本文選題：沙棘籽黃酮 + 葡萄糖攝取��； 參考：《華東師范大學(xué)》2017年碩士論文

【摘要】：沙棘籽黃酮(flavonoids from seed of Hippophae rhamnoides L.,FSH)是從胡頹子科植物沙棘的籽中制備獲得的富集黃酮類成分的活性提取物。實(shí)驗(yàn)室前期研究確立了 FSH的最優(yōu)提取工藝,分析了 FSH的主要成分,明確了其物質(zhì)基礎(chǔ),并制定了質(zhì)量控制標(biāo)準(zhǔn)。通過(guò)動(dòng)物實(shí)驗(yàn)發(fā)現(xiàn)FSH具有明顯的降血糖以及改善胰島素抵抗的活性,但其調(diào)節(jié)糖代謝的細(xì)胞分子機(jī)制尚不清楚,作用靶點(diǎn)及信號(hào)通路也不明確。因此本文選用C2C12骨骼肌細(xì)胞和3T3-L1成熟脂肪細(xì)胞,系統(tǒng)地研究了 FSH對(duì)兩種細(xì)胞糖代謝的影響及其作用機(jī)制,旨在明確FSH發(fā)揮降糖及改善胰島素抵抗作用的分子靶點(diǎn)和靶向的信號(hào)通路,使FSH具備"質(zhì)量可控、功能明確、靶點(diǎn)清晰"的活性提取物的特點(diǎn)。研究首先通過(guò)2-NBDG熒光標(biāo)記法檢測(cè)了 FSH對(duì)2種細(xì)胞葡萄糖攝取的影響,結(jié)果顯示:50-200μg/mL的FSH可顯著促進(jìn)分化成熟的3T3-L1脂肪細(xì)胞和C2C12細(xì)胞的葡萄糖攝取,并有時(shí)間和劑量依賴性。進(jìn)一步的機(jī)制研究發(fā)現(xiàn):FSH可激活基礎(chǔ)狀態(tài)下3T3-L1脂肪細(xì)胞和C2C12細(xì)胞內(nèi)AMPK信號(hào)通路,上調(diào)p-AMPK的蛋白水平,并促進(jìn)GLUT4蛋白轉(zhuǎn)位到細(xì)胞膜上,抑制PPARy及其磷酸化蛋白的表達(dá)。AMPK的抑制劑Compound C可以抑制FSH促進(jìn)的葡萄糖攝取,并能抑制FSH上調(diào)的p-AMPK蛋白表達(dá)以及下游的GLUT4蛋白轉(zhuǎn)位,表明FSH通過(guò)激活A(yù)MPK信號(hào)通路來(lái)促進(jìn)細(xì)胞對(duì)葡萄糖的攝取。胰島素信號(hào)傳導(dǎo)通路是調(diào)節(jié)細(xì)胞糖代謝的重要通路,本文繼續(xù)探究了 FSH對(duì)胰島素信號(hào)傳導(dǎo)通路的影響。FSH處理上述兩種細(xì)胞24 h后,再用100 nM的胰島素處理30 min,激活細(xì)胞胰島素信號(hào)通路,檢測(cè)FSH對(duì)相關(guān)通路的影響。實(shí)驗(yàn)結(jié)果顯示FSH下調(diào)兩種細(xì)胞中IRS-1的表達(dá),抑制胰島素受體底物酪氨酸和絲氨酸磷酸化表達(dá),并降低胰島素信號(hào)通路中PI3K的表達(dá),抑制AKT,AS160的磷酸化水平;FSH還下調(diào)了兩種細(xì)胞中ERK,JNK和P38的磷酸化水平。在骨骼肌細(xì)胞中FSH可明顯降低PKCξ蛋白的表達(dá),對(duì)CAP蛋白水平無(wú)明顯影響;但是在脂肪細(xì)胞中FSH可上調(diào)CAP蛋白水平。上述結(jié)果表明FSH抑制了PI3-K/AKT/AS160信號(hào)通路和MAPK信號(hào)通路。進(jìn)一步評(píng)價(jià)FSH與胰島素聯(lián)合作用時(shí)對(duì)細(xì)胞葡萄糖消耗及AMPK信號(hào)通路的影響,發(fā)現(xiàn)FSH與胰島素聯(lián)合作用時(shí)顯著促進(jìn)了細(xì)胞對(duì)葡萄糖的利用,并增強(qiáng)了胰島素上調(diào)的AMPK磷酸化水平,表明FSH對(duì)胰島素通路的抑制作用不影響細(xì)胞攝取葡萄糖,FSH在胰島素刺激下仍可以通過(guò)AMPK信號(hào)通路攝取葡萄糖。炎癥與糖代謝紊亂和胰島素抵抗關(guān)系密切,激活的單核-巨噬細(xì)胞以及脂肪細(xì)胞均可分泌多種炎癥因子。采用LPS(100 ng/mL)處理RAW264.7單核巨噬細(xì)胞建立體外炎癥模型,首先基于此模型評(píng)價(jià)了 FSH對(duì)炎癥因子表達(dá)及炎癥信號(hào)通路的影響;而后進(jìn)行了 FSH對(duì)脂肪細(xì)胞炎癥因子表達(dá)及炎癥信號(hào)通路的影響分析。結(jié)果顯示FSH顯著地抑制了 LPS誘導(dǎo)的RAW264.7細(xì)胞內(nèi)NF-κB,P38 MAPK信號(hào)通路和CXCL10,IL-6炎癥因子mRNA的表達(dá),促進(jìn)了 TNF-α mRNA的表達(dá);FSH也可以抑制3T3-L1成熟的脂肪細(xì)胞中NF-κB信號(hào)通路及其mRNA表達(dá),促進(jìn)TNF-αmRNA的表達(dá)。提示FSH可能通過(guò)抑制NF-κB,P38MAPK信號(hào)通路,減少部分炎癥因子的表達(dá)來(lái)緩解胰島素抵抗。綜上所述,FSH通過(guò)激活A(yù)MPK信號(hào)通路,促進(jìn)細(xì)胞攝取葡萄糖;FSH可能通過(guò)抑制NF-κB,P38 MAPK信號(hào)通路以及CXCL10,IL-6炎癥因子mRNA的表達(dá),緩解胰島素抵抗作用。
[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.

【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R285

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