王浆主蛋白的抗衰老功能及分子机理研究

发布时间：2018-01-01 13:36

本文关键词：王浆主蛋白的抗衰老功能及分子机理研究　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：随着国内外人口老龄化问题的加剧,国际市场对抗衰老功能食品的需求越来越大,蜂王浆(RJ)作为国际公认的传统抗衰老功能食品,将发挥更加重要的作用。根据新近研究报道,王浆主蛋白(MRJPs)是RJ的关键活性成分,但是否对哺乳动物具有抗衰老作用及作用机理尚未见报道。本研究以从新鲜RJ超滤分离的MRJPs冻干粉为研究对象,以自然衰老大鼠和人体细胞为模型,进行了 MRJPs的抗衰老作用及分子机理系统研究,结果如下:1.MRJPs对大鼠的抗衰老作用及分子机理电泳结果显示MRJPs集中在49-80 kDa,其氨基酸组成与酪蛋白相似,说明MRJPs的功能不是体现在氨基酸水平上,而是通过特殊的形式来表现其生物活性。衰老大鼠的附睾和睾丸指数显著低于青年大鼠,口服MRJPs并不能改变这种脏器的退化,对其它脏器指数和体重也未产生明显影响。苏木精-伊红染色结果表明,MRJPs可以明显改善衰老引起的大鼠肝脏及肾脏等重要器官出现的病理性特征。口服MRJPs能显著提高大鼠血液的CAT活力(P0.05),降低血液的脂质过氧化物MDA水平;MRJPs干预组的GSH含量与GSH-PX活力与青年大鼠表现出类似的调节方式,显示MRJPs具有良好的抗氧化活性。mTOR转录水平与MRJPs干预剂量呈正相关。S6激酶(S6K)通过mTOR径被激活,并出现转录水平上调趋势。青年大鼠的mTOR基因转录水平显著高于自然衰老对照组,而MRJPs干预能提高衰老大鼠的mTOR基因转录水平,类似现象在s6k基因中也有发现。青年大鼠的Foxo1基因转录水平显著高于自然衰老对照组,而MRJPs干预能提高衰老大鼠Foxo1基因转录水平,显示抗衰老活性。此外,MRJPs能显著提高大鼠血液中端粒酶逆转录酶TERT基因的表达,说明其抗衰老机理可能与TERT的转录水平的调节有关。与SOD未出现明显改变一样,Sod1因的转录水平在干预后也未出现明显变化。2.MRJPs对大鼠认知功能的改善作用动物水迷宫实验表明,逃避潜伏期较对照和酪蛋白组分别缩短了 48.5%(P=0.0045)和49.9%(P=0.0036);穿过目标区域的次数较对照和酪蛋白组分别提高了 177.4%和46.1%,说明MRJPs能显著改善老年大鼠的空间认知能力。基于超高效液相色谱四极杆飞行时间质谱(UPLC-QTOF/MS)的大鼠尿液指纹图谱表明,MRJPs干预衰老大鼠后,其代谢特征出现回归青年大鼠的趋势。鉴定了 28个与衰老相关的生物标记物,其中6个标记物,烟酸单核苷酸(1),丙氨酸丁氨酸硒醚(2),CDP-乙醇胺(3),磺基丙氨酸(4),磷酸烯醇式丙酮酸(5)和黄嘌呤核苷(6)在MRJPs干预后发生显著改变。结合文献RJ改善记忆的功能分析,RJ来源的MRJPs有可能是一种新的提高认知功能的补充蛋白质。3.MRJPs及KHC03对大鼠骨密度的改善作用补充MRJPs及KHCO3不会影响大鼠体重。血液肝功能检测结果表明,MRJPs及KHCO3作为膳食补充剂是安全的,且对肝功能有一定调节作用。MRJPs会导致大鼠骨密度轻微降低,推测其通过降低雌激素水平、促进钙流失而引起骨密度的降低。同时补充KHCO3(100 mg/mL)可防止骨密度的降低,并引起雌二醇水平降低,而MRJPs与雌二醇共同作用于成骨细胞时存在拮抗作用,说明MRJPs与KHC03对骨密度的联合保护作用可能是通过降低MRJPs和雌二醇对成骨细胞的拮抗作用实现的。MRJPs与KHCO3的协同作用具有一定的抗氧化性。补充MRJPs和KHC03在一定程度上能降低CTX-Ⅰ,显著提高Ⅰ型原骨胶原(P1NP)和OPG含量,而补充的KHCO3有类似的效果。MRJPs和雌雌醇能上调clock、cry1和cry2,下调bmal1,说明通过时钟节律调控途径可影响骨密度。4.MRJPs对MRC-5细胞的抗衰老作用当MRC-5细胞处于中年期时,补充终浓度25-200 μg/mL的MRJPs对MRC-5虽有一定的促进作用,但不显著。流式细胞检测结果表明,MRJPs对处于中年期MRC-5细胞增殖指数的影响并不明显。但当MRC-5细胞进入衰老期后,细胞增殖速率显著降低,补充MRJPs具有明显的促增殖作用。β-半乳糖苷酶活性检测表明,补充MRJPs能显著降低MRC-5衰老细胞比例,且呈剂量相关性。拉曼检测结果显示,除683 cm-1处外,MRJPs及酪蛋白的拉曼特征峰与对照组高度一致,体现其作为蛋白的共性。
[Abstract]:With the problem of population aging intensifies, the international market demand for anti-aging function food is more and more big, royal jelly (RJ) as a traditional anti-aging function internationally recognized food, will play a more important role. According to recent research reports, royal jelly proteins (MRJPs) is a key active component of RJ, but whether or not has anti-aging effect and mechanism of mammals has not been reported. In this study, isolated from fresh RJ ultrafiltration MRJPs freeze-dried powder as the research object, with the natural aging rats and human cells as a model of the system and the molecular mechanism of MRJPs anti-aging results are as follows: 1.MRJPs on rats and molecular rejuvenation the mechanism of the electrophoresis results showed that MRJPs concentration in 49-80 kDa, and the amino acid composition of casein is similar to that of MRJPs, the function is not reflected in the amino acid level, but through a special form. The biological activity of aging rats testis and epididymis index was significantly lower than that of young rats, oral administration of MRJPs did not change the degradation effect of organs, also did not produce other organ index and weight. Hematoxylin eosin staining results showed that MRJPs can significantly improve the pathological characteristics caused by the decline of old rat liver and the kidney and other important organs. The oral administration of MRJPs can significantly improve the rat blood CAT activity (P0.05), lower blood lipid peroxide levels of MDA; GSH content and GSH-PX activity in young rats and MRJPs intervention group showed a similar regulation, showed that MRJPs had antioxidant activity of.MTOR kinase and MRJPs transcription level intervention dose.S6 good (S6K) by mTOR diameter is activated, and up-regulated transcription. The transcription level of mTOR gene in young rats was significantly higher than that of the natural aging control group, MRJPs The intervention can improve the transcription level of mTOR gene in aging rats, a similar phenomenon is also found in the S6K gene. Foxo1 gene transcription level of young rats was significantly higher than that of the natural aging control group, and MRJPs intervention can improve the transcription level of Foxo1 gene in aging rats, showed anti-aging activity. In addition, MRJPs could significantly increase the expression of gene in the blood of human telomerase reverse transcriptase TERT rats, indicating its anti-aging mechanism may be related to the regulation on the transcription level of TERT and SOD. There was no obvious change, because the transcription level of Sod1 after the intervention did not appear that improve animal change significantly in.2.MRJPs water maze test on cognitive function in rats, shortened 48.5% the escape latency compared with the control group respectively and casein (P=0.0045) and 49.9% (P=0.0036); 177.4% and 46.1% times the increase through the target area compared with the control group and casein respectively, indicating that MRJPs can display To improve the spatial cognitive ability of aged rats. Quadrupole time-of-flight mass spectrometry based on ultra high performance liquid chromatography (UPLC-QTOF/MS) showed that the rat urine fingerprints, MRJPs intervention rats, the metabolic characteristics of young rats appeared regression trend. 28 old biomarkers associated with the decline of the identification. 6 markers, nicotinic acid mononucleotide (1), selenocystathionine (2), CDP- (3) ethanolamine, cysteic acid (4), phosphoenolpyruvate (5) and xanthine nucleotide (6) changed obviously in MRJPs after intervention. Literature analysis improve the memory function with RJ RJ, source of MRJPs may improve the effect of protein.3.MRJPs and KHC03 a new improved cognitive function on bone density in rats of MRJPs and KHCO3 will not affect the body weight of rats. Blood liver function test results show that the MRJPs and KHCO3 as dietary supplements are safe , and there is a certain regulatory role of.MRJPs will lead to bone mineral density in rats decreased slightly on liver function, presumably by reducing estrogen levels, promote calcium loss caused by reduced bone density. At the same time to add KHCO3 (100 mg/mL) can prevent the loss of bone density, and induced by estradiol level decreased, and MRJPs together with the female glycol in osteoblasts when there is antagonism, indicating the protective effect of MRJPs and KHC03 on bone mineral density may be through the synergistic effect of reducing MRJPs and estradiol on.MRJPs and KHCO3 realize the antagonism of the osteoblast has some antioxidant activity. MRJPs and KHC03 can reduce CTX- 1 to a certain extent, significant improve the type I procollagen (P1NP) and the content of OPG, and added KHCO3 have the similar effect of.MRJPs and estrogen estradiol can increase clock, cry1 and CRY2, down BMAL1, the clock rhythm control way can affect bone The density of.4.MRJPs on MRC-5 cell anti-aging effect when MRC-5 cell is in the middle period, adding a final concentration of 25-200 g/mL MRJPs of MRC-5 has a certain role in promoting, but not significant. Flow cytometry results showed that the effect of MRJPs on MRC-5 cell proliferation index is in the middle period is not obvious. But when cells enter MRC-5 after the aging period, the cell proliferation rate was significantly reduced, MRJPs has significantly stimulated the proliferation. Detection indicated that beta galactosidase activity, MRJPs can significantly reduce the aging cell ratio of MRC-5, in a dose-dependent manner. The Raman detection results show that the addition of 683 cm-1, MRJPs and Raman spectra of casein and control group of highly consistent, embodied as a common protein.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS201.21

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