【作者】 张晾；

【导师】 潘杰； 耿越；

【作者基本信息】 山东师范大学 ， 细胞生物学， 2009， 博士

【摘要】 心脑血管疾病是目前危害人类健康的主要杀手,而高脂血症、动脉粥样硬化(atherosclerosis, AS)是上述疾病的病理生理基础。据世界卫生组织2001年统计,全球因心脑血管疾病导致的死亡人数占全部死亡人数的30%,且在逐年递增。血脂异常(高脂血症)是AS和冠心病的主要危险因素之一。日常膳食结构不合理是诱发血脂异常的重要因素。已有的研究表明:炎症贯穿于AS发生发展的各个阶段,炎症机制已被多数学者所认可。多不饱和脂肪酸(polyunsaturated fatty acids, PUFAs)是人体必需脂肪酸,自身不能够合成,必需从外源食物中获得,根据不饱和键的位置分为n-6系和n-3系PUFAs。膳食脂肪酸构成比,尤其是n-6与n-3 PUFAs比值对血脂代谢及免疫功能有不同影响,因此,膳食PUFAs摄入量及两者的最佳比例至关重要。目前各国关于膳食脂肪酸构成比的推荐值不尽相同,而且仍存在争议,其在体内对脂代谢和炎症反应的调控规律尚不清楚。对于高脂血症以及AS患者,他们的日常膳食脂肪组成将影响着病情的发展进程,而对于已经患有高脂血症和AS相关疾病人群的日常膳食n-3与n-6 PUFAs最佳比率仍不清楚。ApoE基因缺失(apoE deficient, apoE-/-)小鼠在普通标准饮食条件下能够自发产生AS病变,且发病过程与人类AS极为相似,是研究人类AS的理想模型,为研究人类AS的发病机理以及干预治疗提供了极大的方便。因此,本研究以8周龄雄性apoE-/-小鼠为模型,模拟人类家族性Ⅲ型高脂蛋白血症和AS患者,通过调整膳食中n-6/n-3 PUFAs不同比率(分别为组1:1.28;组2:5.03;组3:9.98,组4:68.26)干预小鼠6周和13周,从脂代谢、炎症反应等方面探讨不同比率n-6/n-3 PUFAs不同干预时间对AS发展的影响及分子机制,以期为上述患病人群的日常膳食提供理论依据。本研究利用血生化分析、ELISA、气-质联、超速离心、比色法等分析技术,real-time RT-PCR以及Western-Blot等分子生物学技术,冷冻切片、常规病理染色等病理形态学分析实验技术,对不同饮食干预小鼠血脂和血清炎症因子水平、主动脉AS病变、肝脏脂质等进行分析,同时对影响脂代谢相关的含载脂蛋白B100(apolipoprotein B100, apoB100)脂蛋白代谢、高密度脂蛋白(high density lipoprotein, HDL)代谢以及炎症反应等相关基因的表达进行了系统分析,结果如下:1.不同比率n-6/n-3 PUFAs对apoE-/-小鼠AS的影响各组小鼠经不同比率n-6/n-3 PUFAs饮食干预,体重增长无显著差异。小鼠主动脉根部以及全长主动脉AS斑块面积分析表明,饮食干预6周,组3(n-6/n-3 PUFAs为9.98)饮食小鼠具有最低的主动脉根部斑块面积,此时AS处于脂质条纹期。低比率n-6/n-3 PUFAs(组1:1.28)饮食干预13周能够显著抑制apoE-/-小鼠主动脉根部以及全长主动脉AS病变,此时AS处于纤维斑块病变期。高比率n-6/n-3 PUFAs饮食(组4:68.26)主动脉根部及全长主动脉AS病变程度最重。而且随着饮食中n-6 PUFAs含量的增加,病变程度逐渐加重。2.不同比率n-6/n-3 PUFAs对apoE-/-小鼠AS干预机制探讨(1)不同比率n-6/n-3 PUFAs对apoE-/-小鼠脂质代谢的影响:①不同饮食对小鼠血脂水平的影响。小鼠人工饮食干预/喂养后血脂水平结果表明,干预6周后,血清游离胆固醇(free cholesterol, FC)、胆固醇酯(cholesterol easter, CE)和总胆固醇(total cholesterol, TC)水平均没有受饮食成分的改变而发生明显的改变;干预13周后,组3(n-6/n-3 PUFAs为9.98)饮食具有最低的血清TC水平。小鼠饮食干预6周和13周,组3饮食显著降低了低密度脂蛋白胆固醇酯(LDL-C)水平,而组4(n-6/n-3 PUFAs为68.26)饮食显著升高了血清甘油三酯(triglyceride, TG)水平和高密度脂蛋白胆固醇酯(HDL-C)水平。②不同饮食对小鼠肝脏脂质水平的影响:小鼠肝脏脂质分析表明,饮食干预6周,组1(n-6/n-3 PUFAs为1.28)小鼠肝脏组织具有最低的TC、TG水平和脂质积累,且随着饮食中n-6/n-3PUFAs比率的升高而升高,脂质积累逐渐加重。低比率n-6/n-3 PUFAs(组1)能够显著降低肝脏组织中的TC、TG水平以及脂质积累。③不同饮食对血清和肝脏组织中脂肪酸成分的影响:血清和肝脏脂质经脂质提取,甲酯化反应,GC-MS分析检测,结果表明,随着饮食中n-6/n-3 PUFAs比率的增加,血清和肝脏组织中亚油酸(linoleic acid, LA)、花生四烯酸(arachidonic acid, AA)和总n-6 PUFAs含量逐渐增加,而α-亚麻酸(alpha-linolenic acid, ALA)、二十碳五烯酸(eicosapentaenoic acid, EPA)含量逐渐降低。④不同饮食对小鼠含apoB100脂蛋白代谢影响:结果显示,组1(n-6/n-3 PUFAs为1.28)饮食能够显著降低血清ApoB100水平和肝脏β-羟基β-甲基戊二酰辅酶A (β-hydroxy-β-methyl glutaryl CoA, HMG-CoA)还原酶活性。与对照组相比,组1、组2饮食能够显著降低肝脏脂肪酸合成酶(fatty acid synthase, FAS)、HMG-CoA还原酶、乙酰胆固醇乙酰转移酶2(acyl cholesterol acyl transferase 2, ACAT2)、低密度脂蛋白受体(low density lipoprotein receptor, LDLR)、低密度脂蛋白受体相关蛋白(low density lipoprotein receptor related protein, LRP)、固醇调节元件结合蛋白1c(sterol regulatory element binding protein, SREBP1c)、肝X受体α(liver X receptor alpha, LXRα)基因的转录水平,显著升高过氧化物酶体增殖物激活受体(peroxisome proliferator-activated receptor, PPARα)基因的转录水平;组4饮食显著升高apoB100基因的mRNA水平。各组饮食没有影响到肝脏脂蛋白脂酶(lipoprteinlipase, LPL)基因的转录水平。以上结果说明,低比率n-6/n-3 PUFAs饮食可能通过降低肝脏脂肪酸和胆固醇的合成过程中重要酶的表达而降低脂肪酸和胆固醇的合成,但同时降低了肝细胞的LRP和LDLR基因的表达,不利于血清中的极低密度脂蛋白胆固醇酯(very low density lipoprotein cholesterol ester, VLDL-C)和LDL-C进入肝脏。⑤不同饮食对小鼠HDL-C代谢影响:结果表明,高比率n-6/n-3 PUFAs(68.26)饮食能够显著升高血清中载脂蛋白A-I(apolipoprotein A-I, apo A-I)和HDL-C水平。低比率n-6/n-3 PUFAs饮食能够显著升高血清卵磷脂胆固醇脂酰转移酶(lecithin-cholesterol acyltrasferase, LCAT)活性,而且随着饮食中n-3 PUFAs含量的增加而增强。高比率n-6/n-3 PUFAs(组4)饮食明显升高apo A-I、三磷酸腺苷结合盒A1(ATP binding cassette transporter A1, ABCA1)基因的表达,明显抑制apo A-II基因的表达,而低比率n-6/n-3 PUFAs饮食显著降低apo A-I、三磷酸腺苷结合盒A1(ATP binding cassette transporter A1, ABCA1)、LCAT的转录水平。说明,n-6 PUFAs可能通过增强apo A-I和ABCA1的表达升高HDL-C水平。低比率n-6/n-3 PUFAs(组1-组3)饮食能够显著升高主动脉组织中ABCA1基因的表达,而且随着饮食中n-3 PUFAs含量的升高而逐渐增强。说明低比率n-6/n-3 PUFAs(1.28-9.98)饮食有利于主动脉组织的胆固醇外流,起到抗AS的作用。(2)不同比率n-6/n-3 PUFAs对apoE-/-小鼠炎症因子的影响①血清、脾脏及主动脉炎性因子分析结果表明,不同饮食干预6周没有显著影响血清促炎因子白介素(interleukin, IL)1β、IL-6和肿瘤坏死因子(tumor necrosis factor, TNF)α的水平及脾脏和主动脉IL-4基因的转录,但组1(n-6/n-3 PUFAs为1.28)饮食显著抑制了脾脏IL-6和TNFα以及主动脉组织CRP基因的转录水平,组4(n-6/n-3 PUFAs为68.26)饮食显著升高了主动脉TNFα的转录水平以及单核细胞趋化蛋白-1(monocyte chemotactic protein-l, MCP-1)和血管细胞黏附分子-1(vascular cell adhesion molecule-1, VCAM-1)mRNA水平。②对炎性因子C-反应蛋白(C-reactive protein, CRP)及其调控因子PPARγ基因表达分析,结果表明,组1(n-6/n-3 PUFAs为1.28)饮食能够显著降低肝脏CRP基因的mRNA和蛋白质水平,同时升高了肝脏PPARγ的转录水平。综上所述,不同比率n-6/n-3 PUFAs饮食干预apoE-/-小鼠6周,低比率n-6/n-3 PUFAs饮食能够显著降低肝脏脂肪酸和胆固醇的合成以及抑制apoB100的表达,但同时降低了LDLR和LRP的表达,结果导致没有降低血清LDL-C水平。然而其主动脉根部AS病变与血清LDL-C趋势是一致的,组3(n-6/n-3 PUFAs为9.98)饮食具有最低的LDL-C水平和最轻的AS病变,因此我们推测在AS发生早期,血脂水平在AS发生中起重要作用,饮食n-6/n-3 PUFAs比率为9.98时能够通过控制LDL-C水平,并在一定程度上抑制AS的发展。对HDL-C代谢相关因子的分析表明,高比率n-6/n-3 PUFAs(组4:68.26)饮食显著升高HDL-C水平,但没有抑制AS发展,说明由高比率n-6/n-3 PUFAs饮食产生的高水平HDL-C在apoE-/-小鼠中并没有发挥抗AS的作用。然而饮食干预至13周,随着AS的进一步发展,组3饮食干预小鼠仍具有最低的LDL-C水平,但此时处于纤维斑块病变时期的AS病变并未随血清LDL-C水平的降低而减轻,而低比率n-6/n-3 PUFAs(组1:1.28)饮食具有最轻的AS病变,说明在AS发展的中后期,低比率n-6/n-3 PUFAs对AS的抑制作用独立于其对血脂水平的影响,通过主动脉ABCA1基因的表达及肝脏、脾脏、主动脉炎症相关基因的表达分析,推测低比率n-6/n-3 PUFAs饮食抗apoE-/-小鼠AS的机制可能是通过增加主动脉胆固醇外流、抑制机体及主动脉局部炎症反应而实现的。基于本实验研究结果,我们认为:在AS发生的早期(脂质条纹期),n-6/n-3 PUFAs比率为9.98饮食干预能够通过降低血清LDL-C水平减缓apoE-/-小鼠AS的发展,而AS发展的中后期(纤维斑块病变期),n-6/n-3 PUFAs比率为1.28饮食能够通过增加主动脉胆固醇外流和抑制促炎因子的表达而抑制apoE-/-小鼠AS的发展。更多还原

【Abstract】 Cardiovascular disease (including hyperlipidemia and atherosclerosis) continues to be the leading cause of morbidity and mortality in the industrialized world. According to the statistical result of the World Health Organization (WHO), the death toll by cardiovascular disease account 30 percent for total death toll in 2001. Moreover, the quantities will be more and more in future. Abnormal serum lipid (especially hyperlipidemia) is one of the main risk factors for atherosclerosis and cardiovascular disease. Improper dietary is the main factor for abnormal serum lipids. The present researches indicated that inflammation ran through all stages of atherosclerosis, which is authorized by researchers.Polyunsaturated fatty acids (PUFAs) which are not synthesized in vivo are the essential fatty acids for human growth and health. PUFAs can be classified in n-3 (omiga-3) fatty acids and n-6 (omiga-6) fatty acids. Both types of fatty acids are precursors of signaling molecules with opposing effects, that modulate membrane microdomain composition, receptor signaling and gene expression. The predominant n-6 fatty acids and its products are important regulators of cellular functions with inflammatory, atherogenic and prothrombotic effects. The n-3 fatty acids antagonize the pro-inflammatory effects of n-6 fatty acids. n-3 and n-6 fatty acids control various genes of inflammatory signaling and lipid metabolism. So, the mounts and the balance of the n-6 and n-3 fatty acids are important for health. The recommendation ratio of n-6/n-3 PUFAs was inconsistent in many countries at present, and there were some disputations. The regulations of lipid metabolism and inflammatory signaling in vivo by n-6 and n-3 fatty acids were unclear. As for the hyperlipidemia and atherosclerosis patients, dietary fatty acids influenced the progress of diseases. The mounts and the balance of n-6/n-3 PUFAs for above patients were also unclear.Apolipoprotein E deficient (apoE-/-) mice which spontaneously develop atherosclerosis with features similar to those observed in humans are the idea model of human atherosclerosis researches. It provides the advantage for the mechanism and intervention of atherosclerosis researches. Therefore, the present experiment observes the anti-atherogenesis effects by dietary different n-6/n-3 PUFAs in apoE-/- mice which similar similar features to those observed in humans type III familial hyperlipoproteinemia. The male mice were randomly assigned to four experimental groups (fed experimental diets) and one control group (fed control diet) at age of 8 wk old (n=12, respectively) for 6 wk and 13 wk. The experimental diets contained 5% (w/w) fat (11.5% total energy), were made by adding safflower oil and perilla seed oil based on the control diet. The n-6/n-3 PUFAs ratios of the experimental diets, for feeding groups 1-4 mice, were 1.28, 5.03, 9.98 and 68.26, respectively. At the end of the feeding period, all mice were sacrificed. The atherogenesis, lipid metabolism and inflammatory response would be analyzed and discussed. We looked forward to providing the dietary fat for humans.The present experiment analyzed the serum lipids, serum cytokines, aortic en face atherosclerotic lesions, liver lipids and explored the possible mechanisms of the apoB100 containing lipoprotein metabolism, high density lipoprotein cholesterol ester (HDL-C) metabolism by different n-6/n-3 PUFAs. The experiment performed using biochemical analysis, enzyme-linked immunosorbent assays (ELISA), gas chromatograph-mass spectrum (GC-MS), ultracentrifugation etc. analytic techniques; real-time RT-PCR, Western-Blot etc. molecular techniques; frozen section and staining etc. pathological morphology techniques. The results showed as follow:1. Different diets on atherosclerotic lesions.The result showed that no significant difference was observed in the growth of mice fed with different experimental diets. The results of atherosclerotic lesions in root and total en face aortic lesions area analysis indicated that the group 3 mice developed smaller area than other groups for 6 wk intervention. However, dietary low ratio of n-6/n-3 PUFAs inhibited significantly the atherosclerotic lesions in root and total en face aortic lesions area compared with the mice that dietary high ratio of n-6/n-3 PUFAs for 13 wk intervention. Moreover, as the dietary ratio of n-6/n-3 fatty acids ascended, so did the areas of aortic lesions.2. The mechanisms of varying the ratios of n-6/n-3 polyunsaturated fatty acids on atherogenesis(1) Different diets on lipid metabolism.①Different diets on serum lipids. After feeding the apoE-/- mice with the diets for 6 wk, no significant differences were found in the serum concentrations of free cholesterol (FC), total cholesterol (TC) and cholesterol ester (CE) among the apoE-/- mouse groups fed with different diets. After feeding the apoE-/- mice with the diets for 6 wk and 13 wk, the serum LDL-C level of group 3 mice was significant lower than those of other groups. The serum of triglyceride (TG) and HDL-C concentration of group 4 mice were significant higher than other groups.②Different diets on liver lipids. The results of liver lipids contents showed that after feeding with the diets for 6 wk, the TC and TG levels of group 1 mice were significant lower than other groups. Moreover, as the dietary ratio of n-6/n-3 fatty acids ascended, so did the TC and TG contents. Dietary low ratio of n-6/n-3 PUFAs significantly decreased TC and TG levels and lipids aggregation in the liver.③Different diets on serum and liver fatty acids. The results of serum and liver fatty acids analysis indicated that as the dietary ratio of n-6/n-3 fatty acids ascended, so did the concentrations of total n-6 fatty acids, arachidonic acid (AA) and 18: 2 n-6 fatty acids in the serum and the liver tissue. Moreover, as the dietary level of n-3 fatty acids declined, the concentration of total n-3 fatty acids and eicosapentaenoic acid (EPA) were reduced in the serum and the liver.④Different diets on apoB100 containing lipoprotein metabolism. After feeding the apoE-/- mice with the diets for 6 wk, the serum ApoB100 level and liverβ-hydroxy-β-methyl glutaryl CoA (HMG-CoA) reductase activity of group 1 mice were significant lower than other groups. Real-time RT-PCR results showed that the transcription levels of genes involved in apoB100 containing lipoprotein metabolism including fatty acid synthase (FAS), HMG-CoA reductase, acyl cholesterol acyl transferase 2 (ACAT2), low density lipoprotein receptor (LDLR), low density lipoprotein receptor related protein (LRP), sterol regulatory element binding protein (SREBP1c), liver X receptor alpha (LXRα) of group 1 and group 2 mice were inhibited significantly compared with the control mice. The hepatic apoB100 mRNA level was increased by the group 4 diet. These results suggested that dietary low n-6/n-3 PUFAs ratios inhibited the synthesis of fatty acids and cholesterol by decreased the enzymes involved in the synthesis of fatty acids and cholesterol. However, at the same time, the expression of LRP and LDLR were inhibited. Thus, it is not benefit to the uptake of VLDL-C and LDL-C.⑤Different diets on HDL-C metabolism. Varying ratios of n-6/n-3 PUFA on HDL-C concentration was assessed with respect to serum apolipoprotein (apo) A-I concentration, endogenous lecithin-cholesterol acyltransferase (LCAT) activities, and mRNA abundance of genes involved in HDL-C metabolism. The results indicated that the group 4 diet significantly increased the HDL-C and apo A-I concentrations in serum compared with the other groups. LCAT activity in serum increased with decreased ratios of n-6/n-3 PUFA. As the dietary ratio of n-6/n-3 fatty acids ascended, so did the mRNA levels of hepatic apo A-I, scavenger receptor B class-1 (SR-B1), LCAT, ATP binding cassette transporter A1 (ABCA1), ABCG1 and LXRα, however, apo A-II mRNA level had a tendency of decline. Group 4 diet up-regulated the apo A-I and ABCA1 and down-regulated the apo A-II transcriptional levels, whereas, group 1 diet down-regulated the mRNA expressions of apo A-I, LCAT, SR-B1 and ABCG1. Our results indicate that high ratio of n-6/n-3 PUFA increases the HDL-C concentration, possibly due to up-regulating the hepatic apo A-I and ABCA1 and down-regulating the apo A-II mRNA levels. Dietary low ratios of n-6/n-3 PUFAs significantly increased the expression of ABCA1 in aorta. Moreover, as the dietary ratio of n-6/n-3 fatty acids descended, the ABCA1 mRNA level increased. The result suggested that low ratios of n-6/n-3 PUFAs were benefit to the cholesterol efflux progress from the aorta.(2) Varying ratios of n-6/n-3 PUFAs diets on inflammatory cytokines in apoE-/- mice.①Serum concentrations of interleukin (IL) -1β, IL-6 and tumor necrosis factor (TNF)αdid not show any statistical difference among the mice fed the different diets. The results of real-time RT-PCR indicated that the group 1 diet inhibited significantly the transcription of pro-inflammatory cytokine IL-6 and TNFαin spleen and CRP in aorta, however, the group 4 diet increased significantly the expression of TNFα, MCP-1 and VCAM-1 in aorta. The expression of IL-4 in spleen and aorta did not show any significance.②In the present study of dietary varying of n-6/n-3 PUFAs ratios and hepatic CRP expression, we observed statistically significant low at mRNA and protein levels in apoE-/- mice fed the low n-6/n-3 PUFA ratios compared with that of the high n-6/n-3 PUFA ratio. Low ratios of n-6/n-3 PUFAs increased the hepatic PPARγmRNA level compared with the high ratio of n-6/n-3 PUFAs, which presumably inhibited the inflammatory status in the liver of mice fed low ratios of n-6/n-3 PUFAs.In summary, the present study showed that dietary varying the ratios of n-6/n-3 PUFAs for 6 and 13 wk influenced the AS development, lipid metabolism and inflammatory response in apoE-/- mice. After 6 wk intervention, dietary low ratio of n-6/n-3 PUFAs inhibited significantly the synthesis of fatty acids and cholesterol in liver and decreased the expression of hepatic apoB100. However, the expression of hepatic LDLR and LRP were also inhibited. Therefore, dietary low ratio of n-6/n-3 PUFAs did not show decreased serum LDL-C level. The present results indicated that there was the same tendency between serum LDL-C concentration and atherosclerotic lesion in root in different groups. The mice of group 3 showed the smallest lesion areas and the lowest LDL-C level. So it suggested that dietary ratio of n-6/n-3 PUFAs (9.98) could inhibited the early stage of AS development via decreasing the serum LDL-C level.However, the results of HDL-C metabolism showed that dietary high ratio of n-6/n-3 PUFAs (68.26) increased significantly HDL-C concentration and Apo A-I, but did not suppress the AS development, suggesting that HDL-C level did not show the anti-atherogenesis in apoE-/-. After 13 wk intervention, the group 3 diet also showed the lowest serum LDL-C, but did not show the lowest atherosclerotic lesions in root and the total en face lesions. On the contrary, the group 1 (low ratio of n-6/n-3 PUFAs) indicated the lowest lesion areas, suggesting that dietary low ratio of n-6/n-3 PUFAs inhibited the AS development independent on the serum lipids at the middle-late stage of AS. In the present study, we analyzed the expression of ABCA1 in aorta, which was responsible for the cholesterol efflux progress from the artery to the liver. The expression of genes involved in the inflammatory response in liver, spleen and artery tissues were also analyzed by real-time RT-PCR. We speculated on the anti-atherogenesis mechanisms that low ratio of n-6/n-3 PUFAs increased the cholesterol efflux progress from aorta and decreased the inflammatory responses and the local inflammatory responses in aorta on the basis of the present results.更多还原