沙棘籽黄酮促进脂肪和骨骼肌细胞葡萄糖摄取的机制研究

发布时间：2018-04-15 12:10

本文选题：沙棘籽黄酮 + 葡萄糖摄取　；参考：《华东师范大学》2017年硕士论文

【摘要】：沙棘籽黄酮(flavonoids from seed of Hippophae rhamnoides L.,FSH)是从胡颓子科植物沙棘的籽中制备获得的富集黄酮类成分的活性提取物。实验室前期研究确立了 FSH的最优提取工艺,分析了 FSH的主要成分,明确了其物质基础,并制定了质量控制标准。通过动物实验发现FSH具有明显的降血糖以及改善胰岛素抵抗的活性,但其调节糖代谢的细胞分子机制尚不清楚,作用靶点及信号通路也不明确。因此本文选用C2C12骨骼肌细胞和3T3-L1成熟脂肪细胞,系统地研究了 FSH对两种细胞糖代谢的影响及其作用机制,旨在明确FSH发挥降糖及改善胰岛素抵抗作用的分子靶点和靶向的信号通路,使FSH具备"质量可控、功能明确、靶点清晰"的活性提取物的特点。研究首先通过2-NBDG荧光标记法检测了 FSH对2种细胞葡萄糖摄取的影响,结果显示:50-200μg/mL的FSH可显著促进分化成熟的3T3-L1脂肪细胞和C2C12细胞的葡萄糖摄取,并有时间和剂量依赖性。进一步的机制研究发现:FSH可激活基础状态下3T3-L1脂肪细胞和C2C12细胞内AMPK信号通路,上调p-AMPK的蛋白水平,并促进GLUT4蛋白转位到细胞膜上,抑制PPARy及其磷酸化蛋白的表达。AMPK的抑制剂Compound C可以抑制FSH促进的葡萄糖摄取,并能抑制FSH上调的p-AMPK蛋白表达以及下游的GLUT4蛋白转位,表明FSH通过激活AMPK信号通路来促进细胞对葡萄糖的摄取。胰岛素信号传导通路是调节细胞糖代谢的重要通路,本文继续探究了 FSH对胰岛素信号传导通路的影响。FSH处理上述两种细胞24 h后,再用100 nM的胰岛素处理30 min,激活细胞胰岛素信号通路,检测FSH对相关通路的影响。实验结果显示FSH下调两种细胞中IRS-1的表达,抑制胰岛素受体底物酪氨酸和丝氨酸磷酸化表达,并降低胰岛素信号通路中PI3K的表达,抑制AKT,AS160的磷酸化水平;FSH还下调了两种细胞中ERK,JNK和P38的磷酸化水平。在骨骼肌细胞中FSH可明显降低PKCξ蛋白的表达,对CAP蛋白水平无明显影响;但是在脂肪细胞中FSH可上调CAP蛋白水平。上述结果表明FSH抑制了PI3-K/AKT/AS160信号通路和MAPK信号通路。进一步评价FSH与胰岛素联合作用时对细胞葡萄糖消耗及AMPK信号通路的影响,发现FSH与胰岛素联合作用时显著促进了细胞对葡萄糖的利用,并增强了胰岛素上调的AMPK磷酸化水平,表明FSH对胰岛素通路的抑制作用不影响细胞摄取葡萄糖,FSH在胰岛素刺激下仍可以通过AMPK信号通路摄取葡萄糖。炎症与糖代谢紊乱和胰岛素抵抗关系密切,激活的单核-巨噬细胞以及脂肪细胞均可分泌多种炎症因子。采用LPS(100 ng/mL)处理RAW264.7单核巨噬细胞建立体外炎症模型,首先基于此模型评价了 FSH对炎症因子表达及炎症信号通路的影响;而后进行了 FSH对脂肪细胞炎症因子表达及炎症信号通路的影响分析。结果显示FSH显著地抑制了 LPS诱导的RAW264.7细胞内NF-κB,P38 MAPK信号通路和CXCL10,IL-6炎症因子mRNA的表达,促进了 TNF-α mRNA的表达;FSH也可以抑制3T3-L1成熟的脂肪细胞中NF-κB信号通路及其mRNA表达,促进TNF-αmRNA的表达。提示FSH可能通过抑制NF-κB,P38MAPK信号通路,减少部分炎症因子的表达来缓解胰岛素抵抗。综上所述,FSH通过激活AMPK信号通路,促进细胞摄取葡萄糖;FSH可能通过抑制NF-κB,P38 MAPK信号通路以及CXCL10,IL-6炎症因子mRNA的表达,缓解胰岛素抵抗作用。
[Abstract]:Sea buckthorn seed flavonoids (flavonoids from seed of Hippophae rhamnoides L., FSH) is the active extract were obtained from sea buckthorn seed plants 50elaeagnaceae for enrichment of flavonoids. Previous research has established the optimal extraction process of FSH, the main component of FSH is analyzed, the material basis, and to develop a quality control standard. Through animal experiments found that FSH has obvious hypoglycemic activity and improve insulin resistance, but its cellular and molecular mechanisms regulating glucose metabolism is not clear, the target and the signal pathway is not clear. So this paper selects C2C12 skeletal muscle cells and 3T3-L1 adipocytes, systematically studied the effect of FSH on the two cell glucose metabolism and its mechanism of action, to determine FSH signaling pathway to play the role of lowering blood glucose and improving insulin resistance molecular targets and target, make FSH with controllable quality, The function is clear, clear target "characteristics of the activity of the extracts. Study first examined the effects of FSH on the 2 cell glucose uptake by 2-NBDG fluorescence staining results showed that 50-200 g/mL FSH could significantly promote the differentiation of mature 3T3-L1 adipocytes and C2C12 cells glucose uptake, and in a dose and time dependent. Further studies showed that FSH could activate 3T3-L1 signal of fat cells and C2C12 cells in AMPK based state pathway, increase the protein level of p-AMPK, and promote GLUT4 protein translocation to the cell membrane, the expression of.AMPK PPARy inhibited glucose uptake and phosphorylation of protein inhibitor Compound C can inhibit FSH, and inhibit the FSH up-regulated the expression of p-AMPK protein and the downstream GLUT4 protein translocation, showed that FSH to promote the uptake of glucose into cells by activating AMPK signaling pathway through insulin signaling. The road is an important pathway in regulation of glucose metabolism in cells, this paper to explore the effect of FSH on insulin signal transduction pathway of.FSH in the two types of cells after 24 h and 100 nM insulin treatment for 30 min, the activation of insulin signaling pathways in cells, to study the effect of FSH on related pathways. Experimental results show that the down-regulation of FSH expression of two kinds of cells in IRS-1, inhibition of insulin receptor substrate tyrosine and serine phosphorylation, and reduce the expression of PI3K in insulin signaling pathway in the inhibition of AKT phosphorylation of AS160; FSH also has two kinds of cells in ERK, JNK and P38 phosphorylation in skeletal muscle cells. FSH can significantly decrease the expression of PKC. Protein, has no obvious effect on the protein level of CAP; but in the fat cells of FSH can upregulate CAP protein levels. The results showed that FSH inhibited PI3-K/AKT/AS160 and MAPK signal pathways. Further evaluation Effect on cell glucose consumption and AMPK signaling pathway of FSH and insulin combined effects of price, found that the combined effects of FSH and insulin significantly promoted the use of glucose into cells, and enhance the level of AMPK phosphorylation of insulin up-regulated, showed that the FSH of the insulin pathway inhibited by does not affect the uptake of glucose, insulin stimulation in FSH is through the AMPK signaling pathway of glucose uptake. Inflammation and glucose metabolic disorder and insulin resistance is closely related to the activation of macrophages and fat cells can secrete a variety of inflammatory factors. Using LPS (100 ng/mL) treatment of RAW264.7 macrophages in vitro inflammation model, firstly, based on this model to evaluate the effect of FSH on the expression of inflammatory cytokines and the inflammatory signaling pathway; and then analyzed the effects of FSH on adipocyte expression of inflammatory cytokines and inflammatory signaling pathways. Results Showed that FSH significantly inhibited LPS induced RAW264.7 cells NF- kappa B, P38 MAPK and CXCL10 IL-6 signaling pathway, expression of inflammatory cytokines mRNA, promote the expression of TNF- of mRNA; FSH can also inhibit the expression of NF- B signaling pathway and mRNA 3T3-L1 of mature adipocytes, promote the expression of TNF- mRNA. Suggesting that FSH may inhibit the NF- kappa B, P38MAPK signaling pathway, reduce the expression of inflammatory cytokines to improve insulin resistance. To sum up, FSH by activating AMPK signaling pathway to promote cell uptake of glucose; FSH can inhibit NF- kappa B, P38 MAPK and CXCL10 IL-6 signaling pathway, expression of inflammatory cytokines mRNA, alleviate insulin resistance.

【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285

【参考文献】