm and improving sleep. Our previous studies have shown that Neu-P11 can regulate glucose and lipid metabolism and improve insulin resistance and insulin resistance in obese rats induced by high glucose, high fat and high cholesterol. In vitro cell tests also confirmed that Neu-P11 could inhibit the accumulation of triglycerides and improve the IR3 of 3T3-L1 adipocytes. Oxidative stress and mitochondrial dysfunction caused by excessive levels of reactive oxygen in cells are the main factors leading to insulin resistance, based on the relationship between Neu-P11, oxidative stress and IR. On the basis of previous experiments, this study proposed a hypothetical hypothesis that whether or not: Neu-P11 can improve the induction and differentiation of 3T3-L1 adipocytes induced by high glucose and high glucose and high insulin I by inhibiting oxidative stress. Methods the induction and differentiation of 3T3-L1 adipocytes: classic cocktail. "0.5 mmol / L IBM X1 渭 mol/L DEXX 10 渭 g / ml ins) induced adipocytes to differentiate into mature 3T3-L1 adipocytes. Identification of adipocyte morphology by oil red O staining and differentiation of 3T3-L1 preadipocytes. Preparation of IR model: hyperglycemic 25 mmol / L) hyperinsulinemia 1 渭 mol/L ins) incubated adipocytes for 24 h. Glucose oxidase assay was used to detect glucose consumption to verify insulin resistance. Whether the cell model was successfully constructed or not, the fluorescence probe method was used. Rhodamine 123 and glucose oxidase method were used to detect ROS content in adipocytes before and after Neu-P11 or Mel intervention. Changes of Mitochondrial transmembrane potentialMMPs and glucose content in the medium. Western Blot to detect the expression of c-jun amino-terminal kinases Jun N-terminal kinases, nuclear factor kappa Bp65 protein NF- 魏 Bp65, protein kinase B protein in kinase Bnktand Neu-P11 or Mel intervention on these proteins. Results the 3T3-L1 preadipocytes were induced by "cocktail" method for 10 days. Through oil red O staining, it was found that more than 90% cells showed "ring ring" structure: the cells were round. The results showed that 3T3-L1 adipocytes treated with high glucose and high insulin for 24 h could significantly reduce the glucose consumption of 3T3-L1 adipocytes. In addition, the results also showed that compared with Control group, the content of ROS increased significantly and the expression of JNK- / NF- 魏 Bp65 protein increased in IR group. The expression of insulin signal protein Akt was decreased, and the physiological effects induced by high glucose and high insulin could be reversed by Neu-P11 treatment, and the difference was statistically significant. Conclusion Neu-P11 can improve the IR state of 3T3-L1 adipocytes induced by HGI, and its mechanism may be related to Neu-P11 scavenging ROS and improving mitochondrial function and inhibiting the activity of Neu-P11 / NF- 魏 Bp65 protein.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.1

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