高糖膳食对小鼠消化系统氧化还原状态的影响及其机制
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摘要
高糖食品越来越多地出现在人们的生活中,许多流行病学报道了高糖膳食与多种疾病的相关性,但其中的机制还有待研究。氧化应激是多种疾病发生发展的共同作用机制,消化系统是机体重要组成部分,其承担的消化吸收和代谢功能涉及许多物质能量代谢和生理过程,这些过程通常伴随着活性氧的生成。许多研究也证明氧化应激在多种消化系统疾病进程中起着重要作用,然而高糖膳食对消化系统氧化还原状态影响的研究还较为少见。因此本研究拟探究高糖膳食对消化系统氧化还原状态的的影响从而探究高糖膳食与疾病相关性的机制,以便为营养相关疾病的预防和治疗提供参考。
活性氧的化学发光检测法多用于细胞和单层细胞组织的检测,目前还缺乏对多层细胞组织活性氧的测定方法。本研究以过氧化氢为标准品,辣根过氧化物酶为催化剂,鲁米诺为探针,建立了组织活性氧的化学发光检测法。同时用20%(m/v)葡萄糖溶液灌胃昆明种小白鼠,检测了不同时间点血糖浓度和氧自由基水平,发现血糖和活性氧呈正相关。
通过调整玉米淀粉和葡萄糖比例(40%,0；30%,10%；25%,15%；20%,20%；10%,30%；0,40%)配制不同葡萄糖浓度膳食喂饲小鼠2周,观察不同葡萄糖水平对血和消化系统主要组织胃、十二指肠、空肠、肝脏和胰腺氧化还原状态和生长抑素的影响,结果发现20%葡萄糖膳食两周喂饲使得十二指肠和空肠氧化还原状态最优,这说明消化道消化与吸收功能达到平衡有利于机体氧化还原平衡的维持。40%葡萄糖膳食使血和消化系统各组织发生了氧化应激,说明快速吸收会使机体氧化还原状态失衡。分析血和消化系统各组织生长抑素与活性氧的相关性,发现生长抑素可能在活性氧的刺激下参与机体的抗氧化防御。用高糖膳食(含20%葡萄糖)分别喂饲小鼠1、2、4周,观察血及消化系统氧化还原状态和生长抑素的变化,结果显示4周的高糖膳食喂饲导致了小鼠血、胃、肝脏和胰腺氧化应激,而十二指肠和空肠仍然没有发生氧化应激；胃肠道生长抑素和活性氧的变化存在着高度正相关,证实活性氧可能作为信号分子刺激生长抑素参与机体抗氧化防御的推论。
为探究小肠对高糖膳食诱导的氧化应激具有防御能力的机制,本研究取高糖膳食(含20%葡萄糖)4周喂饲下的小鼠空肠组织制备全基因表达谱芯片,全面分析氧化还原酶活性相关基因,发现抗氧化酶基因Prdx1和Prdx4表达显著增高,表明二者的表达产物在空肠中发挥了极强的抗氧化保护功能。用定量PCR检测不同葡萄糖水平膳食两周喂饲和高糖膳食(含20%葡萄糖)不同时间喂饲下十二指肠和空肠Prdx1,Prdx4的基因表达,并分析基因表达变化与生长抑素变化的相关性,发现生长抑素变化和Prdx1,Prdx4基因表达高度正相关；而活性氧变化与Prdx1,Prdx4基因表达并无相关性。综合上述结果得出一个推论：机体ROS可能作为信号分子刺激生长抑素的分泌,生长抑素在小肠可能通过参与Prdx1,Prdx4的表达调控发挥其抗氧化防御功能。
同样为探究四周高糖膳食(含20%)喂饲导致小鼠肝脏发生氧化应激的分子机制,取肝脏组织制备全基因表达芯片,分析了14446条基因的表达变化,发现1102条基因的表达与对照组相比发生了改变。分析活性氧产生与清除相关基因的表达发现,高糖膳食喂饲下肝脏细胞内游离脂肪酸水平增高,其在线粒体的氧化分解并未加强,而在过氧化物酶体的氧化分解加强,此过程伴随着大量活性氧的生成；细胞色素P450家族的基因表达活动也显著加强,这同样会导致活性氧的生成。另外芯片分析也显示高糖膳食主要通过下调硫醇平衡相关基因的表达削弱了小鼠肝脏的抗氧化防御能力。高糖膳食导致的肝脏活性氧增加和硫醇水平的降低导致了肝脏氧化应激的发生。
过量糖的摄入会导致机体脂代谢紊乱,其发生发展过程是否氧化应激的参与,这是本课题的研究内容之一。对脂代谢机制的研究多集中于肝脏,而小肠作为脂吸收的主要场所,其脂代谢的改变在脂代谢紊乱中的作用却常被忽略。本研究用高糖膳食(含20%葡萄糖)喂饲小鼠4周,检测高糖对血脂的影响。取肝脏和空肠制作基因芯片,以分析过量糖摄入导致脂代谢紊乱的分子机制。结果发现20%葡萄糖膳食四周喂饲导致小鼠脂代谢紊乱。与其他研究不同,对肝脏脂代谢相关基因的影响主要为甘油三酯水解相关基因和脂肪酸在过氧化物酶体氧化的相关基因,脂肪酸在过氧化物酶体的氧化伴随活性氧的生成,这一结果证实了脂代谢紊乱过程中氧化应激的参与。而空肠基因芯片的分析则显示,高糖摄入显著影响了小肠脂代谢相关基因的表达,主要为甘油三酯消化吸收和胆固醇沉积相关基因,这些基因的表达改变与肝脏脂代谢相关基因的表达改变以及氧化应激一起参与了脂代谢紊乱的发展形成。
Many researches showed the intake of high sugar diet was related to multiple diseases. But the underlying mechanism is still nof fully clear. Oxidative stress is a well-recognized mechanism in many pathological conditions. Digestion, absorption and metabolism in digestive system involve many energy processes which usually accompanied by the production of reactive oxygen species (ROS). Many research reported oxidative stress played important roles in many diseases in digestive system. However there are few researches on the effect of high sugar diet on digestive system and underlying mechanism. In this research, we explored the effect of high glucose diet on digestive system and underlying mechanism, the relation between oxidative and dyslipidemia, which provided the reference value for the prevention and treatment of diseases related to nutrition.
Chemiluminescence assay is generally applied to detect ROS in cells or monolayer tissues. There are few assays for detection of ROS in multilayer tissues. We established the chemiluminescence assay for ROS in multilayer tissues with hydrogen peroxide as standard, luminol as probe, horseradish peroxidase as catalyst. We did intragastric administration in Kunming mice, detected the blood glucose concentration and ROS level at different times. The results showed positive correlation between blood glucose concentration and ROS.
Mice were fed by diet with different concentration of glucose which were obtained by adjusting the proportion of corn starch and glucose (40%,0;30%,10%;25%,15%;20%, 20%;10%,40%;0,40%). After two weeks, we detected redox status and somatostatin level of blood, stomach, duodenum, jejunum, liver and pancreas in six groups of mice. The results didn't show the correlation between the concentration of glucose and degree of oxidative stress. Diet with 20% glucose induced the optional redox status in duodenum and jejunum. Diet with 40% glucose induced the complete oxidative stress in blood and digestive system. The correlation analysis for somatostatin and ROS suggested somatostatin played important roles in defense against oxidative stress, while ROS was as signal molecule.
Why small intestine could defend against oxidative stress induced by high glucose diet (containing 20% glucose)? We did DNA chip using jejunum of mice on high glucose diet for four weeks, comprehensively analyzed genes related to oxidoreductase, found Prdxl and Prdx4 had higher expressions in group fed with high glucose diet compared with control, which suggested Prdxl and Prdx4 played strong roles in defense against oxidative stress in jejunum. We separately detected the relative expression of Prdxl and Prdx4 in mice on diet with different concentration of glucose for two weeks or diet with 20% glucose for four weeks, found the positive correlation between somatotain and the gene expressions of Prdxl and Prdx4. Sum up the above the results, we concluded that ROS as signal molecule induced the secretion of somatostatin, and then somatostain play its roles in defense against oxidative stress by regulating the expressions of Prdxl and Prdx4.
We also did DNA chip using liver of mice on high glucose diet for four weeks, comprehensively genes related to ROS production and removing, found that ROS in oxidative stress induced by high GI diet were mainly fromβ-andωoxidation in peroxisome and CYPE1 in microsome, which was different from other researches; high glucose diet impaired the antioxidant defense by down-regulating the expression of genes related to thiol redox.
Excess of sugar intake can induce dyslipidemia, whose initiation and development involve oxidative stress. The research on the underlying molecular mechanism of simple sugar inducing deleterious effects on lipid metabolism focused the hepatic gene expression and enzyme activities change related to lipid metabolism. However, there are few researches on the effect of high carbohydrate on lipid metabolism of intestine. In this research, we fed mice with high glucose diet (containing 20% glucose) for four weeks, detected the effect of high glucose diet on blood lipid profiles. The results showed high glucose diet feeding induced dyslipidemia in mice. In order to explore the mechanism of dyslipidemia induced by high glucose diet, we comprehensively analyzed expressions of genes related to lipid metabolism in liver and jejunum using microarray analysis, found expressions of genes related to triglyceride hydrolysis and fatty acid oxidation in peroxisome were different from control. Fatty oxidation in peroxisome was accompanied by the production of ROS, which verified that oxidative stress participated in the initiation and development of dyslipidemia. Microarray analysis for jejunum showed the intake of high glucose diet influenced expressions of genes related to triglyceride digestion, absorption and deposit. The expressions of genes related to lipid metabolism in liver and jejunum, together with oxidative stress, participated in the initiation and development of dyslipidemia induced by high glucose diet.
活性氧的化学发光检测法多用于细胞和单层细胞组织的检测,目前还缺乏对多层细胞组织活性氧的测定方法。本研究以过氧化氢为标准品,辣根过氧化物酶为催化剂,鲁米诺为探针,建立了组织活性氧的化学发光检测法。同时用20%(m/v)葡萄糖溶液灌胃昆明种小白鼠,检测了不同时间点血糖浓度和氧自由基水平,发现血糖和活性氧呈正相关。
通过调整玉米淀粉和葡萄糖比例(40%,0；30%,10%；25%,15%；20%,20%；10%,30%；0,40%)配制不同葡萄糖浓度膳食喂饲小鼠2周,观察不同葡萄糖水平对血和消化系统主要组织胃、十二指肠、空肠、肝脏和胰腺氧化还原状态和生长抑素的影响,结果发现20%葡萄糖膳食两周喂饲使得十二指肠和空肠氧化还原状态最优,这说明消化道消化与吸收功能达到平衡有利于机体氧化还原平衡的维持。40%葡萄糖膳食使血和消化系统各组织发生了氧化应激,说明快速吸收会使机体氧化还原状态失衡。分析血和消化系统各组织生长抑素与活性氧的相关性,发现生长抑素可能在活性氧的刺激下参与机体的抗氧化防御。用高糖膳食(含20%葡萄糖)分别喂饲小鼠1、2、4周,观察血及消化系统氧化还原状态和生长抑素的变化,结果显示4周的高糖膳食喂饲导致了小鼠血、胃、肝脏和胰腺氧化应激,而十二指肠和空肠仍然没有发生氧化应激；胃肠道生长抑素和活性氧的变化存在着高度正相关,证实活性氧可能作为信号分子刺激生长抑素参与机体抗氧化防御的推论。
为探究小肠对高糖膳食诱导的氧化应激具有防御能力的机制,本研究取高糖膳食(含20%葡萄糖)4周喂饲下的小鼠空肠组织制备全基因表达谱芯片,全面分析氧化还原酶活性相关基因,发现抗氧化酶基因Prdx1和Prdx4表达显著增高,表明二者的表达产物在空肠中发挥了极强的抗氧化保护功能。用定量PCR检测不同葡萄糖水平膳食两周喂饲和高糖膳食(含20%葡萄糖)不同时间喂饲下十二指肠和空肠Prdx1,Prdx4的基因表达,并分析基因表达变化与生长抑素变化的相关性,发现生长抑素变化和Prdx1,Prdx4基因表达高度正相关；而活性氧变化与Prdx1,Prdx4基因表达并无相关性。综合上述结果得出一个推论：机体ROS可能作为信号分子刺激生长抑素的分泌,生长抑素在小肠可能通过参与Prdx1,Prdx4的表达调控发挥其抗氧化防御功能。
同样为探究四周高糖膳食(含20%)喂饲导致小鼠肝脏发生氧化应激的分子机制,取肝脏组织制备全基因表达芯片,分析了14446条基因的表达变化,发现1102条基因的表达与对照组相比发生了改变。分析活性氧产生与清除相关基因的表达发现,高糖膳食喂饲下肝脏细胞内游离脂肪酸水平增高,其在线粒体的氧化分解并未加强,而在过氧化物酶体的氧化分解加强,此过程伴随着大量活性氧的生成；细胞色素P450家族的基因表达活动也显著加强,这同样会导致活性氧的生成。另外芯片分析也显示高糖膳食主要通过下调硫醇平衡相关基因的表达削弱了小鼠肝脏的抗氧化防御能力。高糖膳食导致的肝脏活性氧增加和硫醇水平的降低导致了肝脏氧化应激的发生。
过量糖的摄入会导致机体脂代谢紊乱,其发生发展过程是否氧化应激的参与,这是本课题的研究内容之一。对脂代谢机制的研究多集中于肝脏,而小肠作为脂吸收的主要场所,其脂代谢的改变在脂代谢紊乱中的作用却常被忽略。本研究用高糖膳食(含20%葡萄糖)喂饲小鼠4周,检测高糖对血脂的影响。取肝脏和空肠制作基因芯片,以分析过量糖摄入导致脂代谢紊乱的分子机制。结果发现20%葡萄糖膳食四周喂饲导致小鼠脂代谢紊乱。与其他研究不同,对肝脏脂代谢相关基因的影响主要为甘油三酯水解相关基因和脂肪酸在过氧化物酶体氧化的相关基因,脂肪酸在过氧化物酶体的氧化伴随活性氧的生成,这一结果证实了脂代谢紊乱过程中氧化应激的参与。而空肠基因芯片的分析则显示,高糖摄入显著影响了小肠脂代谢相关基因的表达,主要为甘油三酯消化吸收和胆固醇沉积相关基因,这些基因的表达改变与肝脏脂代谢相关基因的表达改变以及氧化应激一起参与了脂代谢紊乱的发展形成。
Many researches showed the intake of high sugar diet was related to multiple diseases. But the underlying mechanism is still nof fully clear. Oxidative stress is a well-recognized mechanism in many pathological conditions. Digestion, absorption and metabolism in digestive system involve many energy processes which usually accompanied by the production of reactive oxygen species (ROS). Many research reported oxidative stress played important roles in many diseases in digestive system. However there are few researches on the effect of high sugar diet on digestive system and underlying mechanism. In this research, we explored the effect of high glucose diet on digestive system and underlying mechanism, the relation between oxidative and dyslipidemia, which provided the reference value for the prevention and treatment of diseases related to nutrition.
Chemiluminescence assay is generally applied to detect ROS in cells or monolayer tissues. There are few assays for detection of ROS in multilayer tissues. We established the chemiluminescence assay for ROS in multilayer tissues with hydrogen peroxide as standard, luminol as probe, horseradish peroxidase as catalyst. We did intragastric administration in Kunming mice, detected the blood glucose concentration and ROS level at different times. The results showed positive correlation between blood glucose concentration and ROS.
Mice were fed by diet with different concentration of glucose which were obtained by adjusting the proportion of corn starch and glucose (40%,0;30%,10%;25%,15%;20%, 20%;10%,40%;0,40%). After two weeks, we detected redox status and somatostatin level of blood, stomach, duodenum, jejunum, liver and pancreas in six groups of mice. The results didn't show the correlation between the concentration of glucose and degree of oxidative stress. Diet with 20% glucose induced the optional redox status in duodenum and jejunum. Diet with 40% glucose induced the complete oxidative stress in blood and digestive system. The correlation analysis for somatostatin and ROS suggested somatostatin played important roles in defense against oxidative stress, while ROS was as signal molecule.
Why small intestine could defend against oxidative stress induced by high glucose diet (containing 20% glucose)? We did DNA chip using jejunum of mice on high glucose diet for four weeks, comprehensively analyzed genes related to oxidoreductase, found Prdxl and Prdx4 had higher expressions in group fed with high glucose diet compared with control, which suggested Prdxl and Prdx4 played strong roles in defense against oxidative stress in jejunum. We separately detected the relative expression of Prdxl and Prdx4 in mice on diet with different concentration of glucose for two weeks or diet with 20% glucose for four weeks, found the positive correlation between somatotain and the gene expressions of Prdxl and Prdx4. Sum up the above the results, we concluded that ROS as signal molecule induced the secretion of somatostatin, and then somatostain play its roles in defense against oxidative stress by regulating the expressions of Prdxl and Prdx4.
We also did DNA chip using liver of mice on high glucose diet for four weeks, comprehensively genes related to ROS production and removing, found that ROS in oxidative stress induced by high GI diet were mainly fromβ-andωoxidation in peroxisome and CYPE1 in microsome, which was different from other researches; high glucose diet impaired the antioxidant defense by down-regulating the expression of genes related to thiol redox.
Excess of sugar intake can induce dyslipidemia, whose initiation and development involve oxidative stress. The research on the underlying molecular mechanism of simple sugar inducing deleterious effects on lipid metabolism focused the hepatic gene expression and enzyme activities change related to lipid metabolism. However, there are few researches on the effect of high carbohydrate on lipid metabolism of intestine. In this research, we fed mice with high glucose diet (containing 20% glucose) for four weeks, detected the effect of high glucose diet on blood lipid profiles. The results showed high glucose diet feeding induced dyslipidemia in mice. In order to explore the mechanism of dyslipidemia induced by high glucose diet, we comprehensively analyzed expressions of genes related to lipid metabolism in liver and jejunum using microarray analysis, found expressions of genes related to triglyceride hydrolysis and fatty acid oxidation in peroxisome were different from control. Fatty oxidation in peroxisome was accompanied by the production of ROS, which verified that oxidative stress participated in the initiation and development of dyslipidemia. Microarray analysis for jejunum showed the intake of high glucose diet influenced expressions of genes related to triglyceride digestion, absorption and deposit. The expressions of genes related to lipid metabolism in liver and jejunum, together with oxidative stress, participated in the initiation and development of dyslipidemia induced by high glucose diet.
引文
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