电针干预胰岛素抵抗状态下AD模型大鼠的实验研究

发布时间：2018-05-18 03:42

本文选题：针刺 + 胰岛素抵抗　；参考：《广州中医药大学》2017年博士论文

【摘要】：本研究是以PI3K胰岛素信号转导途径为基础,设计实验观察针刺对胰岛素抵抗(IR)的干预作用;以自发性胰岛素抵抗模型-OLETF大鼠为动物模型,用R灯-PCR、Western blotting等检测方法,检测指标空腹血糖(FPG)、血浆胰岛素(FINS)、C肽、脑脊液胰岛素,以观察针刺干预对OLETF大鼠海马中IRS-2、PI3K、Akt、GSK的mRNA和蛋白表达的改变,以及大鼠海马中Aβ的沉淀以及Tau蛋白mRNA改变的情况,由分子生物学水平探讨针刺改善胰岛素抵抗的机制原理。目的:以自发性胰岛素抵抗模型OLETF大鼠为研究对象,在此动物模型基础上建立AD模型,观察针刺(穴选百会、肾俞、内关、足三里、三阴交)对AD模型大鼠中枢胰岛素抵抗状态的影响,初步探讨针刺对AD模型大鼠中枢胰岛素信号转导PI3K通路各组成蛋白(IRS-2,PI3K,Akt,IDE,GSK-3α/β,Aββ,Tau)表达的作用,从中枢胰岛素信号转导通路的角度阐释针灸治疗AD的机理。方法:选用自发性胰岛素抵抗OLETF大鼠和同品系LETO大鼠,以OLETF大鼠为基础建立AD模型。造模方法采用腹腔注射D-半乳糖和灌胃A1C13溶液的方法建立AD 模型。D-半乳糖采用 60mg · kg-1 · d-1,A1C13 采用 50mg · kg-1 · day-1,按照1Oml/kg溶液体积配置溶液。连续造模60天,造模完成后用Morris水迷宫进行行为学测试,剔除不合格模型。以LETO大鼠为正常组,将造模成功的OLETF大鼠随机分为模型组和针刺组,每组8只。针刺组给予针刺干预,穴选百会、肾俞(双)、内关(双)、足三里(双)、三阴交(双),连续治疗三周。治疗结束后,检测所有大鼠空腹血糖(FPG)、血浆胰岛素(FINS)、血清C-肽(C-P),和海马中的IRS-2、PI3K-p85α、Akt2、IDE、GSK-3α/β,以及海马中的 Aβ 含量、tau 蛋白。结果:针刺干预的EA-AD-OLETF组大鼠的FPG、FINS、C-P较模型AD-OLETF组显著降低(P0.01)。Morris水迷宫实验中,模型AD-OLETF组较正常LETO组穿越原平台象限时间占总时间百分比减小(P0.01);针刺EA-AD-OLETF组较模型组则显增大(P0.05)。海马中的 IRS-2、PI3K-p85α、Akt2、IDE、GSK-3α/β 针刺作用后,其mRNA变化不明显,但是其相关的p-IRS-2、IDE、p-GSK-3 β蛋白在干预后有了明显的提升(P0.01,P0.05),p-PI3K-p85α、p-Akt2 蛋白较干预前有所降低(P0.05)。在针刺干预后,针刺组EA-AD-OLETF组大鼠海马中Aβ沉淀细胞的个数较AD-OLETF组明显减少(P0.05)。针刺干预后的EA-AD-OLETF大鼠海马Tau mRNA表达比 AD-OLETF 升高(P0.05)。结论:从针刺干预胰岛素抵抗角度治疗AD,可以明显改善模型大鼠胰岛素抵抗状态,并且改善其认知行为,可以增加对A β的降解,改善A β在海马的沉积,抑制Tau蛋白表达。总结提出"针刺影响胰岛素IRS-PI3K-Akt-GSK信号转导途径可能是针刺干预胰岛素抵抗影响AD状态的关键环节之一",为临床运用针刺治疗胰岛素抵抗提供新的科学证据和理论基础。
[Abstract]:On the basis of PI3K insulin signal transduction pathway, we designed an experiment to observe the intervention effect of acupuncture on insulin resistance (IRR), and used spontaneous insulin resistance model (-OLETF) as animal model, using R-PCRX Western blotting, and so on. To observe the effect of acupuncture intervention on the expression of mRNA and protein in the hippocampus of OLETF rats, as well as the precipitation of A 尾 and the changes of Tau mRNA in hippocampus of OLETF rats. To explore the mechanism of acupuncture in improving insulin resistance from molecular biological level. Objective: to establish AD model on the basis of spontaneous insulin resistance (OLETF) model in rats and observe acupuncture (points Xanbaihui, Shenshu, Neiguan, Zusanli). The effect of acupuncture on the expression of GSK-3 伪 / 尾 A 尾 Tau3 in central insulin signal transduction PI3K pathway of AD model rats was studied in order to explore the effect of acupuncture on the expression of GSK-3 伪 / 尾 A 尾 Taug in the central insulin signal transduction pathway of AD rats, and the expression of GSK-3 伪 / 尾 A 尾 Taug in the central insulin signal transduction pathway of AD model rats. To explain the mechanism of acupuncture and moxibustion in treating AD from the angle of central insulin signal transduction pathway. Methods: the AD model of spontaneous insulin resistance (OLETF) rats and homologous LETO rats was established on the basis of OLETF rats. The AD model was established by intraperitoneal injection of D-galactose and intragastric administration of A1C13 solution. 60mg kg-1 d-1A1C13 was used for 50mg kg-1 day-1, and the solution was configured according to the volume of 1Oml/kg solution. After 60 consecutive days of modeling, Morris water labyrinth was used to conduct behavioral tests to eliminate the unqualified model. LETO rats were randomly divided into model group and acupuncture group with 8 rats in each group. Acupuncture group was given acupuncture intervention, point selection Baihui, Shenshu (Shuangzheng, Neiguan (Shuangzhu, Zusanli), Sanyinjiao (Shuangzhuo, Shuangjingjiao), continuous treatment for three weeks. At the end of treatment, fasting blood glucose (FPG), plasma insulin (FINSN), serum C- peptide (C- peptide) and PI3K-p85 伪 in hippocampus (IRS-2PI3K-p85 伪), GSK-3 伪 / 尾, and A 尾 content in hippocampus were measured in all rats. Results: compared with the model AD-OLETF group, the time of crossing the original platform quadrant in the model AD-OLETF group was significantly lower than that in the normal LETO group, and that in the acupuncture EA-AD-OLETF group was significantly higher than that in the model group. Results: compared with the model AD-OLETF group, the percentage of the total time of crossing the original platform quadrant in the model AD-OLETF group was significantly lower than that in the model AD-OLETF group, and that in the acupuncture EA-AD-OLETF group was significantly higher than that in the model group. The mRNA of IRS-2PI3K-p85 伪 and Akt2I3K-p85 伪 after acupuncture did not change significantly, but the related protein of p-IRS-2IDEP-GSK-3 尾 increased significantly after intervention, and the protein of p-PI3K-p85 伪 -p-Akt2 was lower than that of P0.05p85 伪 -PI3K-p85 伪. After acupuncture intervention, the number of A 尾 -precipitated cells in hippocampus of EA-AD-OLETF group was significantly lower than that of AD-OLETF group. The expression of Tau mRNA in hippocampus of EA-AD-OLETF rats after acupuncture intervention was higher than that of AD-OLETF. Conclusion: the treatment with acupuncture on insulin resistance can obviously improve the insulin resistance state and cognitive behavior of the model rats, increase the degradation of A 尾, improve the deposition of A 尾 in the hippocampus and inhibit the expression of Tau protein. It is concluded that "acupuncture affecting insulin IRS-PI3K-Akt-GSK signal transduction pathway may be one of the key links of acupuncture intervention in insulin resistance affecting AD state", which provides a new scientific evidence and theoretical basis for clinical treatment of insulin resistance.
【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R245;R-332

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