趋化因子受体CCR7介导高脂饮食小鼠下丘脑胰岛素抵抗的机制研究

发布时间：2019-06-04 22:15

【摘要】：慢性炎症反应介导的胰岛素抵抗是肥胖发展为二型糖尿病(type 2 diabetes,T2DM)的主要原因,因此,肥胖被认为是诱发T2DM的重要风险因子之一。大量临床资料显示,当人体摄入的能量远远超过新陈代谢所需要的能量时,会造成机体脂肪堆积,使机体内能量代谢平衡失调,导致肥胖。已有研究表明,肥胖组织含有大量的脂肪细胞,这些细胞含有的游离脂肪酸(free fatty acids,FFA)会促进大量炎性因子产生和释放,长期作用就形成了机体的炎性环境,下丘脑(hypothalamus)是机体的饮食调节中枢,也是代谢调控中心,研究表明,下丘脑炎症反应可以降低机体对胰岛素和瘦素的敏感性,导致胰岛素和瘦素抵抗,打破机体对能量摄入的调控平衡,最终诱发T2DM。因此,炎症反应在下丘脑胰岛素抵抗(Insulin resistance,IR)和瘦素抵抗(Leptin resistance,LR)中发挥重要作用。趋化因子(Chemokines)是一类能够促进白细胞迁移到感染部位的小分子量蛋白质,它通过与其受体的特异性结合,促进各类炎症因子的释放,在机体炎症反应中发挥重要作用。我们的前期研究表明,趋化因子受体CCR7基因在下丘脑中高表达,这一结果提示CCR7可能参与下丘脑IR的病理生理学发生过程,因此,本研究着重探索CCR7在高脂饮食诱导的T2DM小鼠下丘脑IR的分子机制。实验选取6周龄雄性ICR小鼠,随机分为两组,一组饲喂含11.4%脂肪的普通饲料,设为对照组(Ctrl组);另一组饲喂含60%脂肪的高脂饲料(HFD组),设为模型组;两组小鼠分别处理16周,期间每周固定时间测量并记录各组小鼠的体重、血糖及摄食量,第16周对两组小鼠进行葡萄糖耐受(OGTT)与胰岛素耐受(ITT)评价,结果显示HFD组小鼠的c均显著高于Ctrl组(P0.001),且HFD组小鼠产生了葡萄糖不耐受和胰岛素抵抗。综合分析实验数据后,评估T2DM模型小鼠的构建,于第17周,将上述Ctrl组和HFD组两组小鼠进一步分为四组,总体分组如下:Ctrl Scramble sgRNA组、HFD Scramble sgRNA组、HFD CCR7sgRNA组、Ctrl CCR7 sgRNA组,利用CRISPR-Cas9基因编辑技术敲除小鼠下丘脑CCR7基因(利用脑立体定位仪分别向下丘脑双侧基底部VMH区域,前囟点后1.58mm,中间线外侧0.3mm,颅骨表面下方5.68mm处注射慢病毒包装的CCR7sgRNA颗粒(病毒滴度为1x106TU/ml),Scramble组注射空载慢病毒颗粒);术后动物处理与术前相同,继续每周测量并记录各组小鼠的体重、血糖及摄食量,待各项生理指标稳定后,即脑立体定位注射手术5周左右,利用蛋白免疫印迹技术(western blotting technology,WB)和免疫荧光组织化学技术(immunofluorescence histochemistry technology,IF)对四组小鼠下丘脑中CCR7蛋白表达量进行测定,评价CCR7基因在相应处理组小鼠下丘脑中的敲除情况。各组小鼠生理生化指标检测结果显示:与Ctrl Scramble sgRNA组相比,HFD Scramble sgRNA组小鼠的体重和血糖水平显著提高(P0.001);与HFD Scramble sgRNA组相比,HFD CCR7 sgRNA组小鼠体重和血糖水平明显下降,并存在显著性差异(P0.001),而与Ctrl Scramble sgRNA组相比,Ctrl CCR7 sgRNA组小鼠的体重和血糖水平无显著性差异;这表明HFD CCR7 sgRNA组小鼠的葡萄糖不耐受与胰岛素抵抗得到改善。此外,各组小鼠在实验各阶段摄食量稳定且无显著性组别差异。进一步利用WB和IF的方法检测不同处理组小鼠下丘脑中NLRP3炎症小体介导的炎症信号通路关键蛋白,如NLRP3,ASC,Caspase 1,IL-1β等,及胰岛素信号通路相关蛋白IRS-1和Akt的表达变化情况,结果显示:HFD组小鼠下丘脑中CCR7蛋白表达显著高于Ctrl组,同时NLRP3,ASC,Caspase-1,IL-1β,等蛋白表达量也显著增多,胰岛素受体底物1(IRS-1)磷酸化增强;特异性敲除CCR7基因后,HFD CCR7 sgRNA组与HFD Scramble sgRNA组相比,以上炎症通路蛋白表达量均显著下降(P0.001),IRS-1蛋白磷酸化程度降低;而Ctrl CCR7sgRNA组与Ctrl Scramble sgRNA组相比,炎症通路相关蛋白表达量及IRS-1蛋白酸化情况均没有明显变化,IF检测结果与WB检测结果一致。综上所述,CCR7介导高脂饮食小鼠下丘脑中NLRP3炎症小体的活化,激活Caspase-1蛋白,促进IL-1β和TNF-α等炎性因子的成熟和释放,放大下丘脑炎症反应,负调节下丘脑胰岛素信号和瘦素信号,最终导致IR和LR,这些结果提示CCR7是治疗T2DM下丘脑IR的关键候选靶点。
[Abstract]:Chronic inflammatory response-mediated insulin resistance is a major cause of obesity development for type 2 diabetes mellitus (T2DM), and therefore, obesity is considered one of the key risk factors for inducing T2DM. A lot of clinical data show that when the energy consumed by the human body far exceeds the energy required by the metabolism, the body fat accumulation can be caused, the energy metabolism in the body is balanced, and the obesity is caused. The research has shown that the obesity tissue contains a large amount of fat cells, free fatty acids (FFA) contained in these cells can promote the production and release of a large number of inflammatory factors, and the long-term effect forms an inflammatory environment of the body, and the hypothalamus (hythaloamus) is the food regulation center of the body. It is also the metabolic control center. The research shows that the hypothalamic inflammation reaction can reduce the sensitivity of the body to the insulin and the leptin, lead to the resistance of the insulin and the leptin, break the regulation and balance of the body to the energy intake, and finally induce the T2DM. Thus, the inflammatory response plays an important role in the hypothalamic insulin resistance (IR) and leptin resistance (LR). Chemokines are a class of small-molecular-weight proteins that can promote the migration of leukocytes to the site of infection, which can promote the release of various inflammatory factors through the specific binding with their receptors, and play an important role in the inflammation reaction of the body. Our early studies have shown that the chemokine receptor CCR7 gene is highly expressed in the hypothalamus, which suggests that CCR7 may be involved in the pathophysiology of the hypothalamic IR. Therefore, this study focuses on the molecular mechanism of the hypothalamic IR in the T2DM mouse induced by high-fat diet. The 6-week-old male ICR mice were randomly divided into two groups. One group was fed with a common feed containing 11.4% fat, which was set as the control group (Ctrl group), and the other group was fed with a high-fat diet (HFD group) containing 60% of fat, which was set as the model group, and the two groups were treated for 16 weeks, respectively. The body weight, blood glucose and food consumption of each group of mice were measured and recorded during the weekly fixed time, and the glucose tolerance (OGTT) and the insulin resistance (ITT) evaluation were performed on the two groups at Week 16, and the results showed that the c of the mice in the HFD group was significantly higher than that in the control group (P 0.001). And the HFD group mice produced glucose intolerance and insulin resistance. After comprehensive analysis of the experimental data, the construction of the model mice with T2DM was evaluated. In the first week, two groups of mice in the control group and the HFD group were further divided into four groups. The overall group was as follows: Ctrl Sramble sgRNA group, HFD Sramble sgRNA group, HFD CCR7sgRNA group, and Ctrl CCR7 sgRNA group. The CCR7 gene (virus titer of 1 x 106TU/ ml) was injected at 5.68 mm below the skull surface by using the CRISPR-Cas9 gene editing technique to knock out the CCR7 gene of the hypothalamus of the mouse (using the brain stereolocator, respectively, to the VMH region at the bottom of the bilateral basal part of the hypothalamus, 1.58 mm after the anterior and the outer side of the middle line, and 5.68 mm below the surface of the skull. The weight, blood sugar and food consumption of each group of mice were measured and recorded on a weekly basis, and the weight, blood sugar and food consumption of each group of mice were measured and recorded on a weekly basis. After the physiological indexes were stable, the brain three-dimensional positioning and injection operation was performed for about 5 weeks, and western blotting technology was used. WB and IF were used to measure the expression of CCR7 in the hypothalamus of four groups of mice, and the knockout of CCR7 gene in the hypothalamus of the corresponding treatment group was evaluated. The results showed that the body weight and blood glucose level of the mice in the HFD Sramble sgRNA group were significantly higher than that of the control Sramble sgRNA group (P 0.001), and the body weight and blood glucose level of the HFD CCR7 sgRNA group decreased significantly compared with that of the HFD Sramble sgRNA group (P 0.001). There was no significant difference in body weight and blood glucose levels in the control CCR7 sgRNA group compared to the Ctrl Sramble sgRNA group; this indicated that the glucose intolerance and insulin resistance of the mice in the HFD CCR7 sgRNA group were improved. In addition, that food consumption in each group was stable and there was no significant group difference in each stage of the experiment. The expression of NLRP3, ASC, Caspase-1, IL-1 and the expression of IRS-1 and Akt in the hypothalamus of mice with different treatment groups, such as NLRP3, ASC, Caspase-1, IL-1, and the expression of IRS-1 and Akt, were detected by WB and IF. The results showed that: The expression of CCR7 protein in the hypothalamus of the HFD group was significantly higher than that of the control group, while the expression of the protein, such as NLRP3, ASC, Caspase-1, IL-1, and the like, increased significantly, and the phosphorylation of the insulin receptor substrate 1 (IRS-1) was enhanced. After the specific knockout of the CCR7 gene, the HFD CCR7 sgRNA group compared with the HFD Sramble sgRNA group. The expression of the above-mentioned inflammatory pathway protein decreased significantly (P0.01), and the phosphorylation of the IRS-1 protein was decreased; while the expression of the inflammation pathway-related protein and the expression of the IRS-1 protein did not change significantly compared with the control Sramble sgRNA group, and the results of the IF detection were consistent with the WB detection results. In conclusion, CCR7 mediates the activation of an NLRP3 inflammatory body in the hypothalamus of a high-fat diet mouse, activates the Caspase-1 protein, promotes the maturation and release of the inflammatory factors such as IL-1 and TNF-1, amplifies the hypothalamic inflammation reaction, negatively regulates the hypothalamic insulin signal and the leptin signal, and finally leads to IR and LR, These results suggest that CCR7 is a key candidate for the treatment of the hypothalamic IR of T2DM.
【学位授予单位】：江苏师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1

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