【作者】 杨瑞丽；

【导师】 乐国伟；

【作者基本信息】 江南大学 ， 粮食、油脂及植物蛋白工程， 2008， 博士

【摘要】 消化系统是营养物质消化、吸收和代谢的主要器官。长期摄入高脂食物会使机体自由基生成增加,氧化还原状态的改变可能会影响消化系统腺体功能和基因表达,进而影响机体糖、脂代谢。生长抑素作为消化吸收功能的抑制性调节激素对保持机体物质能量代谢平衡具有重要的调节作用。本论文对高能摄入对消化系统氧化还原状态、糖、脂代谢、生长抑素分泌、基因表达的影响以及抗氧化剂硫辛酸的干预作用进行研究。1.高脂日粮与硫辛酸对小鼠氧化还原状态和自由基水平的影响72只C57BL/6小鼠随机分为3组,分别饲喂正常日粮、高脂日粮、高脂日粮+0.1%LA。研究随饲喂时间的延长高脂日粮与硫辛酸对小鼠消化系统氧化还原状态和自由基水平的影响,结果表明:高脂日粮喂养小鼠1周,小鼠消化系统自由基水平有所升高,差异不显著(P>0.05),随着进食高脂日粮时间的延长,到6周时,小鼠胰腺、肝脏、十二指肠的ROS水平和MDA含量显著提高(P<0.05),抗氧化能力显著降低(P<0.05),造成机体氧化应激。抗氧化剂LA有效抑制机体氧化还原状态失衡。2.油酸与硫辛酸对大鼠胃粘膜细胞氧化还原状态和生长抑素分泌的影响体外分离培养大鼠胃黏膜细胞,研究油酸与硫辛酸对大鼠胃粘膜细胞氧化还原状态和生长抑素分泌的影响。结果表明:低浓度的OA(0.1 mmol)能刺激胃粘膜细胞分泌生长抑素(P<0.05),ROS含量和胞内Ca2+浓度都有所升高,同时添加LA降低ROS的生成量,SS的分泌量也显著降低;高浓度的OA(1 mmol)显著提高细胞ROS水平(P<0.05),GSH/GSSG比值显著降低,MDA含量显著提高,导致细胞氧化损伤,生长抑素的分泌水平也随之降低。去除高浓度(1 mmol)油酸造成细胞氧化损伤组外,SS的分泌量与ROS水平呈非线性回归关系,y=25.645Ln(X)-159.93, R~2=0.8467,结果表明:在一定的范围内ROS可能是OA刺激生长抑素分泌的信号,高浓度的OA氧化损伤胃粘膜细胞,生长抑素分泌量显著降低(P<0.05)。3.高脂日粮与硫辛酸对小鼠生长抑素分泌及血糖、血脂代谢的影响72只C57BL/6小鼠随机分为3组,分别饲喂正常日粮、高脂日粮、高脂日粮+0.1%LA。研究随饲喂时间的延长高脂日粮与硫辛酸对小鼠生长抑素表达分泌水平以及血糖、血脂代谢的影响,结果表明:在高脂日粮喂养小鼠1周时,小鼠血糖、血脂水平没有明显改变(P>0.05)。随着进食高脂日粮时间延长,到3周时,小鼠TG、TC、LDL-C和血糖水平显著升高(P<0.05),到6周时,小鼠胰岛素和HOMA-IR指数显著升高(P<0.05),出现明显的糖、脂代谢紊乱。添加LA可以显著降低同期高脂日粮小鼠血糖、血脂、胰岛素和HOMA-IR指数。高脂日粮小鼠血浆、胰腺和十二指肠中生长抑素水平有相似的变化趋势:进食高脂日粮1周时,SS显著升高(P<0.05),此时十二指肠、胰腺组织的自由基水平有所升高,但差异不显著(P>0.05)。随时间的延长,到6周时,高脂日粮小鼠胰腺和十二指肠自由基含量显著升高(P<0.05),SS表达分泌水平显著降低(P<0.05),表明:短期SS能够通过控制营养物质的消化、吸收而控制消化道自由基水平。但随高脂饲喂时间的延长,高水平的自由基损害SS表达分泌系统,减少分泌量,机体自由基水平剧增。添加LA显著降低长期高脂日粮小鼠血液、组织自由基水平,提高SS表达水平,抑制了机体氧化还原状态的失衡。4.人高脂血症与血清生长抑素水平和氧化应激关系的探讨选择性别、年龄相当的高脂血症患者和正常个体各28例。测定血清生长抑素水平和抗氧化指标。结果表明:高脂血症患者血糖、胰岛素、HOMA-IR指数、动脉粥样硬化指数和MDA均显著高于正常对照组,SOD、GSH-Px显著低于对照组,存在明显的氧化应激和胰岛素抵抗现象。高脂血症患者血清SS水平显著低于正常对照组(P<0.05),SS水平与动脉粥样硬化指数呈显著负相关(r =-0.33, P=0.007)。说明SS分泌降低可能与血脂代谢紊乱密切相关。5.高脂日粮与硫辛酸对小鼠肠道氧化还原和消化吸收相关功能基因表达的影响利用affymetrix MOE430A基因芯片研究高脂日粮与硫辛酸对小鼠肠道基因表达的影响。结果表明,高脂饲喂引起肠道大量基因表达改变。利用GenMAPP研究高脂和高脂+LA饲喂对小鼠肠道基因表达的影响,结果发现高脂和高脂+LA响应基因显著相关的GO定义相似,主要分为以下几个大类:氧化应激、DNA修复、细胞凋亡、消化吸收、物质转运、免疫反应和信号转导。利用Mappfinder分析了高脂和高脂+LA饲喂显著影响的基因通路,结果表明,高脂日粮小鼠抗氧化酶、物质消化吸收、JAK-STAT信号通路和免疫反应等相关功能基因的表达显著下调,生长抑素的表达水平也显著降低。抗氧化剂LA清除自由基,上调高脂日粮小鼠肠道抗氧化相关功能基因的表达水平,显著提高6周高脂日粮小鼠生长抑素表达水平,进而缓解高脂日粮小鼠肠道氧化损伤。恢复物质消化转运、免疫反应等相关功能基因的表达水平,抑制细胞凋亡通路相关基因表达水平,保持肠道正常生理功能。6.高脂日粮与硫辛酸对小鼠肝脏氧化还原和脂代谢相关功能基因表达的影响利用Genmapp分析了正常、高脂和高脂+LA饲喂小鼠肝脏显著改变的基因,研究了相关的显著性GO定义,结果表明,高脂饲喂肝脏响应基因主要与脂类代谢、糖代谢、代谢酶和生物转化、应激反应、炎症/免疫反应、AMPK和NF-kB信号通路有关。利用Mappfinder分析了高脂和高脂+LA饲喂小鼠肝脏显著影响的基因通路。结果表明,抗氧化剂LA清除自由基,上调高脂日粮小鼠肝脏抗氧化相关功能基因的表达水平,抑制肝脏氧化还原状态失衡,进而影响AMPK和NF-kB通路基因表达水平,上调脂肪酸?氧化、并下调胆固醇合成通路相关功能基因的表达水平,参与脂类代谢过程的调节。氧化应激抑制AMPK通路相关基因表达,LA解除肝脏氧化应激上调AMPK通路基因表达水平,AMPK促进脂肪酸氧化,并抑制肝内的成脂过程。氧化应激激活NF-kB通路,提高TNF-α的表达水平。TNF-α是肝脏内抑制胰岛素信号传导的关键物质。硫辛酸显著下调NF-kB通路基因的表达,降低TNF-αmRNA,进而改善高脂日粮小鼠糖、脂代谢代谢紊乱。更多还原

【Abstract】 Digestive system is the main digestive, absorptive and metabolic organ in animal. High fat diet (HFD) increased ROS level and may have an effect on the function and gene expression of digestive system, which in turn modulate nutrient metabolism. Somatostatin (SS) may protect organism from overnutrition-induced oxidative stress by inhibiting pancreatic endocrine and exocrine secretion, gastrointestinal digestion and absorption. The aim of our experiment was to study the effect and regulation mechanism of HFD and antioxidant LA on gastrointestinal redox status, function, somatostatin secretion, lipid metabolism, glycometabolism and gene expression of liver and intestine.1. Effect of HFD and LA on the redox status and ROS level of miceMale C57BL/6 mice were fed an ordinary diet, HFD (21.45% fat, w/w) and HFD plus 0.1% LA respectively for 1, 3 or 6 weeks. The redox status was examined. The results showed that there was not significant difference in ROS level and MDA content for 1 week (P>0.05). A significant increase in the level of ROS in panscreas, liver and intestine was observed in the group fed the HFD for 3 weeks and there was a further increase after 3 weeks. A progressively increase (P<0.05) in the level of plasma MDA was observed in the group fed the HFD for 6 weeks with a similar pattern of increase in the panscreas, liver, and intestine. Treatment with LA brought about a significant improvement in redox homeostasis of HFD-fed mice.2. Effect of OA and LA on redox status and secretion of SS in rat GMCsThe effect of the different dose oleic acid (OA) on redox status and SS secretion of rat gastric mucosal cells (GMCs) in vitro was studied. The results showed that low OA (0.1 mmol) increased SS secretion by 2.85-fold and cellular ROS by 2.71-fold at 5 h. LA at 0.5 mmol significantly inhibited OA-induced SS secretion and cellular ROS in GMCs. Both of ROS and MDA increased gradually with the increase of OA concentration (P<0.05). 1 mmol OA significantly increased cellular ROS by 8.08-fold, MDA content by 3.69-fold and significantly decreased the GSH/GSSG ratio and the activity of SOD and GSH-Px (P<0.05). These results strongly suggested that GMCs exposed to high OA could induce its oxidative stress and injury. High OA concentration decreased SS levels. LA (0.5 mmol) partially restored SS secretion levels through inhibition of high OA-induced oxidative stress. A nonlinear regression relationship between SS level and ROS was observed except high OA group (y=25.645Ln(X)-159.93, R2=0.8467).The results suggested that properly increased ROS may as messenger induced SS secretion, while high ROS brought about oxidative damage.3. Effect of HFD and LA on the secretion and expression of SS, lipid metabolism and glycometabolismThe objective of this study was to investigate the effect of HFD and antioxidant LA on the secretion and expression of SS, lipid metabolism and glycometabolism. The results showed that there was not significant difference in lipid and glucose level for 1 week (P>0.05). A marked increase in the levels of lipid, glucose, insulin and homeostasis model assessment (HOMA) index was detected in the group fed the HFD for 6 weeks. Hyperlipidemia and glycometabolism disorders, accompanied by a depressed antioxidant defense system, were observed in HFD-fed mice for 6 weeks. These changes were partially restored in the LA-treated group. A significant increase (P<0.05) in the levels of SS in plasma, panscreas and intestine, was observed in the group fed with HFD for 1 week compared with the control group. However, after 3 and 6 weeks, significant decrease in the SS level was observed in the group fed with HFD. No change of the steady state level of SS mRNA expression in intestine was observed in mice fed the HFD for 1 week. However, significant decrease in the SS expression level was observed for 6 weeks (P<0.05). Treatment with LA partially restored the SS mRNA and its protein levels to that of control levels for 6 weeks. These results suggested that SS can modulate ROS level by controlling digestive system function for 1 week. Then, oxidative stress brought about damage to SS secretion, which in turn aggravated redox imblanace and metabolic dysfunction.4. Reduced serum somatostatin levels in hyperlipidemic subjectsWe measured serum SS level and antioxidant status in 28 hyperlipidemic (total cholesterol>4.5 mmolol/L, triglycerides >1.7 mmolol/L) and the age- and sex-matched control subjects. We found that subjects with hyperlipidemia have insulin resistance and high levels of oxidative stress. Median somatostatin (18.28±7.42 vs. 23.25±8.69 pg/mL; P<0.05) levels were lower in hyperlipidemic than in normolipidemic subjects. A significant inverse relationship between SS level and AI (r =-0.33, P=0.007) was observed. These results suggest a possible protective role of endogenous SS at least on hyperlipidemia and atherosclerosis that are attributed to excess energy intake and oxidative stress. Of course these preliminary results should be supported by prospective studies.5. Effects of high fat diet and LA supplement on redox, digestive and transport related gene expression of intestine in C57BL/6 miceEffects of high fat diet and LA supplement on gene expression of intestine in C57BL/6 mice were analyzed using the Affymetrix MOE430A GenChip. Significant changed GO terms were studied by Genmapp, which revealed that the differentially expressed genes were mainly related to reactive oxygen species metabolism, DNA repair, induction of apoptosis, transport, digestive enzyme, signal transduction and immolune response. HFD and HFD+LA significantly influenced biological process was analyzed using Mappfinder based on KEGG and GenMAPP database, which revealed that LA up-regulated the expression of genes related to free-radical scavenger enzymes and SS. Then, the transport, digestive enzyme, DNA repair, JAK-STAT cascade and immolune response related gene were up-regulated, while those involved in apoptosis were down-regulated. These results suggested LA can improve intestinal function of HFD fed mice.6. Effects of high fat diet and LA supplement on redox and lipid metabolism related gene expression of liver in C57BL/6 miceEffects of high fat diet and LA supplement on gene expression of liver in C57BL/6 mice were analyzed using the Affymetrix MOE430A GenChip. Significant changed GO terms were studied by Genmapp, which revealed that the differentially expressed genes were mainly related to lipid metabolism, glucose metabolism, metabolic enzyme, stress response, immolune response and signal transduction. HFD and HFD+LA significantly influenced biological process was analyzed using Mappfinder based on KEGG and GenMAPP database, which revealed that LA ingestion up-regulated the expression of genes related to free-radical scavenger enzymes, ?-oxidation and AMPK cascade, while those involved in NF-kB cascade and cholesterol synthesis were down-regulated. LA activated AMPK cascade by improving oxidative stress. AMPK plays an important role in lipid metabolism acting as a sensor of cellular energy status. LA decreased TNF-αexpression level by inactivating NF-kB cascade, and improved insulin resistance.更多还原