燕麦葡聚糖抗炎与降脂功能的评估及其分子机理研究

发布时间：2017-12-28 20:14

本文关键词：燕麦葡聚糖抗炎与降脂功能的评估及其分子机理研究　出处：《中南林业科技大学》2017年博士论文　论文类型：学位论文

【摘要】：燕麦(Avenasativa)是禾本科燕麦属草本植物,一年生,是一种古老的粮食作物,已经被人类种植了近两千年之久,一般被直接作为食物或者作为饲料给动物使用。燕麦分为皮燕麦和裸燕麦两种,裸燕麦在我国种植历史悠久,《本草纲目》中称之为雀麦、野麦子。燕麦性味甘平。能益脾养心、敛汗。有较高的营养价值。古代医书《救荒本草》中提到,燕麦可用于体虚自汗、盗汗或肺结核病人。煎汤服,或“春去皮作面蒸食及作饼食”。燕麦能促进肠胃蠕动,利于排便,热量低,具有升糖指数低,降脂降糖的功效。早期人们对燕麦的研究较少,认为燕麦具有增加饱腹感和降低营养物质吸收的能力,近年来,燕麦有益于健康的生理功能,使得燕麦成为了食疗与健康新的研究热点,研究发现,燕麦具有非常广泛的生理功能,包括降低胆固醇的作用,降低血糖水平的能力,调节免疫,降低肠癌风险等。研究还发现,燕麦的这些营养作用主要来源于燕麦麸皮中的一种营养组分——燕麦葡聚糖,燕麦葡聚糖主要存在于燕麦胚乳和糊粉层细胞壁中,主要是由许多单糖按照β-(1,3)和β-(1,4)糖苷键连接形成一种不可消化的β-D-葡聚糖,而这一特殊结构也使得燕麦葡聚糖具有特殊的有益健康生理功能。1997年美国食物与药物监督管理局(FDA)建议每天食用3克以上含燕麦葡聚糖的食品,并允许在食品上明确标识。1.燕麦葡聚糖对DSS诱导小鼠溃疡性结肠炎的抑制作用小鼠自由饮用3%硫酸葡聚糖钠(DSS)连续7天构建小鼠溃疡性肠炎模型,在实验开始前3天于保护组灌胃500mg/kg.bw和1000mg/kg.bw燕麦葡聚糖直至实验结束。结果发现DSS损伤组小鼠逐渐出现活动力减弱、厌食、腹泻、毛色凌乱等情况。并出现体重下降、腹泻、便血等症状,燕麦葡聚糖保护组DAI评分明显降低(P0.01)处死小鼠后发现,DSS损伤组小鼠肠道长度显著低于正常组小鼠,肠道肿胀,变粗,肠腔中未见成型粪便。而正常小鼠肠道长而紧致,肠腔中可见颗粒状成型粪便。燕麦葡聚糖保护组小鼠的平均肠道长度介于正常组与损伤组之间,肠炎小鼠脾脏体积和重量均大于正常组,而葡聚糖保护组小鼠脾脏有所改善。苏木素伊红(HE)染色结果发现,正常组小鼠肠道结构完整,肠道壁厚度均匀,肠绒毛及隐窝结构清晰,而DSS损伤组小鼠肠道横切面则显示出强烈的炎症反应,如肠道壁增厚,结肠上皮粘膜糜烂、隐窝结构被破坏、粘膜下水肿、淋巴及中性粒细胞浸润、肠道内壁不规则增生等。燕麦葡聚糖保护组小鼠肠道镜下观测效果优于损伤组。小鼠肠道研磨后检测髓过氧化物酶(MPO),丙二醛(MDA)以及一氧化氮(NO)的量,损伤组小鼠都显著高于正常组,葡聚糖保护组的这些指数显著低于损伤组。提取肠道RNA和蛋白质分别进行荧光定量PCR和Western blot实验,结果损伤组炎症因子肿瘤坏死因子α(TNF-α),白细胞介素1和6(IL-1β、IL-6)以及诱导型一氧化氮合酶(iNOS)的mRNA以及蛋白表达量显著上调,保护组炎症因子表达量显著低于损伤组。且基本呈现浓度依赖性。从免疫组织化学(IHC)图中可以看出炎症因子表达量较高,且在肠道中大量聚集,但保护组的表达量较低。2.燕麦葡聚糖抑制LPS诱导巨噬细胞的炎症及炎症因子表达巨噬细胞RAW264.7在脂多糖(LPS)刺激下的可以诱导为炎症因子体外模型,通过使用燕麦葡聚糖观察其对炎症状态的抑制效果。研究发现,20 μg/mL和40 μg/mL的燕麦葡聚糖对巨噬细胞的活力无显著影响,因而可以适用于该模型的研究。LPS组对比对照组,促炎因子TNFα、IL-6、IL-1β、iNOS和IFN-γ的mRNA表达量和蛋白表达明显上升,对比LPS组,燕麦葡聚糖组能够降低这些mRNA和蛋白质表达,且结果呈现出一定量效关系。3.燕麦葡聚糖预防小鼠高脂血症的发生及其分子机制通过给小鼠饲喂高脂饲料,建立了高脂血症小鼠模型,并通过灌胃的方式给小鼠饲喂500mg/kg.bw和1000mg/kg.bw燕麦葡聚糖直至实验结束。高脂组小鼠体重从第一周开始增加,其后维持稳定,而葡聚糖保护组小鼠体重则低于高脂组小鼠,且结果呈浓度依赖性。解剖小鼠后取器官分析,结果发现,正常组小鼠肝脏呈暗红色,体积较小,而高脂组小鼠肝脏重量和体积都较大,色泽发白或者发黄,表面有厚重的油腻感和颗粒感。低浓度葡聚糖保护组肝脏颜色较高脂组颜色深,且白色颗粒状较少,体积较小。高浓度葡聚糖保护组小鼠肝脏颜色接近正常组,重量显著小于高脂组和低浓度葡聚糖保护组。高脂组小鼠脾脏体积和重量显著高于正常组,而保护组小鼠脾脏相对较小。高脂组的总甘油三酯、总胆固醇、低密度脂蛋白胆固醇显著高于正常组,而葡聚糖保护组的具有显著降低脂肪与胆固醇、同时还包含有低密度脂蛋白胆固醇的作用。然而,高脂组的高密度脂蛋白胆固醇低于对照组,燕麦葡聚糖在一定程度上具有增加高密度脂蛋白胆固醇的作用,但并未具有显著性差异。肝脏HE染色,结果发现,正常组小鼠肝细胞排列比较整齐,未见脂肪空泡。高脂组小鼠肝细胞排列松散,肝细胞肿大,胞质内可见许多大小不一的脂肪空泡,低保护燕麦葡聚糖组小鼠脂肪有少量脂肪泡,高浓度保护组细胞排列紧密,基本与正常组无区别。脂肪垫HE染色结果发现,正常组小鼠脂肪细胞直径较小,排列紧密,而高脂组小鼠脂肪细胞直径显著大于正常组,直径约为正常组小鼠脂肪细胞的两倍。燕麦葡聚糖保护组小鼠脂肪细胞直径显著低于高脂组。荧光定量PCR结果发现,脂肪代谢相关基因过PPARα在高脂组表达量显著低于正常对照组,而燕麦葡聚糖组的PPARα基因表达量显著提高,大大高于高脂组,提示燕麦葡聚糖可以通过刺激能量的消耗,减少能量物质堆积于肝脏。PPARy、脂肪酸合成酶(Fatty acid synthesis,FAS)和胆固醇调节元件结合蛋白(Sterol-regulatory element binding protein 1C,SREBP-1C)都与脂肪和胆固醇的合成有关,高脂组小鼠肝脏中的PPARγ、FAS和SREBP-1C的mRNA表达量都显著增加,而葡聚糖保护组小鼠的这三个基因都有所下降,提示燕麦葡聚糖能减少脂肪和胆固醇合成,降低其沉积于体内的量,从而达到降脂的目的。同时高脂组小鼠肝脏炎症因子TNF-α、IL-6和诱导型一氧化氮合酶(inducible nitric oxide synthesis,iNOS)都有所上调,两个浓度的燕麦葡聚糖都能显著的降低炎症因子的表达量。减少高脂血症伴随的炎症发生。
[Abstract]:Oats (Avenasativa), a grass herb belonging to Gramineae, is an ancient food crop, which has been planted for nearly two thousand years in the world. It is usually used as food or feed for animals. Oat is divided into two kinds of oat and oat, oat cultivated in China has a long history, "Compendium of Materia Medica" called Brome, wild wheat. Sexual flavour Ganping oats. It can nourish the spleen and raise the sweat. It has high nutritional value. Mentioned in ancient books "for materia medica", can be used for oat body empty spontaneous, night sweats or pulmonary tuberculosis patients. Jiantang clothes, or "spring surface and peeled steamed cakes". Oats can promote intestinal peristalsis, which is beneficial to defecation, low heat, low rising sugar index and reducing fat and reducing sugar. Study on the early people of oat that has less oats increase satiety and reduce the ability of nutrient absorption in recent years, oat is beneficial to healthy physiological function, which become the diet and health of oat new research focus, the study found that the physiological function of oat is very extensive, including cholesterol lowering effect ability to reduce blood glucose levels, regulating immunity, reduce the risk of colorectal cancer. The study also found that the nutritional effects of oat mainly derived from oat bran in a nutrient component of oat glucan, oat glucan mainly exists in oat endosperm and aleurone cell wall, is mainly composed of many monosaccharides in accordance with beta (1,3) and beta (1,4) glycosidic bond can not form a digestion of beta -D- dextran, and this special structure makes the oat glucan has beneficial physiological function of special health. In 1997, the United States Food and Drug Administration (FDA) recommended to eat more than 3 grams of oat glucan a day and allow a clear label on the food. 1. oat glucan on DSS induced ulcerative colitis in mice the mice drinking 3% dextran sulfate sodium (DSS) for 7 consecutive days of ulcerative colitis mice, 3 days before the experiments began in 500mg/kg.bw and 1000mg/kg.bw protection group fed oat glucan until the end of the experiment. The results showed that the mice in the DSS injury group gradually weakened, anorexia, diarrhea, and hair color disorder and so on. And the emergence of weight loss, diarrhea, hematochezia and other symptoms, oat glucan protection group DAI scores were significantly decreased (P0.01) mice were killed after the discovery, the DSS injury group mice intestinal length was significantly lower than normal group mice, intestinal swelling, thickening of the intestinal cavity, not forming stool. In the normal mice, the intestinal tract was long and compact, and the granule shaped feces were found in the intestinal cavity. The average length of intestinal tract in oats dextran protection group was between normal group and injury group. The spleen volume and weight of enteritis mice were all larger than those of normal group, but the spleen of dextran protection group improved. Hematoxylin and eosin (HE) staining results showed that normal mice intestinal structural integrity, intestinal wall thickness, villus and crypt structure is clear, and the injury of DSS mice intestinal cross section showed a strong inflammatory response, such as bowel wall thickening, colonic mucosal erosion, the crypt structure was destroyed, submucosal edema, lymphocytes and neutrophil infiltration, intestinal wall irregular hyperplasia. The observation effect of oat dextran protective group under intestinal microscope was better than that of injury group. After murine intestinal lapping, myeloperoxidase (MPO), malondialdehyde (MDA) and nitric oxide (NO) levels were detected. The injury group mice were significantly higher than those of the normal group, and those indexes of dextran protection group were significantly lower than those of the injury group. The extraction of intestinal RNA and protein were PCR and Western fluorescence quantitative blot test results in injury group inflammatory cytokines tumor necrosis factor alpha (TNF- alpha), interleukin 1 and 6 (IL-1 P, IL-6) and inducible nitric oxide synthase (iNOS) and mRNA protein expression was significantly up-regulated, protection group inflammation gene expression was significantly lower than the injury group. And it is basically dependent on concentration. It was found that the expression of inflammatory factors was higher and accumulated in the intestinal tract, but the expression of the protective group was low in IHC. 2., oatmeal dextran inhibited LPS induced inflammatory and inflammatory cytokines expression in macrophages. The macrophage RAW264.7 could be induced by lipopolysaccharide (LPS) as an inflammatory factor in vitro, and its inhibitory effect on inflammation was observed by oatmeal dextran. It was found that oat dextran of 20 g/mL and 40 mu g/mL had no significant effect on the viability of macrophages, so it could be applied to the study of the model. Compared with control group, mRNA expression and protein expression of proinflammatory cytokines TNF, IL-6, IL-1 beta, iNOS and IFN- gamma increased significantly in group LPS, compared with LPS group, oatmeal dextran group could reduce these mRNA and protein expression, and the results showed a dose-dependent relationship. 3. oatmeal dextran prevents the occurrence and molecular mechanism of hyperlipidemia in mice. By feeding high fat diet to mice, a mouse model of hyperlipidemia was established, and 500mg/kg.bw and 1000mg/kg.bw oat dextran were fed to mice through the way of gavage until the end of the experiment. The body weight of the high fat group increased from the first week, then maintained stable, while the body weight of the dextran protection group was lower than that of the high-fat group, and the result was concentration dependent. After dissection of the mice, the organs were analyzed. It was found that the liver of the normal group was dark red and small, while the liver weight and volume of the high-fat group were bigger, the color was whitish or yellowed, and the surface had thick greasy and granular feelings. In the low concentration dextran protection group, the color of the liver with higher color was deep, and the white granules were less and the volume was smaller. The liver color of the high concentration dextran protective group was close to the normal group, and the weight was significantly lower than that of the high fat group and the low concentration dextran protective group. The volume and weight of spleen in the high fat group were significantly higher than that in the normal group, while the spleen in the protective group was relatively small. Total triglyceride and total bilirubin in high fat group
【学位授予单位】：中南林业科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS210.1

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