794ap（昔美酸沙美特罗）改善胰岛素抵抗机制的初步研究

发布时间：2018-03-14 09:15

本文选题：胰岛素抵抗　切入点：794ap　出处：《重庆理工大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景Ⅱ型糖尿病(T2DM)是一种复杂的内分泌代谢紊乱性疾病,已成为继心血管疾病、恶性肿瘤之后第三大严重威胁人类健康的非传染性疾病。而胰岛素抵抗是Ⅱ型糖尿病的发病基础,并贯穿于Ⅱ型糖尿病的整个病理过程中。过氧化物酶体增殖物激活受体(Peroxisome proliferators activated receptors,PPARs)是人类代谢相关疾病防治药物的重要靶点,属于非甾体类核受体超家族。其中PPARγ是研究胰岛素抵抗的关键靶点之一,参与众多的复杂的信号通路进而发挥其生物学效应,比如PPARγ与PI3K-AKT激酶信号通路,PPARγ在PI3K信号通路中可以促进AKT的表达,增强胰岛素敏感性,并促进Glu T4基因的表达,促进对葡萄糖的摄取;PPARγ与ADIPOQ,脂联素可以增加胰岛素敏感性,具有抗炎保护内皮等功能,是PPARγ激动剂作用的新靶点,能刺激AMPK磷酸化,许多效应是通过AMPK途径实现。PPARγ激动剂主要是能通过作用于脂肪组织,促进葡萄糖向脂肪组织转运、促进脂肪细胞的分化等环节,实现其胰岛素增敏从而降低血糖的药理作用。大量文献表明高抑制CDK5介导的p-PPARγSer273的配体化合物是最佳的候选抗糖尿病药物。前期研究发现794ap(昔美酸沙美特罗)能降低Ⅱ型糖尿病SD大鼠的血糖,后进行分子动力学研究发现其与PPARγ受体结合度比较高。因此本文通过建立胰岛素抵抗细胞模型并结合Western Blot、IHC、ICC和q RT-PCR实验方法对794ap降糖并改善胰岛素抵抗的作用机制进行初步的探究。目的初步探讨794ap改善胰岛素抵抗及降糖的作用机制。方法运用分子对接技术找出794ap与PPARγ的蛋白晶体结合的最佳构象,采用分子动力学模拟PPARγ受体激动剂794ap与PPARγ受体的结合模式,用Amber做动力学研究并使用MM-PBSA法计算其结合自由能。探索胰岛素诱导的最佳浓度最佳浓度和时间,建立Hep G2胰岛素抵抗模型。并用MTT法检测794ap作用于Hep G2胰岛素抵抗模型后,计算细胞的存活率并选择合适的药物浓度梯度并结合文献报道作为接下来实验的药物浓度;将794ap作用于Hep G2胰岛素抵抗模型并做相应的葡萄糖检测试验,以反应药物的效果;Western blot检测蛋白含量,以罗格列酮作为阳性药物,观察794ap对PPARγ、AMPKα1、AKT2、ADIPOQ、JAK2的影响;免疫细胞化学(ICC)和免疫组化组织(IHC)水平研究PPARγ等各蛋白在胰岛素抵抗的Hep G2细胞及大鼠肝脏和胰腺组织中宏观的表达;q RT-PCR方法检测PPARγ、ADIPOQ、AMPKα1 m RNA表达。结果794ap与受体2XKW和4EMA的复合物在进行MD 30ns后都达到动态平衡,形成稳定的复合物结构且794ap与受体2XKW结合的效果最好,范德华力在结合过程中起了关键作用。成功建立Hep G2细胞胰岛素抵抗模型,且WB实验显示794ap能抑制p-PPARγSer273的表达,上调p-PPARγSer112、AMPKα1、p-AMPKα1、ADIPOQ、p-AKT2的表达,但794ap并不上调JAK2、p-JAK2、ERK1/2和p-ERK1/2蛋白的表达;加入抑制剂T0070907后,794ap对PPARγ等蛋白表达的影响依旧不变;794ap对SD大鼠肝脏、胰腺组织和IR-Hep G2细胞PPARγ等蛋白的影响与WB结果一致;794ap上调ADIPOQ和AMPKα1的m RNA表达,但并不增加PPARγm RNA表达水平。结论经过实验室前期的实验研究证明794ap(昔美酸沙美特罗)能降低Ⅱ型糖尿病SD大鼠的血糖,改善胰岛素抵抗。分子动力学模拟结果验证了794ap与PPARγ的受体结合紧密,理论上证明是PPARγ的激动剂,且Western blot、IHC、ICC和q RT-PCR结果显示794ap可以通过抑制p-PPARγSer273,上调AMPKα1、AKT2和ADIPOQ的表达来改善IR-SD大鼠和IR-Hep G2细胞的胰岛素抵抗,增加其对胰岛素的敏感性,从而产生降糖的作用。
[Abstract]:On the background of type II diabetes mellitus (T2DM) is a kind of complicated endocrine metabolic disorders, cardiovascular disease has become the third largest after malignant tumor, a serious threat to human health and non communicable disease. Insulin resistance is the basis of type II diabetes mellitus, and throughout the whole pathological process of type II diabetes mellitus. Peroxisome proliferation activated receptor (Peroxisome proliferators activated receptors, PPARs) is an important target for drug prevention and treatment of diseases related to human metabolism, which belongs to non steroidal nuclear receptor superfamily. PPAR gamma is one of the key target in the research of insulin resistance in complex, numerous signaling pathways and its biological effects, such as PPAR gamma and PI3K-AKT kinase signal pathway, PPAR gamma could promote the expression of AKT in the PI3K signaling pathway, enhance insulin sensitivity, and the expression of Glu T4 gene, promote In the uptake of glucose; PPAR gamma and ADIPOQ, adiponectin can increase insulin sensitivity, has anti-inflammatory and protect endothelial function, is a new target for PPAR gamma agonist, can stimulate the phosphorylation of AMPK, many effect is through the AMPK pathway.PPAR gamma agonist is mainly through effects on adipose tissue, promote glucose to promote the differentiation of adipose tissue transfer, and other aspects of fat cells, the insulin sensitizing and reduce blood glucose. The pharmacological effects of a large number of literatures show that compounds p-PPAR gamma Ser273 mediated suppression of CDK5 is the best candidate for anti diabetes drugs. Previous study found that 794ap (with beauty acid Shah Mette Lo) can reduce type II diabetes SD the blood sugar, after the study found that the molecular dynamics and PPAR gamma receptor binding is very high. So through the establishment of a cell model of insulin resistance and the combination of Western Blot, IHC, IC Preliminary study of mechanism of C and Q RT-PCR experimental method of 794ap glucose and improving insulin resistance. Objective to investigate the effects of 794ap on the insulin resistance and the hypoglycemic mechanism. The best conformation of protein crystal method using molecular docking technique to identify 794ap and PPAR gamma binding, binding mode using the molecular dynamics simulation of PPAR gamma receptor agonist 794ap with the PPAR gamma receptor, and calculate the binding energy using MM-PBSA method with Amber dynamics. To explore the best concentration of insulin induced the optimal concentration and time, the establishment of Hep G2 model of insulin resistance. And MTT was used to detect the effect of 794ap on the Hep G2 model of insulin resistance, calculate the cell survival rate and drug concentration gradient the right and the literatures as the next experimental drug concentration; 794ap in Hep G2 model of insulin resistance and the corresponding grape Sugar test, in response to the effects of drugs; protein content detection of Western blot, taking rosiglitazone as positive drug, the effect of 794ap on PPAR AMPK gamma, alpha 1, AKT2, ADIPOQ, JAK2; Immunocytochemistry (ICC) and immunohistochemistry (IHC) to study the expression of PPAR protein in insulin and other gamma G2 resistant Hep cells and rat liver and pancreas in macroscopic; RT-PCR method for detection of PPAR Q ADIPOQ, AMPK gamma, alpha 1 m RNA. Results the expression of 794ap complex and 2XKW receptor and 4EMA in MD 30ns after they reach a dynamic balance, the formation of a stable complex structure and the 794ap and 2XKW receptor binding the best, van Edward force plays a key role in the binding process. Establish insulin resistance cell model Hep G2, and WB test showed that 794ap could decrease the expression of p-PPAR gamma Ser273, upregulation of p-PPAR gamma Ser112, AMPK alpha 1, alpha 1, p-AMPK, ADIPOQ, p-AKT2 expression, but 7 94ap is not the upregulation of JAK2, p-JAK2, ERK1/2 and p-ERK1/2 protein expression; adding inhibitor T0070907, effects of 794ap on the expression of PPAR gamma protein remains unchanged; the 794ap of SD rat liver, pancreas and IR-Hep effect of G2 cells PPAR protein gamma etc. The results are consistent with the WB; 794ap AMPK m RNA upregulated ADIPOQ and alpha 1 the expression of PPAR, but did not increase the expression level of RNA gamma m. Conclusion through laboratory experiment proved that 794ap (early in beauty acid Shah Mette Lo) can reduce type II diabetes SD rats blood glucose, improve insulin resistance. The molecular dynamics simulation results prove the combination of 794ap and PPAR gamma receptor theory that is PPAR gamma agonist, Western and blot, IHC, ICC and Q RT-PCR results showed that 794ap can inhibit p-PPAR gamma Ser273, upregulation of AMPK alpha 1, expression of AKT2 and ADIPOQ to improve IR-SD rats and IR-Hep G2 cell insulin resistance, increase Its sensitivity to insulin, resulting in a hypoglycemic effect.

【学位授予单位】：重庆理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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