EPA及其前体ALA对草鱼脂肪蓄积的调控作用及机制分析

发布时间：2018-01-10 23:31

本文关键词：EPA及其前体ALA对草鱼脂肪蓄积的调控作用及机制分析　出处：《西北农林科技大学》2017年博士论文　论文类型：学位论文

【摘要】：脂肪组织被认为是机体最大的能量储存器官,同时也是机体一个重要的内分泌器官。其在能量平衡、动物繁殖、器官保护、性别选择等多个方面具有重要作用。当前,随着鱼粉、鱼油资源量的下降和价格的不断攀升,大豆油、红花油、葵花籽油等富含n-6系列脂肪酸而相对缺乏n-3系列脂肪酸的植物性油脂源被广泛应用于草鱼的商业饲料中。除此之外,为了应对巨大的商业压力,高能商业饲料也广泛被使用以促进养殖鱼类的生长速度,加速其上市时间。然而,高能以及n-6/n-3比率失衡的日粮极易导致养殖鱼类脂肪的过度蓄积,引发诸多问题,如死亡率上升、摄食量下降、免疫力下降、品质降低等,最终影响经济效益。因此,对脂肪过度蓄积的调控显得尤为重要。目前,已有学者通过在体与离体两种模型,对脂肪蓄积的调控进行了一定的研究,发现二十碳五烯酸(Eicosapentaenoic acid,EPA)可以通过促进脂肪细胞凋亡、抑制脂肪合成等途径降低脂肪蓄积,但其具体机制尚不明确。Alpha-亚麻酸(Linolenic acid,ALA)是重要的n-3 PUFAs(polyunsaturated fatty acids)之一,也是草鱼的必需脂肪酸之一。有研究指出,日粮中添加一定量的ALA可以降低脂肪蓄积。鉴于EPA有限的来源而导致的使用限制,以及草鱼具有将C18的ALA转化成EPA的能力,本论文旨在探讨EPA及其前体ALA这两种重要n-3 PUFAs对草鱼脂肪蓄积调控的作用及机制,以期为淡水鱼类脂质代谢提供一定的理论参考。主要研究结果如下:1.EPA影响草鱼腹腔脂肪组织脂质蓄积的在体与离体研究。采用EPA的在体饲喂,结合草鱼脂肪细胞的离体培养,对试验鱼生长、生物学性状、血清生化、脂肪酸组成等进行评估,并采用HE染色、RT-PCR、流式细胞术等检测方法确定了EPA对草鱼脂肪蓄积的具体作用及机制。结果表明:用富含0.52%EPA的日粮饲喂草鱼,其生长并未受到影响。经过3周的饲养,试验鱼的腹腔脂肪指数显著降低,而当饲养时间延长到6周,腹腔脂肪指数无显著差异。同时,在体和离体结果均显示EPA显著促进了脂肪组织/细胞脂肪组织甘油三酯脂酶(ATGL)、激素敏感性脂酶(HSL)-a、过氧化物酶体增殖激活物受体(PPAR)-γ、脂蛋白脂酶(LPL)、caspase 3a、caspase 3b的mRNA水平以及caspase 3的酶活性,下调了肝X受体(LXR)-α、固醇调节原件结合蛋白(SREBP)-1c、脂肪酸合成酶(FAS)的基因表达以及Bcl-2/Bax的比率。离体结果表明,EPA以剂量和时间依赖型的方式抑制草鱼前体脂肪细胞的活力,且当其浓度达到650μM,作用4天时,处理组细胞活力只有对照组的约50%。此时,流式细胞术检测凋亡的结果显示,处理组凋亡率高达37%,显著高于对照组。且ROS含量也显著上升。而抗氧化剂α-生育酚的使用可显著缓解EPA诱导的凋亡。另一方面,EPA的使用可显著促进脂肪组织/细胞的EPA含量。2.epa促进草鱼脂肪细胞表达甘油激酶基因。根据试验一的研究结果,为深入研究epa短期饲喂能降低脂肪含量,而长期饲喂则无此作用的内在机制。首先,克隆了草鱼甘油激酶(gyk)基因部分序列,发现其在肝胰脏、脂肪组织和肾脏中表达最高。随着脂肪细胞的发现,其表达呈现上升的趋势。用100μm的epa处理细胞6、12、24h,发现其能显著促进gyk的基因表达和酶活性,同时atgl和pparγ的基因表达也显著上调。为进一步探究epa诱导草鱼脂肪细胞表达gyk基因的潜在机制,分别以atgl和pparγ的抑制剂hy-15859、gw9662孵育细胞,结果显示此两种抑制剂均能减弱epa对gyk的诱导作用。此外,对细胞进行饥饿24h处理,虽然atgl显著上调,但gyk的酶活性并未发生变化。而包括α-亚麻酸(18:3n-3,ala)、二十二碳六烯酸(22:6n-3,dha)、亚油酸(18:2n-6,lna)在内的pparγ的其余配体脂肪酸,均能促进gyk的基因表达和酶活性。3.日粮添加α-亚麻酸对草鱼稚鱼脂质代谢,脂肪酸组成及腹腔脂肪细胞凋亡的影响。分别用富含0.0%、1.0%、2.0%ala的日粮饲喂草鱼稚鱼8周。结果发现,日粮中添加ala对试验草鱼的生长无负面影响。同时,腹腔脂肪指数在2.0%组最低,而caspase3、caspase8、caspase9的酶活性此组最高。与其他两组相比,2.0%的ala显著下调了fas、srebp-1c的基因表达以及bcl-2/bax的比率。同时,2.0%组试验鱼腹腔脂肪组织出现更多的凋亡样线粒体,主要表现为线粒体肿胀、嵴消失等。另一方面,对脂肪酸生物合成途径的基因表达检测发现,肌肉和肝胰脏中,ala组脂肪酸延长酶(elo)和脂肪酸去饱和酶(fad)的表达显著高于对照组,而在腹腔脂肪组织中也呈现出相反的趋势。然而,日粮中添加ala均能显著促进肌肉、肝胰脏和腹腔脂肪组织的epa和dha的含量。4.α-亚麻酸对草鱼脂肪细胞凋亡的影响及其机制分析。利用草鱼离体脂肪细胞培养平台,用ala分别对草鱼前体以及成熟脂肪细胞进行处理。结果表明,ala(0-800μm)并未对细胞活力产生抑制作用,且当处理时间为2和4天时,ala促进细胞活力。此外,不同分化阶段的前体细胞具有相同的应答模式。分别用100、650、1300μm的ala处理前体细胞2和4天,随着ala浓度的升高,caspase3a、caspase3b和caspase8的mrna水平显著下降,而bcl-2/bax则呈现出相反的变化趋势。而当处理时间延长到8天,caspase9、caspase3b和caspase8的mrna水平在1300μm组最高,而bcl-2/bax在此组最低。对于成熟脂肪细胞,经过2天的处理,随着ala浓度的升高,caspase3a、caspase3b、caspase8和caspase9的基因表达显著下调,而bcl-2/bax则呈现出相反的变化趋势。然而,经过8天的处理,caspase3a、caspase3b、caspase8和caspase9的基因表达显著上调,而bcl-2/bax则呈现出先上升后下降的趋势,且最低值出现在1300μm组。av/pi凋亡染色结果也表明,当处理时间为2和4天时,ala主要表现为抗凋亡,而当处理时间延长到8天,650和1300μm组的凋亡率显著高于其他两组。对于脂肪酸组成的检测发现,用1300μm的ala处理8天,前体脂肪细胞处理组的ala和dha含量显著高于对照组,而成熟脂肪细胞处理组只有ALA的含量显著高于对照组。综上所述,1)日粮中添加适量的EPA在一定条件下可以通过促进甘油三酯水解、促进脂肪细胞凋亡、抑制脂肪酸从头合成等途径降低草鱼腹腔脂肪蓄积;2)不同于哺乳动物,草鱼脂肪组织可以表达GyK基因。EPA通过诱导GyK的表达,促进甘油三酯的重新酯化,从而出现补偿性合成。且EPA对GyK的诱导受到PPARγ的调控,ATGL也参与调控这一过程;3)日粮中2.0%的ALA可以过促进脂肪细胞凋亡、抑制脂肪酸的从头合成等途径降低草鱼腹腔脂肪蓄积。且ALA的添加可以显著促进鱼体肌肉n-3长链多不饱和脂肪酸的沉积,从而提高鱼体的品质;4)ALA本身可以促进成熟脂肪细胞凋亡,而对于前体脂肪细胞,ALA可能通过其生物合成产物发挥作用。草鱼前体和成熟脂肪细胞在利用ALA合成DHA的能力上可能具有差异。
[Abstract]:Adipose tissue is considered to be the biggest energy storage organ, the body is also an important endocrine organ. The balance of energy, animal breeding, protection of organs, plays an important role in the aspects of gender selection. At present, with fish meal, fish oil resources volume decline and prices rising, soybean oil, safflower oil. Sunflower seed oil rich in n-6 fatty acids and relative lack of commercial feed n-3 series of fatty acids in vegetable oil source is widely used in grass carp. In addition, in order to cope with the huge commercial pressure, high commercial feed is also widely used to promote the growth of farmed fish, accelerate their time to market. However, dietary energy and n-6/n-3 ratio imbalance can easily lead to excessive accumulation of fat fish, causing many problems, such as increased mortality, decreased food intake, decreased immunity, reducing the quality of the most. It affects economic benefits. Therefore, to control excessive accumulation of fat is particularly important. At present, some scholars through in vivo and in vitro two models, regulation of fat accumulation was studied, found that twenty of five carbon acid (Eicosapentaenoic acid, EPA) through promoting fat cell apoptosis, inhibit fat synthesis other ways to reduce fat accumulation, but the specific mechanism is not clear.Alpha- linolenic acid (Linolenic acid ALA) is an important n-3 PUFAs (polyunsaturated fatty acids) is one of the essential fatty acids is one of grass carp. Studies have shown that ALA was added in the diet can reduce fat accumulation. In view of the restriction of the use of source EPA due to the limited, and grass carp has the C18 ALA into EPA, this thesis aims to discuss the EPA and its precursor ALA these two important n-3 PUFAs regulation of fat accumulation and the role of grass carp The mechanism, in order to provide a theoretical reference for freshwater fish lipid metabolism. The main results are as follows: the accumulation effect of 1.EPA grass carp abdominal adipose tissue lipid in vivo and in vitro studies. Using EPA in vitro were fed with grass carp fat cells, the test fish growth, biological traits, serum biochemical. The fatty acid composition and evaluated by HE staining, RT-PCR, flow cytometry detection method to determine the specific role and mechanism of EPA on grass carp lipopexia. The results showed that: with the 0.52%EPA rich diet of grass carp, its growth is not affected. After 3 weeks of feeding, fish abdominal fat index test significantly, when the feeding time is prolonged to 6 weeks, there was no significant difference between abdominal fat index. At the same time, in vivo and in vitro results showed that EPA significantly promoted the adipose tissue / cell adipose triglyceride lipase (ATGL), hormone Sensitive lipase (HSL) -a, peroxisome proliferator activated receptor gamma (PPAR), lipoprotein lipase (LPL), caspase 3a, caspase 3B enzyme activity level of mRNA and caspase 3, reduced liver X receptor (LXR) alpha, sterol regulatory element binding protein (SREBP) -1c, fatty acid synthase (FAS) gene expression and Bcl-2/Bax ratio. In vitro results show that grass carp preadipocyte cell viability inhibition of EPA in a dose and time dependent manner, and when the concentration reached 650 M, 4 days, about 50%.. The cell viability in the control group only at this time, flow type detection of apoptosis FCM results showed that the apoptosis rate of treatment group reached 37%, significantly higher than the control group. And the content of ROS also increased significantly. The use of antioxidants, tocopherol can significantly relieve the apoptosis induced by EPA. On the other hand, the use of EPA can significantly promote the.2.epa EPA content of adipose tissue / cells Grass carp to promote fat cell expression of glycerol kinase gene. According to the results of a test, for further research on EPA short-term feeding can reduce the fat content, the intrinsic mechanism and long-term feeding is no effect. First, cloning grass carp glycerol kinase (gyk) gene, found in hepatopancreas, the highest expression of adipose tissue and kidney. As the fat cells found that its expression showed a rising trend. EPA cells treated with 6,12,24h for 100 m, it can significantly promote gene expression and enzyme activity of gyk, ATGL and PPAR gamma gene expression was also up-regulated. To further explore the potential mechanisms for EPA induced grass carp fat cells gyk gene expression, respectively by ATGL and PPAR gamma inhibitor hy-15859, GW9662 cells were incubated. The results show that the two inhibitors can reduce the induction effect of EPA on gyk. In addition, the cells were hungry at 24h, while ATGL A significant increase of the gyk activity did not change. Including alpha linolenic acid (18:3n-3, ALA), twenty-two carbon (22:6n-3, DHA) six acid, linoleic acid (18:2n-6, LNA), PPAR gamma ligand remaining fatty acids, can promote the gyk gene expression and enzyme activity of.3. diet add alpha linolenic acid on the metabolism of juvenile grass carp lipid, fatty acid composition and apoptosis of peritoneal fat cells were used with 0%, 1%, 2.0%ala diet for 8 weeks. The results showed that the larvae of grass carp, no negative effects of ALA supplementation on the growth of grass carp test. At the same time, abdominal fat index in 2% groups the lowest, while Caspase3, caspase8, caspase9 activity of this group was the highest. Compared with the other two groups, 2% ala decreased FAS, SREBP-1c gene expression and bcl-2/bax ratio. At the same time, 2% groups of experimental fish abdominal adipose tissue mitochondria appear more apoptosis, mainly Mitochondrial swelling, cristae disappeared. On the other hand, detected expression of fatty acid biosynthesis pathway genes, muscle and hepatopancreas of group ala, fatty acid elongase (ELO) and fatty acid desaturase (FAD) expression was significantly higher than the control group, while in the abdominal adipose tissue is also showing on the contrary trend. However, dietary supplementation of ALA could significantly promote the muscle, liver and pancreas and abdominal adipose tissue of the EPA and DHA.4. content of alpha linolenic acid on apoptosis of adipocytes and its mechanism. The grass carp platform in vitro fat cells by grass carp, grass carp respectively with ALA precursor and mature fat cells were treated. The results showed that ALA (0-800 m) did not have inhibitory effect on cell viability, and when the treatment time was 2 and 4 days, ALA promotes cell viability. In addition, the same should be a model of progenitor cells with different differentiation stages. By 100, Ala 6501300 m precursor cells 2 and 4 days, with the increase of ALA concentration, caspase3a, caspase3b and caspase8 mRNA were significantly decreased, while bcl-2/bax decreased. When the treatment time was prolonged to 8 days, caspase9, mRNA caspase3b and caspase8 of the water level was highest in 1300 m in this group, and bcl-2/bax group was the lowest. The mature fat cells, after 2 days treatment, with the increase of the concentration of ala, caspase3a, caspase3b, caspase8 and caspase9 gene expression were significantly reduced, while bcl-2/bax decreased. However, after 8 days of treatment, caspase3a, caspase3b, caspase8 and caspase9 gene expression was significantly up-regulated, while bcl-2/bax showed a downward trend after the first rise, and the lowest value appeared in 1300 m group,.Av/pi staining results also show that when the treatment time was 2 and 4 days, ALA mainly for anti apoptosis, and When the treatment time was extended to 8 days, 650 and 1300 m group, the apoptosis rate was significantly higher than that in other two groups. For the detection of fatty acid composition, with 1300 m ala for 8 days, ALA and DHA in preadipocytes treated group was significantly higher than the control group, the content of mature fat cells in treatment group only ALA was significantly higher than the control group. In summary, 1) EPA addition in diets can promote triglyceride hydrolysis under certain conditions, promote fat cell apoptosis, inhibition of fatty acid biosynthesis and other ways to reduce abdominal fat accumulation grass carp; 2) different from the mammalian animal, grass carp can adipose tissue expression of GyK gene by.EPA the expression of GyK, promote the re esterification of triglycerides, and compensatory synthesis. The induction of GyK and EPA is regulated by PPAR gamma, ATGL is also involved in the regulation of this process; 3) the 2% diets can promote fat cells ALA Apoptosis, inhibition of de novo synthesis of fatty acids and other ways to reduce abdominal fat accumulation and grass carp. The addition of ALA can significantly promote the deposition of fish muscle n-3 long chain polyunsaturated fatty acids, thereby improving the quality of fish body; 4) ALA itself can promote the mature adipose cell apoptosis in preadipocytes, ALA may play a role in the biosynthesis of the product. The precursor and mature fat cells may have differences in the ability of using grass carp ALA DHA synthesis.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S917.4

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