EPA和DHA对小鼠非酒精性脂肪肝治疗作用的研究

发布时间：2018-08-30 09:00

【摘要】：鱼油中的二十碳五烯酸(Eicosapentaenoic acid,EPA)和二十二碳六烯酸(Docosahexaenoic acid,DHA)多不饱和脂肪酸具有多种生物活性,例如调节血脂、改善记忆力、调节脂代谢、增强免疫等功效。目前由于多数针对于非酒精性脂肪肝(Non-alcoholic fatty liver diseases,NAFLD)治疗的研究都是采用EPA和DHA的混合物如鱼油胶囊或者含有的鱼油饮食而不是单一的脂肪酸EPA和DHA,所以仍旧不清楚EPA和DHA对NAFLD是否有独立的生理活性而,这两种多不饱和脂肪酸EPA和DHA可能对NAFLD产生截然的不同的效果。并且这些研究大部分采用的的是市场上比较多的乙酯型或者游离型的n-3多不饱和脂肪酸(Omega-3 polyunsaturated fatty acids,n-3PUFA),但乙酯型的吸收率仅为2l%,游离型的EPA和DHA虽然吸收率为95%,但容易氧化,很不稳定,且对消化道有刺激作用。甘油酯型是鱼油的天然存在形式,符合人体吸收的油脂形式,其人体吸收率明显高于乙酯型鱼油,吸收可以达到60%左右,性质相对稳定。本实验室采用酶法制备了高纯度甘油三酯型EPA(trieicosapentaenoyl glycerol,tri EPA)和DHA(tridocosahexaenoyl glycerol,tri DHA),两者纯度纯度都达到了80%。本论文以上两种高纯度鱼油为研究对象,采用小鼠为动物模型探究了其对NAFLD的调节作用,并探讨其治疗机制。通过在饲料中添加1%的胆固醇和20%的饱和长链脂肪酸建立小鼠NAFLD的模型,研究tri EPA和tri DHA对小鼠非酒精性脂肪的治疗作用。结果显示,tri EPA和tri DHA补充高脂饲料诱导的NAFLD模型小鼠8w后,显著降低了小鼠的肝重系数、血清谷丙转氨酶(Alanine transaminase,ALT)和谷草转氨酶(Aspartate transferase,AST)活性,血清游离脂肪酸(Free fatty acids,FFA)含量,显著降低胰岛素指数,显著降低了肝脏总甘油三脂(Total triglyceride,TG)和总胆固醇(Total cholesterol,TC)水平,显著降低肝脏的脂变程度。由此,tri EPA和tri DHA能够减轻胰岛素抵抗,减少肝脏脂肪堆积,NAFLD有积极的预防和改善作用,而两者在这些方面并未出现显著性差异。维生素E(α-生育酚,a-tocopherol)作为天然的抗氧化剂能够清除自由基和抑制脂质过氧化作用。为进一步加强tri EPA和tri DHA对小鼠NAFLD脂肪肝的治疗效果,在本次研究中小鼠额外加入维生素E。结果显示,补充8w后,相对于tri EPA,tri DHA对肝脏谷胱甘肽过氧化物酶(Glutathione peroxidase,GSH-Px)和谷胱甘肽(Glutathione,GSH)的提高更明显,具有更好的抗氧化能力。相对于n-3 PUFA,n-3 PUFA+维生素E补充液对小鼠GSH、GSH-Px和SOD(超氧化物歧化酶,Superoxide dismutase)均有显著提高,ALT和AST显著降低。在其他指标上未存在显著性差异。单独维生素E的补充并未改善以上指标而与模型组较为接近,但维生素E可与n-3 PUFA协同治疗NAFLD。进一步了解n-3 PUFA对小鼠NAFLD的治疗机理,本论文研究了tri DHA和tri EPA对小鼠肝脏脂代谢相关基因的表达以及脂肪酸组成的的影响。RT-PCR结果显示,补充tri EPA和tri DHA的小鼠,肝脏SREBP-1c表达量显著下降,但PPARα并未显著性升高,说明tri EPA和tri DHA并未在PPARα转录水平上其改善作用,但有研究显示PUFA主要是通过结合PPARα,改变其构象使其对应的调控的转录产物提高。肝脏脂肪酸组成分析显示,肝脏脂肪酸中多不饱和脂肪酸总量明显提高,而花生四烯酸含量显著下降。tri EPA和tri DHA补充组的DHA含量显著高于EPA含量,因此tri EPA和tri DHA可通过调节肝脏脂肪酸组成和脂代谢相关基因的表达来改善小鼠NAFLD,而EPA可能主要作为DHA的合成的前体。
[Abstract]:Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) polyunsaturated fatty acids in fish oil have a variety of biological activities, such as regulating blood lipids, improving memory, regulating lipid metabolism, and enhancing immune function. Both EPA and DHA have been studied in combination with fish oil capsules or diets containing fish oil rather than single fatty acids EPA and DHA, so it is still unclear whether EPA and DHA have independent physiological activity to NAFLD and the two polyunsaturated fatty acids EPA and DHA may have distinct effects on NAFLD. Most of these studies were carried out using ethyl-3 polyunsaturated fatty acids (Omega-3 polyunsaturated fatty acids, n-3PUFA), which were widely used in the market. However, the absorptivity of ethyl-3 polyunsaturated fatty acids (EPA and DHA) was only 2 l%. Although the absorptivity of EPA and DHA was 95%, they were easily oxidized, unstable and irritating to the digestive tract. Glycerol ester is a natural form of fish oil, which conforms to the form of oil absorbed by human body. The absorption rate of glycerol ester is higher than that of ethyl ester fish oil. The absorption rate of glycerol ester can reach about 60% and its properties are relatively stable. High purity EPA (trieicosapentaenoyl glycerol, tri EPA) and DHA (tridocosahexaenoyl glycerol) were prepared by enzymatic method in our laboratory. Rol, tri DHA, the purity of both reached 80%. In this paper, the above two kinds of high purity fish oil as the research object, using mice as animal models to explore its regulatory effect on NAFLD, and explore its therapeutic mechanism. By adding 1% cholesterol and 20% saturated long-chain fatty acids in the diet to establish mice NAFLD model, tri EPA and t were studied. The results showed that tri-EPA and tri-DHA supplementation significantly decreased the liver weight coefficient, the activities of serum Alanine transaminase (ALT) and aspartate transferase (AST), and serum free fatty acid (Free fatty acid) in NAFLD mice induced by high-fat diet for 8 weeks. The contents of CIDS and FFA decreased the insulin index, the levels of total triglyceride (TG) and total cholesterol (TC) in the liver, and the degree of hepatic steatosis. Vitamin E (a-tocopherol) as a natural antioxidant can scavenge free radicals and inhibit lipid peroxidation. In order to further enhance the therapeutic effect of tri EPA and tri DHA on NAFLD fatty liver in mice, vitamin E was added to the mice in this study. Compared with tri-EPA, tri-DHA had more significant effects on liver glutathione peroxidase (GSH-Px) and glutathione (GSH) and had better antioxidant capacity. Compared with n-3 PUFA, n-3 PUFA + vitamin E supplement, tri-DHA had better antioxidant capacity on mice GSH, GSH-Px and SOD (superoxide dismutase, Superoxide dismu). There was no significant difference in other indicators. Vitamin E supplementation alone did not improve the above indicators, but vitamin E and n-3 PUFA could cooperate in the treatment of NAFLD. To further understand the mechanism of n-3 PUFA in the treatment of NAFLD in mice, tri DHA and tri EPA were studied in this paper. The results of RT-PCR showed that the expression of SREBP-1c in liver of tri-EPA and tri-DHA supplemented mice decreased significantly, but PPARa did not increase significantly, indicating that tri-EPA and tri-DHA did not improve the transcription of PPARa, but some studies showed that PUFA was mainly PUFA. The content of polyunsaturated fatty acids in liver fatty acids was significantly increased, while the content of arachidonic acid was significantly decreased. The content of DHA in tri EPA and tri DHA supplementation groups was significantly higher than that in EPA, so tri EPA and tri DHA could pass through. Mouse NAFLD was improved by regulating fatty acid composition and lipid metabolism related gene expression in liver, and EPA may be the main precursor of DHA synthesis.
【学位授予单位】：浙江海洋学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R575.5

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