短期灌服丁酸钠缓解小鼠肥胖的功效与机制研究

发布时间：2017-12-27 00:31

本文关键词：短期灌服丁酸钠缓解小鼠肥胖的功效与机制研究　出处：《南京农业大学》2016年博士论文　论文类型：学位论文

【摘要】：丁酸是膳食纤维在结肠经发酵而产生的短链脂肪酸之一,它天然存在于黄油和奶酪中。丁酸不仅可以为机体提供能量,还可以作为信号分子通过激活G蛋白耦联受体41 (G protein-coupled receptor 41,GPR41)和 43 (G protein-coupled receptor 43, GPR43 )或抑制组蛋白去乙酰化酶(Histone deacetylase,HDAC )来发挥生物学功能。丁酸具有抗炎、抗癌、抗氧化和免疫调节等生理作用。目前,大多数研究都是在诱导肥胖的模型过程中将丁酸长期添加到高脂日粮中,研究其对肥胖的预防作用,丁酸对已建立的肥胖模型的治疗作用仅在一项研究报道中简单的提及,并且缺乏深入的分子机制研究。因此,我们以高脂诱导的肥胖小鼠为模型,研究短期灌服丁酸钠对肥胖的缓解作用,并对其作用机制进行了研究。1短期灌服丁酸钠缓解肥胖的功效3周龄SPF级雄性C57BL/6J小鼠(n = 36),购于扬州大学比较医学中心,饲养于江苏省中西医结合医院实验动物中心,适应一周后,小鼠随机分为两组,高脂组小鼠(High-fat diet, HF, n = 24)饲喂高脂日粮(脂肪供能45%)诱导肥胖,对照组小鼠(Con,n= 12)饲喂正常脂肪含量日粮(脂肪供能10%), 12h光照,12h黑暗,恒温恒湿,自由采食和饮水,记录每周体重增长情况。实验发现,饲喂8周后,高脂组小鼠相对于对照组小鼠体重极显著升高(P 0.01 ),并且高脂组相比对照组体重增加20%以上,达到了肥胖小鼠模型建立的基本要求。此外,葡萄糖耐量实验(Glucose tolerance test, GTT)测试结果显示相比Con组,HF组中小鼠无论是空腹血糖(0min)还是在腹腔注射葡萄糖15 min、30min、60min、90 min和120 min血糖含量均极显著升高(P 0.01 )。说明高脂日粮饲喂8周后小鼠发生了葡萄糖不耐受。饲喂8周肥胖模型建立成功后,将高脂日粮饲喂小鼠随机均分为2组:处理组灌胃 1 mL 水含 80 mg 丁酸钠(High-fat diet with sodium butyrate,HFB);高脂对照组和空白对照组灌服等体积的水。每两天灌胃一次,灌胃时间下午5点,共灌胃5次。在丁酸钠灌胃的10天里,小鼠采食的日粮仍然与处理前保持一致。实验发现,与HF组小鼠相比,HFB组中小鼠的体重(P 0.05 )和肝脏重(P 0.05 )显著降低;腓肠肌重、腓肠肌重/体重和肝重/体重没有变化。HFB组中小鼠无论是空腹血糖(P= 0.001),还是在腹腔注射葡萄糖15 min (P= 0.163)、30 min(P= 0.091)、60 min (P = 0.263 )、90 min (P = 0.105 )和 120 min (P = 0.000)的血糖含量均有一定程度的降低,表明短期丁酸钠灌胃能够一定程度缓解小鼠高脂日粮造成的葡萄糖不耐受。与HF组相比,HFB组小鼠血清中葡萄糖(P0.05)、胰岛素(P0.05)、瘦素(P0.05)的水平显著降低,但血清中总甘油三酯、总胆固醇、高低密度脂蛋白胆固醇和游离脂肪酸的含量没有变化。此外,与HF组相比,HFB组小鼠肝脏中总甘油三酯和总胆固醇的含量没有显著变化。以上结果表明,高脂日粮饲喂小鼠8周后造肥胖模型成功。当丁酸钠短期灌服后,缓解了高脂日粮导致的肥胖和葡萄糖不耐受。2 丁酸钠对脂肪组织的影响及作用机制丁酸钠短期灌服后,HE染色结果显示,与HF组相比,HFB组中脂肪组织脂肪细胞的大小(P0.05)、附睾脂重(P0.05)以及附睾脂重/体重(P0.05)显著降低。实验发现,与HF组相比,HFB组中脂肪分解的关键酶激素敏感酯酶(Hormone sensitive lipase, HSL)和甘油三酯水解酶(Adipose triglyceride lipase, ATGL)的 mRNA表达水平没有变化,但HSL (P0.05)和ATGL (P0.05)的蛋白表达水平显著升高。HFB组小鼠脂肪组织中13个线粒体自身编码基因中的4个(P 0.05 X ND2, ND4,ND4L和COX1)的mRNA表达显著升高,另外3个(ND6, CYTB和ATP6 )有升高的趋势。与HF组相比,HFB组中编码线粒体解耦联蛋白的基因(Uncoupling protein 2,UCP2 )和(Uncoupling protein 3, UCP3 ),脂肪棕色化的标志基因(PR domain containing 16,PRDM16)以及线粒体 β 氧化相关基因(Peroxisomal acyl-coenzyme A oxidase 1,ACOX1)的mRNA表达水平没有变化,却显著升高了调控线粒体功能的关键基因(Peroxisome proliferator-activated receptor gamma coactivator 1-alpha, PGC1α) (P 0.05 )的mRNA和蛋白表达水平以及与线粒体功能相关的蛋白(Cytochrome c oxidase IV,COX4)(P 0.05 )的蛋白表达水平。此外,HFB组中蛋白激酶A (Protein kinase A,PKA)(P 0.05)的蛋白表达水平以及环磷腺苷效应元件结合蛋白(cAMP-response element binding protein, CREB )(P 0.05 )的磷酸化水平显著升高。HFB组脂肪组织中GPR43的mRNA表达水平没有变化,GPR43 (P 0.05 )的蛋白表达水平显著升高,对 β3 肾上腺素受体(β3-adrenergic receptor,AR-β3 ), HFB 组中其 mRNA (P 0.05 )和蛋白(P0.05)表达水平同时显著升高。染色质免疫共沉淀技术(Chromatin Immunoprecipitation, ChIP)检测 GPR43 和AR-β3启动子上的乙酰化水平,结果发现,与HF组相比,HFB组中GPR43基因启动子上的H3K9Ac的含量没有影响,却显著提高了 AR-β3基因启动子上的H3K9Ac富集程度(P 0.05)。以上结果说明,在脂肪组织中丁酸钠通过增强GPR43和AR-β3信号通路,促进脂肪组织的脂解作用和增强线粒体功能,从而降低脂肪含量。3 丁酸钠对骨骼肌的影响及作用机制丁酸钠短期灌服后,显著降低了 HFB组小鼠骨骼肌中甘油三酯(P 0.05)和胆固醇(P 0.05)的含量,显著升高了 HFB组小鼠骨骼肌中二磷酸腺苷(Adenosine diphosphate,ADP)(P 0.05)和一磷酸腺苷(Adenosine monophosphate,AMP)(P 0.05)的含量,但对三磷酸腺苷(Adenosine tiphosphate,ATP)的含量和能荷值没有影响。实验发现,与HF组相比,HFB组骨骼肌中脂解的关键酶HSL (P0.05)和脂蛋白脂酶(Lipoprotein lipase,LPL)(P 0.05 )的mRNA表达水平显著升高,并且与线粒体功能相关基因UCP2 (P 0.05)、UCP3 (P 0.05)、肉毒碱棕榈酰转移酶(Carnitine palmitoyl transferase Ib,CPT1b)(P 0.05 )和 PGC1a (P 0.05 )的 mRNA和蛋白表达水平都显著升高,同时骨骼肌中13个线粒体自身编码基因中的12个基因(P 0.05 )的mRNA表达水平和与线粒体功能相关的蛋白COX4 (P 0.05 )的蛋白表达水平均显著升高。与HF组相比,HFB组骨骼肌中过氧化物酶体增殖激活受体α(Peroxisome proliferator-activated receptor alpha, PPARα)(P 0.05)的 mRNA 表达水平显著升高,但其蛋白的表达没有影响,并且骨骼肌中蛋白激酶(Adenosine 5'-monophosphate (AMP)-activated protein kinase, AMPK)的蛋白磷酸化水平(P 0.05)显著升高。此外,HFB组骨骼肌中瘦素受体(Leptinreceptor,LeptinR) (P0.05)的mRNA表达水平显著升高了,但其蛋白水平没有变化,同时脂联素受体1 (Adiponectin receptor 1,AdipoR1)和 2 (Adiponectin receptor 2, AdipoR2)的 mRNA (P 0.05 )和蛋白(P 0.05)表达水平显著升高,但血液和骨骼肌中脂联素(Adiponectin)的蛋白表达水平没有变化。HFB组骨骼肌中丁酸的两个主要受体GPR41和GPR43的蛋白表达都没有变化,但组蛋白去乙酰化酶1 (Histone deacetylase 1, HDAC1)的蛋白表达水平(P0.05)显著降低。染色质免疫共沉淀技术(ChIP)检测发现,与HF组相比,HFB组中UCP2 (P 0.05)、UCP3 (P0.05)、AdipoR1 (P0.05)和 AdipoR2 (P0.05)基因启动子上H3K9Ac的含量显著提高。以上结果说明,在骨骼肌中丁酸钠通过促进脂联素信号通路和增强线粒体β氧化来促进脂肪酸的代谢。综上所述,丁酸钠短期灌服可以通过促进脂肪组织和骨骼肌中的能量代谢以及线粒体功能来缓解高脂日粮诱导的肥胖。
[Abstract]:Butyric acid (butyric acid) is one of the short chain fatty acids produced by the dietary fiber in the colonic fermentation. It is naturally found in butter and cheese. Butyric acid can not only provide energy for the body, but also can be used as a signal molecule through the activation of G protein coupled receptor 41 (G protein-coupled 41 receptor, GPR41) and 43 (G protein-coupled 43 receptor, GPR43) or inhibition of histone deacetylase (Histone deacetylase, HDAC) to play the biological functions. Butyric acid has some physiological functions, such as anti-inflammatory, anticancer, antioxidation and immunoregulation. At present, most of the studies are in the process of the long-term model of obesity induced by butyrate added to high fat diet, to study its preventive effect on obesity, therapeutic effect of butyrate on obesity model has been established only in a study reported in the simple mention, and lack of in-depth study on the molecular mechanism. Therefore, we used high fat induced obese mice as a model to study the effect of sodium butyrate on obesity in short term, and the mechanism of its action was studied. 1 short term gavage of sodium butyrate to alleviate the obesity effect SPF 3 week old male C57BL/6J mice (n = 36), purchased from Yangzhou University medical center, raised in Jiangsu province combining traditional Chinese and Western Medicine Experimental Animal Center Hospital, after one week adaptation, mice were randomly divided into two groups, high fat group (High-fat diet, HF n = 24), fed with high fat diet (45% fat) induced obese mice in control group (Con, n= 12) with a normal fat diet (10% fat), 12h light, 12h dark, constant temperature and humidity, free feeding and drinking water, record weekly weight gain. The experiment found that after 8 weeks of feeding, the body weight of the high-fat group was significantly higher than that of the control group (P 0.01), and the body weight of the high-fat group increased by more than 20% compared with the control group, which reached the basic requirement of establishing the obesity mouse model. In addition, the results of Glucose tolerance test (GTT) test showed that compared with the Con group, the fasting plasma glucose (0min) or the intraperitoneal injection of glucose 15 min, 30min, 60min, 90 min and 120 60min blood glucose levels in the HF group were significantly increased (0.01). It was suggested that glucose intolerance occurred in mice after 8 weeks of high fat diet feeding. After feeding for 8 weeks, the obesity model was established successfully, and the mice fed high-fat diet were randomly divided into 2 groups: the treatment group was gavaged 1 mL water containing 80 mg sodium butyrate (High-fat diet with sodium butyrate, HFB); the high-fat control group and the blank control group were given equal volume of water. Every two days, the stomach was gavage, and the time of gavage was 5 p.m. and gavage was 5 times. In the 10 day of sodium butyrate gavage, the diet of the mice was still consistent with that before treatment. It was found that compared with group HF, body weight (P 0.05) and liver weight (P 0.05) of mice in HFB group were significantly reduced, gastrocnemius muscle weight, gastrocnemius muscle weight / body weight and liver weight / weight did not change. The mice in the HFB group both fasting blood glucose (P= 0.001), or in the intraperitoneal injection of glucose 15 min (P= 0.163), 30 min (0.091 P=), 60 min (P = 0.263), 90 min (P = 0.105) and 120 min (P = 0) reduced blood sugar content to a certain extent, the short-term intragastric administration of sodium butyrate can alleviate to some extent of high-fat diet mice caused by glucose intolerance. Compared with group HF, the levels of serum glucose, P0.05, P0.05 and leptin (P0.05) in group HFB were significantly decreased, but the total triglycerides, total cholesterol, high-density lipoprotein cholesterol and free fatty acids in serum did not change. In addition, there was no significant change in total triglyceride and total cholesterol in the liver of HFB mice compared with the HF group. The above results showed that high fat diet fed mice 8 weeks after the successful model of obesity. When sodium butyrate was administered in a short period of time, obesity and glucose intolerance caused by high fat diet were relieved. 2, the effect and mechanism of sodium butyrate on adipose tissue. After short-term administration of sodium butyrate, HE staining showed that the adipocyte size (P0.05), epididymal fat weight (P0.05) and epididymal fat / body weight (P0.05) in HFB group were significantly lower than those in HF group. Experiments show that, compared with the HF group, the key enzyme hormone sensitive lipase lipolysis in group HFB (Hormone sensitive lipase HSL (Adipose triglyceride) and triglyceride hydrolase lipase, ATGL) did not change the expression level of mRNA, but HSL (P0.05) and ATGL (P0.05) protein expression levels were significantly increased. MRNA expression of 13 mitochondrial self coding genes (P 0.05 X ND2, ND4, ND4L and COX1) in HFB adipose tissue of group 4 increased significantly, and the other 3 (ND6, CYTB and ATP6) increased. Compared with group HF, the gene encoding mitochondrial uncoupling protein in the HFB group (Uncoupling protein 2, UCP2 (Uncoupling) and protein 3, UCP3), the brown fat mark gene (PR domain containing 16, PRDM16) and mitochondrial beta oxidation related genes (Peroxisomal acyl-coenzyme A oxidase 1, ACOX1) did not change the expression level the mRNA was significantly increased in the key genes regulating mitochondrial function (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha, PGC1 alpha) (P 0.05) mRNA and protein expression level and protein related to mitochondrial function (Cytochrome c oxidase IV, COX4) (P 0.05) protein expression levels. In addition, the protein expression level of protein kinase A (Protein kinase A, PKA) (P 0.05) and the phosphorylation level of cyclic adenosine effect element binding protein (cAMP-response element binding protein, binding 0.05) increased significantly in group HFB. The expression level of GPR43 mRNA in adipose tissue of HFB group did not change. The protein expression level of GPR43 (P 0.05) increased significantly, and the expression level of mRNA (P 0.05) and protein (HFB) increased significantly in beta 3 adrenergic receptor (3-adrenergic receptor, AR- beta 3) and HFB group. Detection of GPR43 and AR- beta 3 by chromatin immunoprecipitation (Chromatin Immunoprecipitation, ChIP)
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S859.7

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