【作者】 张磊；

【导师】 刘雄； 陈宗道；

【作者基本信息】 西南大学 ， 食品科学， 2013， 博士

【摘要】 高胆固醇血症是导致动脉粥样硬化及心脑血管疾病主要的危险因素之一,对人们的生命健康构成了严重威胁。因此,控制和降低血中胆固醇水平是防治心脑血管疾病的重要环节。降低胆固醇的功能食品和保健品在控制和改善血脂水平中起到了重要的作用,尤其适用于患者血液中的胆固醇水平略高,但还没有高到足以使用降胆固醇处方药物的情况。辣椒碱是存在辣椒中的一类辣味物质,是辣椒主要的活性成分,具有多种生理功能。近年来研究表明,辣椒碱具有促进能量消耗,减少脂肪在体内的聚集,降低血压,保护心血管健康等作用。还有研究表明辣椒碱具有抗氧化活性,能阻止人类LDLs的氧化。这些结果均表明辣椒碱具有降低胆固醇的潜在效果。但是,在辣椒碱降低血浆胆固醇方面的研究较少,其降胆固醇的效果还不明确,尤其是作用机制还不清楚。因此,本研究分别于在体动物实验和离体细胞培养中开展了对辣椒碱降低血浆胆固醇效果及机理的探索。研究的内容分为四个部分,主要研究结果如下：1.辣椒碱对喂食高胆固醇饲料雌性大鼠血浆及肝脏胆固醇代谢的影响研究不同剂量的辣椒碱喂养不同时间对大鼠血脂和肝脂的影响,探讨辣椒碱对喂食高胆固醇饲料大鼠血浆胆固醇的降低效果。以96只雌性SD大鼠为研究对象,喂食高胆固醇饲料,辣椒碱的灌胃剂量选取每日0mg kg-1·bw、4mg kg-1·bw、8mg kg-1·bw和12mg kg-1·bw,试验期分为4周、8周和12周。结果表明：与空白组相比,每日喂食4mg kg-1·bw辣椒碱8周才能显著降低大鼠血浆中Non-HDL-C、肝脏中TC含量和TG/HDL-C的比值；喂食12周后才能显著降低大鼠血浆和肝脏中的胆固醇。而喂食8mg kg-1·bw和12mg kg-1·bw辣椒碱4周就能显著降低血浆中Non-HDL-C和肝脏中TC含量；喂食8周就能显著调节大鼠血浆和肝脏胆固醇含量,降低大鼠的致动脉硬化指数。另外,各剂量的辣椒碱喂食4周就能显著增加大鼠小肠内容物中总胆汁酸含量,但只有喂食12mg kg-1·bw辣椒碱才能显著增加大鼠粪便中总胆汁酸和中性固醇含量。说明辣椒碱喂食辣椒碱对大鼠体重、血脂和肝脂的降低作用存在剂量效应和时间效应。2.辣椒碱对卵巢切除大鼠胆固醇代谢及机理的研究人们在日常生活中主要通过食入辣椒摄取辣椒碱。本节主要研究了辣椒中的其它化学成分对辣椒碱降胆固醇效果的影响,以及辣椒碱和全辣椒降低机体胆固醇的作用机理。将32只雌性SD大鼠随机分为两组,24只进行双侧卵巢切除手术,8只进行伪切除手术(Sham-CON)。恢复一周后,24只双侧卵巢切除大鼠按体质量随机分成3组,每组8只,分别为卵巢切除空白组(OVX-CON)、卵巢切除辣椒碱组(OVX-CAP)和卵巢切除朝天椒组(OVX-RP)。Sham-CON组和OVX-CON组喂食高胆固醇饲料；OVX-CAP喂食添加了0.01%辣椒碱的高胆固醇饲料；OVX-RP畏食添加了2%朝天椒粉末的高胆固醇饲料(与OVX-CAP含相同量的辣椒碱)。结果表明：卵巢切除术能显著增加卵巢切除大鼠血浆中TG和TC,尤其是LDL-C浓度增加最明显；另外,卵巢切除大鼠肝脏中甘油三酯和胆固醇的积聚也显著增加。然而,在饲料中添加辣椒碱和朝天椒能通过降低血浆TC、LDL-C和TG含量,很好的改善卵巢切除大鼠血浆中的脂蛋白水平；并能降低卵巢切除大鼠肝脏中胆固醇和甘油三酯的浓度；阻止脂滴在卵巢切除大鼠肝脏中的积聚。这些数据表明,辣椒碱和朝天椒能阻止雌激素缺乏和高胆固醇饲料引发的高脂血症。辣椒碱和朝天椒的摄入对肝脏中HMG-CoA还原酶、回肠中IBABP和ASBT的蛋白及mRNA的表达没有显著性影响,但能显著上调卵巢切除大鼠肝脏中CYP7A1和TRPV1蛋白及mRNA的表达,并且能增加小肠内容和粪便中总胆汁酸的含量,增加粪便中中性固醇的含量。辣椒碱和朝天椒降低机体胆固醇的可能机制是：促进肝脏中胆固醇合成胆汁酸,并增加粪便中总胆汁酸和中性固醇的排泄。全辣椒降胆固醇的效果优于辣椒碱。3.膳食纤维对辣椒碱生理功效的协调作用辣椒中含有丰富的纤维素,为探讨纤维素对辣椒碱降低胆固醇效果中的协调作用,研究了辣椒碱与纤维素对大鼠血脂、肝脂的降低效果,及肠道发酵环境的影响。以32只双侧卵巢切除SD大鼠为研究对象,大鼠分为空白组(CON)：喂食高胆固醇饲料；高辣椒碱组(HCAP):喂食添加0.01%的辣椒碱的高胆固醇饲料；纤维素组(DF)：喂食添加5%的纤维素的高胆固醇饲料；纤维素+辣椒碱组(DF+LCAP):喂食添加5%的纤维素+0.005%的辣椒碱的高胆固醇饲料。结果表明：饲料中添加DF+LCAP能显著降低卵巢切除大鼠体重增加量、血脂和肝脂水平,增加粪便胆汁酸和中性固醇的分泌；其对大鼠胆固醇降低效果优于HCAP。另外,0.01%的辣椒碱能显著增加大鼠盲肠内容物的pH值和游离氨含量,并能显著增加有害微生物大肠杆菌和肠球菌的数量,减少有益微生物乳酸杆菌和双歧杆菌的数量。而DF+LCAP能显著增加大鼠盲肠内容物中短链脂肪酸含量和有益微生物数量,降低盲肠内容物pH值和游离氨含量。说明纤维素和辣椒碱在卵巢切除大鼠胆固醇代谢中具有协同作用,纤维素能降低辣椒碱的食用剂量及其对肠道健康潜在的损伤。4.辣椒素受体在辣椒碱降胆固醇中的调节作用肝脏是胆固醇代谢的主要器官。为探讨辣椒素受体TRPV1在辣椒碱影响机体胆固醇代谢环节中的作用,选用人肝L02细胞作为研究对象,以TRPV1激动剂(辣椒素)及其特异性拮抗剂(辣椒卓平)作为干预手段,研究了0、10nmol/L、100nmol/L、1μmol/L和10μmol/L辣椒素对人肝L02细胞中胆固醇及胆固醇代谢相关基因表达的影响。结果表明：辣椒素处理48h可呈剂量依赖性的降低人肝L02细胞和培养基中的总胆固醇,增加培养基中的总胆汁酸含量,其中1μmol/L和10μmol/L的辣椒素效果较好。同时,10nmol/L和100nmol/L的辣椒素对HMG-CoA还原酶的表达无显著影响；而1μmol/L和10μmol/L的辣椒素能显著下调HMG-CoA还原酶的表达。各辣椒素剂量均能显著上调肝脏CYP7A1和TRPV1的蛋白和mRNA表达。在辣椒卓平阻断TRPV1的实验中发现,1μmol/L的辣椒素可显著上调CYP7A1的表达,抑制HMG-CoA还原酶的表达；应用TRPV1受体拮据剂辣椒卓平则明显阻断辣椒素的作用。说明辣椒素降低人肝细胞中胆固醇可能的机制是：低剂量的辣椒素主要通过促进胆固醇转化为胆汁酸；中、高剂量的辣椒素不仅促进胆固醇转化为胆汁酸,还能减少肝脏中胆固醇的合成。辣椒素可能是通过激活辣椒素受体TRPV1调节胆固醇代谢的相关基因。更多还原

【Abstract】 Hypercholesterolemia is the main risk factor of atherosclerosis and coronary heart disease, which threaten life and health of human being severely. Therefore, control and decrease of blood cholesterol level is important for preventing the development of coronary heart disease. The cholesterol-lowering functional foods and nutraceuticals are possible alternative therapies for lowering plasma total cholesterol, especially for patients, whose blood cholesterol level is marginally high but not high enough to warrant the prescription of cholesterol-lowering medication.Capsaicinoids refer to a group of pungent compounds and the main active components of chili peppers. They are the focus of numerous studies because of their extensive biological and physiological properties. Recently, some studies reported that capsaicinoids can promote energy metabolism and suppress the accumulation of body fat, prevent hypertension and improve cardiovascular function. In addition, capsaicinoids possess antioxidant activities that can prevent the oxidation of human LDL. These data suggest that capsaicinoids exhibit cholesterol-lowering effects. However, very little information is available on the effect of administration of capsaicinoids on different aspects of cholesterol metabolism in Vivo and Viro. What’s more, the mechanisms by which capsaicinoids affect cholesterol homeostasis have not been completely clarified yet. The present study aims to investigate the influence of capsaicinoids on the change in cholesterol metabolism and its mechanism. Four parts of work were conducted and the corresponding results are as following:1. The effect of capsaicinoids on plasma and hepatic lipids in female rats fed with high cholesterol dietThis study investigated the doses effect and time effect of capsaicinoids on plasma lipid and hepatic lipid in the rats. Female Sprague-Dawley rats were fed with high-cholesterol diet and orally administered0mg kg-1·bw,4mg kg-1·bw,8mg kg-1·bw and12mg kg-1·bw capsaicinoids daily for4weeks,8weeks and12weeks.The results demonstrated that the plasma Non-HDL-C, TG/HDL-C, and hepatic cholesterol were significantly decreased after treating the rats with4mg kg-1·bw capsaicinoids for8weeks, the plasma lipids and hepatic lipids were remarkably decreased after12weeks, compare with control group. However, the plasma Non-HDL-C and hepatic cholesterol were significantly decreased administrated with8mg kg-1·bw or12mg kg-1·bw capsaicinoids only for4weeks, and effective cholesterol-lowering actions were exhibited after8weeks. What’more, the total bile acid levels in small intestinal contents were significantly increased by treated with each dose of capsaicinoids after4weeks. While only12mg kg-1·bw capsaicinoids could elevate the amount of total bile acid and sterol in feces. In conclusion, the hypocholesterolemic effect of capsaicinoids exhibited dose effect and time effect.2. The influence of capsaicinoids on the change in cholesterol metabolism associated with estrogen deficiency in ovariectomized ratsPeople ingest capsaicin mainly through eating chili in daily life. This study investigated the effect and mechanism of action of capsaicinoids and red pepper on cholesterol metabolism in ovariectomized (OVX) rats. Female Sprague-Dawley rats were subjected to sham operation or ovariectomy. After recovery,24OVX rats were randomly divided into three dietary groups consisting of8rats each using a randomized block design. The sham group (sham-CON) and OVX control group (OVX-CON) were fed with high-cholesterol diets, whereas the capsaicinoids group (OVX-CAP) fed with high-cholesterol plus0.01%capsaicinoids diets, and the red pepper group (OVX-RP) fed with high-cholesterol plus2%red pepper powder diets for3weeks.The results demonstrated that the plasma triacylglycerol and cholesterol concentrations, particularly the LDL-C level, were significantly increased, and triacylglycerol and cholesterol accumulations in the liver were elevated in the OVX rats. However, supplementation of capsaicinoids in diet can favorably modify the plasma lipoprotein profile by decreasing plasma TC, LDL-C, and TG, and reducing the concentrations of hepatic cholesterol and triacylglycerol in OVX rats. The hepatic fat-droplet accumulation was ameliorated by the capsaicinoids-supplemented diet in the OVX-CAP group. These data suggest that capsaicinoids can prevent ovarian hormone deficiency induced hypercholesterolemia. Western blot and real-time PCR analyses revealed that capsaicinoids and red pepper significantly enhanced the expressions of hepatic CYP7A1and TRPV1but did not affect the expression of HMG-CoA reductase, IBABP and ASBT in the OVX rats. In conclusion, Capsaicinoids have cholesterol-lowering effects in OVX rats. The hypocholesterolemic activity of capsaicinoids is caused by the stimulating conversion of cholesterol to bile acids by up-regulation of CYP7A1expression and the increase in fecal total bile acid excretion. Red pepper had a greater efficacy in producing beneficial anti-hyperlipidemic effects in the cholesterol-fed OVX rats.3. The synergistic effect of capsaicinoids and dietary fiber in regulation of cholesterol homeostasisThis study investigated the synergistic effect of capsaicinoids and dietary fiber on cholesterol metabolism in ovariectomized rats. Female Sprague-Dawley rats were performed to ovariectomy. After recovery,32OVX rats were randomly divided into four dietary groups consisting of8rats each using a randomized block design. The OVX control group (CON) was fed with high-cholesterol diets, the dietary fiber group (DF) was fed with high-cholesterol plus5%fiber diets, the high capsaicinoids group (HCAP) was fed with high-cholesterol plus0.01%capsaicinoids diets, and the capsaicinoids+dietary fiber group (DF+LCAP) was fed with high-cholesterol plus5%fiber and0.005%capsaicinoids diets for4weeks.The results demonstrated that body weight gain, plasma lipids and hepatic lipids levels were significantly decreased, while the amount of total bile acid and sterol in feces were dramatically increased in DF+LCAP group rats. What’ more, the concentration of free ammonia and pH of cecal content were significantly increased in HCAP group rats. The0.01%dose of capsaicinoids could promote the growth of Escherichia coli and Enterococcus, and inhibit Bifidobacterium and Lacticacidbacteria in cecal content. However, the administration of5%dietary fiber+0.05%capsaicinoids remarkable increased the concentration of SCFA and the amount of Bifidobacterium and Lacticacidbacteria, and decreased the pH and free ammonia of cecal content in the OVX rats. In conclusion, the combination of dietary fiber and capsaicinoids work synergistically in regulating the homeostasis of cholesterol in OVX rats. In addition, dietary fiber also modifies the injury caused by0.01%capsaicinoids.4. Regulating mechanism of TRPV1on cholesterol metabolism in human hepatocyteLiver is the main organ for endogenous cholesterol synthesis. This study investigated the interaction of administrated0,10nmol/L,100nmol/L,1μmol/L and10μmol/L capsaicin with the mRNA and protein expressions of key receptors and enzymes involved in cholesterol metabolism in human hepatocyte L02. We also used the TRPV1 antagonist capsazepine to explore the involvement and hypocholesterolemic mechanism of TRPV1.The results demonstrated that after treating the human hepatocyte L02with capsaicin48h, the concentration of cholesterol in cell and medium were decreased, while the total bile acid in medium was significantly increased compared with the control group as the dose of capsaicin increased. The1μmol/L and10μmol/L capsaicin have the best effect on the cholesterol-lowering action. In addition, Western blot and real-time PCR analyses revealed that10nmol/L and100nmol/L capsaicin had no effect on the protein and mRNA expression of HMG-CoA reductase, while the1μmol/L and10μmol/L cpacaicin could significantly enhance its expression. The protein and mRNA expression of CYP7A1and TRPV1were dramatically elevated as the capsaicin dose increased.The blockage of TRPV1was obtained by antagonist of TRPV1named capsazepin. In the human hepatocyte L02, the protein and mRNA levels of CYP7A1were up-regulated while the levels of HMG-CoA reducatase were down-regulated by1μmol/L capsaicin, but not in the presence of capsazepine. In conclusion, the cholesterol-lowering effect low dose of of capsaicin in human hepatocyte L02ascribed to stimulating conversion of cholesterol to bile acid. The1μmol/L and10μmol/L capsaicin inhibited the endogenous cholesterol synthesis and promoted cholesterol metabolism. Activation of TRPV1by capsaicin could modulate the expressions of genes involved in cholesterol metabolisms.更多还原