肝肠间氧化应激的相关性及食品的调节作用
发布时间：2018-08-03 21:04
【摘要】：肝脏和肠道是生物体进行食物代谢和吸收的重要器官。近30年来,大量临床数据和流行病学调查显示肝脏疾病和肠道疾病二者存在强烈的相关性。尽管肝肠同时病变或先后病变的发生机制还未被阐明,但已有许多研究发现氧化应激在肝肠疾病相关性上起关键作用。因此,本论文开展了系列研究,通过显示肝肠间活性氧(ROS)分布的关联性,解析肝肠间氧化应激的相关性,并探讨生物体摄入抗氧化食品对肝肠氧化应激乃至整体氧化应激系统的调节作用。首先,探讨了肝肠间氧化应激在构造上的相关性。使用ROS探针二氯荧光素双醋酸盐(DCFH-DA,25mg/kg体重)尾静脉注射SD大鼠,其整体荧光成像中胆总管和十二指肠显现ROS的特征荧光；紧接着,对胆总管和十二指肠进行组织切片,二者荧光成像显示,ROS的特征荧光均集中分布在结缔组织层上——胆总管的ROS特征荧光集中分布在外膜层上,十二指肠的ROS特征荧光集中分布在粘膜下层,即从胆总管至十二指肠的结缔组织层形成了荧光通道,说明了从胆总管外膜层这一结缔组织层连通到十二指肠粘膜下层的结缔组织层,形成了ROS集中分布的通道。由此,我们推测肝脏氧化应激状态有可能通过胆管发生传导。为进一步证实上述猜测,实验通过对新西兰兔肝脏左肝叶中部注射20μL过氧化氢(30%,w/v)的方式诱导肝脏氧化应激,监测该氧化应激所致流经胆总管的电流变化。结果发现,注射引起流经胆总管的电流迅速上升,电子走向为从肝脏至十二指肠,且电流的上升趋势在心脏停止跳动后依然持续一段时间,该电流有可能是线粒体在氧化应激时呼吸链漏出的电子的迁移所形成的,说明肝脏发生的氧化应激可能以电子传递的方式通过胆管传导至十二指肠壁。上述结果,为肝肠间氧化应激的相关性提供了新的证据。为进一步证实上述肝肠间氧化应激相关性的结果,我们将专一性清除超氧阴离子自由基(O2.-)的跨膜蛋白酶(Tat-SOD融合蛋白)作用于肠道,分析肠道的抗氧化干预对肝脏氧化应激状态的调节作用。实验以腹腔注射四氯化碳(CCl4) (1mL/kg体重,每周两次,持续6周给药)诱导肝纤维化的SD大鼠为模型,Tat-SOD融合蛋白(25000U/kg体重,每天两次,持续4周给药)以经口灌服方式进行抗氧化干预治疗。结果发现,肠道上Tat-SOD融合蛋白的抗氧化干预均可显著降低SD大鼠的血清谷丙转氨酶(ALT)和谷草转氨酶(AST)以及肝脏组织中丙二醛(MDA)含量至正常水平(P0.01),显著升高肝脏组织中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)活力(P0.01),并使呈弥漫性纤维化的肝脏得以逆转再生,肝脏组织结构得以显著修复。上述结果说明,经口灌服Tat-SOD融合蛋白的抗氧化干预可以达到逆转肝纤维化的治疗作用。我们推测该Tat-SOD融合蛋白可能透皮进入肠壁疏松结缔组织清除该部位所富集或传递的02-,进而可能通过胆管的桥连缓解肝脏的氧化应激状态,从而起到对肝纤维化的修复。由于氧化应激状态可能以电子传递方式在结缔组织层传导,且人体经络系统在解剖学上与筋膜等结缔组织系统重叠,经络系统有可能是与之相连脏器氧化应激的传导和调控系统,因此经线上两个穴位间电位的测定有可能精确反应沿经线氧化应激的传递,以及反应与经线相连脏器的氧化应激状态。由此我们使用穴位间电位实时监测法,进一步在人体上检视结缔组织与氧化应激的关系。实验选取胃经的下巨虚穴位(ST39)和内庭穴位(ST44)为穴位体表电位监测点,足三里穴位(ST36)为穴位干预点,分别给予足三里穴位单纯按摩、0.2mL空白药膏按摩、0.2mL Tat-SOD (3000U/mL)药膏按摩、注射100μL还原型谷胱甘肽钠(0.12g/mL)和针灸五种干预,考察其对胃经下游相邻两穴位(下巨虚与内庭)间电位的影响。结果显示：下巨虚与内庭间电位随足三里上按摩的施加或停止出现上升或回落；在按摩停止后抗氧化干预组的穴位间电位依然在高处波动,且在干预48小时后抗氧化干预组的穴位间电位显著低于物理干预组,说明穴位间电位可准确反映经线上氧化应激的传导。该监测方法有可能成为实时监测与经线相连脏器的工作状态。最后,我们采用穴位间电位实时监测方法,跟踪红茶(金骏眉)、乌龙茶(水仙岩茶)和绿茶(高山绿茶)三种茶汤的摄入对人体手少阴心经、手太阴肺经、足少阴肾经、足太阴脾经、足厥阴肝经、手太阳小肠经、足少阳胆经和足阳明胃经这八条经络的体表电位影响和即刻生理效应。结果显示,三种茶汤对手太阳小肠经的体表电位的影响最为显著；三种茶汤均可产生即刻生理效应,可出现打嗝、生津、出汗、打哈欠、肠道蠕动、手心生热等生理变化,其中又以红茶的即刻效应最明显,作用时间最长；经络体表电位的波动都有相应的生理反应。其次,我们还测定了三种茶汤的体外抗氧化活力和粒径分布,以及跟踪了动物模型饮茶的长期生理效应(三种茶汤的摄入对以高脂饲料诱导的C57BL/6小鼠肝肠组织氧化应激等指标的影响),并将其与茶汤的人体经络体表电位变化和即刻生理效应进行对比分析,结果表明,红茶茶汤的粒径最小最均一,其即刻生理效应和引起的人体经络穴位间电位的变化也最明显,可在一定程度上反应茶汤的颗粒大小可影响其即刻效应；红茶茶汤的体外抗氧化活性和茶多酚含量最低,但其引起的经络体表电位变化却最明显,说明了体外实验的抗氧化活力的分析结果,不一定能在体内实验得到相应的表现；三种茶汤均具有抑制肥胖形成和缓解肝肠间氧化应激的作用。我们推测,茶汤的抗氧化纳米颗粒通过清除肠壁疏松结缔组织层的ROS,引起肠道平滑肌缩收,产生打嗝等生理反应,并可能借助胆管的桥连进一步解除肝脏的氧化应激,从而预防脂肪肝及肥胖的发生；同时由于人体生物电网络系统的存在,肝肠间氧化应激的传递可能引发与各脏器相连经线上的电位变化。以上研究结果,为肝肠间氧化应激的相关性提供一个有力的证据,初步说明了肠道可能为肝脏ROS的新处理场；证实通过穴位间电位监测与经线相连器官氧化应激状态的可行性；揭示了食品除了提供营养和能量外还具有调节人体氧化应激处理体系的作用；为消化道、肠道疾病等提供崭新的视角和方向,将对食品与人体相互作用的认识提高到新的层次。
[Abstract]:The liver and intestines are important organs for the metabolism and absorption of food. In the past 30 years, a large number of clinical data and epidemiological investigations have shown that there is a strong correlation between the two groups of liver diseases and intestinal diseases. Although the pathogenesis of liver and intestinal lesions or successively pathological changes has not been explained, many studies have found that oxidative stress has been found. The correlation of liver and intestinal diseases plays a key role. Therefore, a series of studies have been carried out in this paper to analyze the correlation between the distribution of active oxygen (ROS) in the liver and intestine, to analyze the correlation between the oxidative stress in the liver and the intestine, and to explore the regulation of the antioxidative food on the oxidative stress of liver and the whole body and the whole body oxidation stress system. The correlation of the interintestinal oxidative stress on the structure. SD rats were injected with the ROS probe two chlorofluorescein diacetate (DCFH-DA, 25mg/kg weight) tail vein. The overall fluorescence imaging of the common bile duct and duodenum showed the characteristic fluorescence of ROS; then, the common bile duct and duodenum were sectioning, and the two fluorescence imaging showed that ROS was special. The ROS characteristic fluorescence of the choledochus is concentrated on the outer membrane layer. The ROS characteristic fluorescence of the duodenum is concentrated in the submucosa, that is, the fluorescent channel is formed from the connective tissue layer of the common bile duct to the duodenum, indicating that the connective tissue layer from the outer membrane layer of the choledochus is connected to ten. Two refers to the connective tissue layer of the submucosa of the intestinal mucosa, which forms a ROS concentrated channel. Thus, we speculate that the oxidative stress in the liver may be transmitted through the bile duct. To further confirm the above speculation, the experimental liver oxidative stress was induced by injecting 20 mu hydroperoxide (30%, w/v) into the central left hepatic lobe of New Zealand rabbit. The current changes in the common bile duct caused by the oxidative stress. It was found that the current caused by the injection of the common bile duct increased rapidly, the electron trend was from the liver to the duodenum, and the trend of the current continued to continue for a period after the heart stopped beating. The current could be the electricity that the mitochondria leaked out of the respiratory chain during oxidative stress. The migration of the children indicates that the oxidative stress in the liver may be transmitted through the bile duct to the duodenal wall electronically. The above results provide new evidence for the correlation of interintestinal oxidative stress. To further confirm the results of the above interhepatic oxidative stress correlation, we will specifically eliminate superoxide anion. The free radical (O2.-) transmembrane protease (Tat-SOD fusion protein) acts on the intestinal tract and analyzes the regulating effect of antioxidant intervention on the oxidative stress state of the liver. The experimental SD rats with SD (1mL/kg weight, two times a week, 6 weeks of administration) were used to induce liver fibrosis in SD rats, and the Tat-SOD fusion protein (25000U/kg body). The results showed that the antioxidant intervention of Tat-SOD fusion protein in the intestinal tract could significantly reduce the serum alanine transaminase (ALT) and glutamic aminotransferase (AST) and the content of malondialdehyde (MDA) in the liver tissue to normal level (P0.01) and significantly increase the liver of SD rats. In the dirty tissue, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activity (P0.01), and the diffuse fibrosis of the liver can be reversed and regenerated. The liver tissue structure can be restored significantly. The results show that the antioxidant intervention of the Tat-SOD fusion protein by oral administration can reverse the liver fiber. We speculate that the Tat-SOD fusion protein may be transdermal into the loose connective tissue of the intestinal wall to remove the 02- that is enriched or transmitted by the site, and thus may relieve the oxidative stress state of the liver through the bridge of the bile duct, thus repairing the liver fibrosis. The tissue layer is transmitted, and the human meridian system overlaps anatomically with the fascia and other connective tissue systems. The meridian system may be the conduction and regulation system of the oxidative stress associated with the organs. Therefore, the measurement of the potential of the two acupoints on the meridian may accurately reflect the transmission of the oxidative stress along the meridian, and the reaction and the meridian viscera. The relationship between the connective tissue and oxidative stress is further examined in the human body by the method of real-time monitoring of the acupoint potential. The test points for the surface potential of the acupoint (ST39) and the acupoint (ST44) of the stomach meridian are selected as the monitoring point of the acupoint body surface potential, and the Zusanli point (ST36) is the point intervention point of the acupoint, and the point single point is given to the Zusanli point single point, respectively. Pure massage, 0.2mL blank ointment massage, 0.2mL Tat-SOD (3000U/mL) ointment massage, injection of 100 micron L, glutathione sodium (0.12g/mL) and acupuncture and moxibustion, to investigate its effect on the potential of two acupoints (lower giant deficiency and inner atrium) adjacent to the lower reaches of the stomach meridian. The results show that the lower giant deficiency and the interting potential with the application or stop of the massage on the Zusanli. The acupoint potential of the anti oxidation intervention group still fluctuates at high level after the stop of the massage, and the point potential of the acupoint in the antioxidant intervention group is significantly lower than that in the physical intervention group after 48 hours of intervention, indicating that the acupoint potential can accurately reflect the conduction of oxidative stress on the meridian. The meridian line connects the working state of the viscera. Finally, we use the real-time monitoring method of the acupoint potential, tracking the three kinds of tea soup of black tea (Jin Jun eyebrow), oolong tea (Narcissus rock tea) and green tea (alpine green tea) to the human hand less Yin heart meridian, hand Taiyin lung meridian, foot less Yin kidney meridian, foot Taiyin splenic meridian, foot syncope Yin liver meridian, hand sun small intestine meridian foot, foot Shaoyang gallbladder The effect of body surface potential and immediate physiological effect on the eight meridians of the meridian and Foot Yangming Meridian showed that the body surface potential of the three kinds of tea soup was the most significant. The three kinds of tea soup could produce immediate physiological effects, and there were some physiological changes such as hiccup, birth, sweat, Kazakhstan, intestinal peristalsis, hand heart and other physiological changes. The immediate effect of black tea was most obvious, the action time was the longest, the fluctuation of the surface potential of the meridians and collaterals had corresponding physiological responses. Secondly, we also measured the antioxidant activity and particle size distribution of the three kinds of tea soup in vitro, and tracked the long-term physiological effects of the animal model drinking tea (three kinds of tea soup intake to the high fat diet induced C57BL/6 The effects of oxidative stress on the liver and intestinal tissue of mice were compared with the changes of the body surface potential and immediate physiological effects of the body and collaterals of the tea soup. The results showed that the smallest size of the black tea tea soup was the smallest, the immediate physiological effect and the changes of the potential of the meridian acupoints caused by the human body were the most obvious, and to a certain extent, it could be reversed to a certain extent. The particle size of the tea soup can affect its immediate effect; the antioxidant activity and the content of tea polyphenols in the tea tea soup in vitro are the lowest, but the changes in the surface potential of the meridians and collaterals are the most obvious. The results of the analysis of the antioxidant activity in the experiment in vitro may not have the corresponding performance in the experiment in the body; the three kinds of tea soup all have the inhibition. Obesity forms and alleviates the effect of interintestinal oxidative stress. We speculate that the antioxidant nanoparticles of the tea soup can reduce the oxidative stress of the liver by removing the ROS of the loose connective tissue layer of the intestinal wall, causing the contraction of the intestinal smooth muscle, producing hiccups, and preventing the fatty liver and obesity by using the bridge of the bile duct to further relieve the oxidative stress of the liver. At the same time, due to the existence of the human bioelectrical network system, the transmission of interintestinal oxidative stress may lead to potential changes on the lines connected to the organs. The above results provide a powerful evidence for the correlation of the hepatic interintestinal oxidative stress, which preliminarily indicates that the intestinal tract may be a new treatment field for the liver ROS; it is confirmed through the acupoint The feasibility of interposition potential monitoring and the oxidative stress state of the interconnected organs of the meridians revealed that food in addition to providing nutrition and energy also has the role of regulating the system of human oxidative stress treatment, providing new perspectives and directions for digestive tract and intestinal diseases, and raising the understanding of the interaction between food and human body to a new level.
【学位授予单位】：浙江工商大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R151
本文编号：2162989
[Abstract]:The liver and intestines are important organs for the metabolism and absorption of food. In the past 30 years, a large number of clinical data and epidemiological investigations have shown that there is a strong correlation between the two groups of liver diseases and intestinal diseases. Although the pathogenesis of liver and intestinal lesions or successively pathological changes has not been explained, many studies have found that oxidative stress has been found. The correlation of liver and intestinal diseases plays a key role. Therefore, a series of studies have been carried out in this paper to analyze the correlation between the distribution of active oxygen (ROS) in the liver and intestine, to analyze the correlation between the oxidative stress in the liver and the intestine, and to explore the regulation of the antioxidative food on the oxidative stress of liver and the whole body and the whole body oxidation stress system. The correlation of the interintestinal oxidative stress on the structure. SD rats were injected with the ROS probe two chlorofluorescein diacetate (DCFH-DA, 25mg/kg weight) tail vein. The overall fluorescence imaging of the common bile duct and duodenum showed the characteristic fluorescence of ROS; then, the common bile duct and duodenum were sectioning, and the two fluorescence imaging showed that ROS was special. The ROS characteristic fluorescence of the choledochus is concentrated on the outer membrane layer. The ROS characteristic fluorescence of the duodenum is concentrated in the submucosa, that is, the fluorescent channel is formed from the connective tissue layer of the common bile duct to the duodenum, indicating that the connective tissue layer from the outer membrane layer of the choledochus is connected to ten. Two refers to the connective tissue layer of the submucosa of the intestinal mucosa, which forms a ROS concentrated channel. Thus, we speculate that the oxidative stress in the liver may be transmitted through the bile duct. To further confirm the above speculation, the experimental liver oxidative stress was induced by injecting 20 mu hydroperoxide (30%, w/v) into the central left hepatic lobe of New Zealand rabbit. The current changes in the common bile duct caused by the oxidative stress. It was found that the current caused by the injection of the common bile duct increased rapidly, the electron trend was from the liver to the duodenum, and the trend of the current continued to continue for a period after the heart stopped beating. The current could be the electricity that the mitochondria leaked out of the respiratory chain during oxidative stress. The migration of the children indicates that the oxidative stress in the liver may be transmitted through the bile duct to the duodenal wall electronically. The above results provide new evidence for the correlation of interintestinal oxidative stress. To further confirm the results of the above interhepatic oxidative stress correlation, we will specifically eliminate superoxide anion. The free radical (O2.-) transmembrane protease (Tat-SOD fusion protein) acts on the intestinal tract and analyzes the regulating effect of antioxidant intervention on the oxidative stress state of the liver. The experimental SD rats with SD (1mL/kg weight, two times a week, 6 weeks of administration) were used to induce liver fibrosis in SD rats, and the Tat-SOD fusion protein (25000U/kg body). The results showed that the antioxidant intervention of Tat-SOD fusion protein in the intestinal tract could significantly reduce the serum alanine transaminase (ALT) and glutamic aminotransferase (AST) and the content of malondialdehyde (MDA) in the liver tissue to normal level (P0.01) and significantly increase the liver of SD rats. In the dirty tissue, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activity (P0.01), and the diffuse fibrosis of the liver can be reversed and regenerated. The liver tissue structure can be restored significantly. The results show that the antioxidant intervention of the Tat-SOD fusion protein by oral administration can reverse the liver fiber. We speculate that the Tat-SOD fusion protein may be transdermal into the loose connective tissue of the intestinal wall to remove the 02- that is enriched or transmitted by the site, and thus may relieve the oxidative stress state of the liver through the bridge of the bile duct, thus repairing the liver fibrosis. The tissue layer is transmitted, and the human meridian system overlaps anatomically with the fascia and other connective tissue systems. The meridian system may be the conduction and regulation system of the oxidative stress associated with the organs. Therefore, the measurement of the potential of the two acupoints on the meridian may accurately reflect the transmission of the oxidative stress along the meridian, and the reaction and the meridian viscera. The relationship between the connective tissue and oxidative stress is further examined in the human body by the method of real-time monitoring of the acupoint potential. The test points for the surface potential of the acupoint (ST39) and the acupoint (ST44) of the stomach meridian are selected as the monitoring point of the acupoint body surface potential, and the Zusanli point (ST36) is the point intervention point of the acupoint, and the point single point is given to the Zusanli point single point, respectively. Pure massage, 0.2mL blank ointment massage, 0.2mL Tat-SOD (3000U/mL) ointment massage, injection of 100 micron L, glutathione sodium (0.12g/mL) and acupuncture and moxibustion, to investigate its effect on the potential of two acupoints (lower giant deficiency and inner atrium) adjacent to the lower reaches of the stomach meridian. The results show that the lower giant deficiency and the interting potential with the application or stop of the massage on the Zusanli. The acupoint potential of the anti oxidation intervention group still fluctuates at high level after the stop of the massage, and the point potential of the acupoint in the antioxidant intervention group is significantly lower than that in the physical intervention group after 48 hours of intervention, indicating that the acupoint potential can accurately reflect the conduction of oxidative stress on the meridian. The meridian line connects the working state of the viscera. Finally, we use the real-time monitoring method of the acupoint potential, tracking the three kinds of tea soup of black tea (Jin Jun eyebrow), oolong tea (Narcissus rock tea) and green tea (alpine green tea) to the human hand less Yin heart meridian, hand Taiyin lung meridian, foot less Yin kidney meridian, foot Taiyin splenic meridian, foot syncope Yin liver meridian, hand sun small intestine meridian foot, foot Shaoyang gallbladder The effect of body surface potential and immediate physiological effect on the eight meridians of the meridian and Foot Yangming Meridian showed that the body surface potential of the three kinds of tea soup was the most significant. The three kinds of tea soup could produce immediate physiological effects, and there were some physiological changes such as hiccup, birth, sweat, Kazakhstan, intestinal peristalsis, hand heart and other physiological changes. The immediate effect of black tea was most obvious, the action time was the longest, the fluctuation of the surface potential of the meridians and collaterals had corresponding physiological responses. Secondly, we also measured the antioxidant activity and particle size distribution of the three kinds of tea soup in vitro, and tracked the long-term physiological effects of the animal model drinking tea (three kinds of tea soup intake to the high fat diet induced C57BL/6 The effects of oxidative stress on the liver and intestinal tissue of mice were compared with the changes of the body surface potential and immediate physiological effects of the body and collaterals of the tea soup. The results showed that the smallest size of the black tea tea soup was the smallest, the immediate physiological effect and the changes of the potential of the meridian acupoints caused by the human body were the most obvious, and to a certain extent, it could be reversed to a certain extent. The particle size of the tea soup can affect its immediate effect; the antioxidant activity and the content of tea polyphenols in the tea tea soup in vitro are the lowest, but the changes in the surface potential of the meridians and collaterals are the most obvious. The results of the analysis of the antioxidant activity in the experiment in vitro may not have the corresponding performance in the experiment in the body; the three kinds of tea soup all have the inhibition. Obesity forms and alleviates the effect of interintestinal oxidative stress. We speculate that the antioxidant nanoparticles of the tea soup can reduce the oxidative stress of the liver by removing the ROS of the loose connective tissue layer of the intestinal wall, causing the contraction of the intestinal smooth muscle, producing hiccups, and preventing the fatty liver and obesity by using the bridge of the bile duct to further relieve the oxidative stress of the liver. At the same time, due to the existence of the human bioelectrical network system, the transmission of interintestinal oxidative stress may lead to potential changes on the lines connected to the organs. The above results provide a powerful evidence for the correlation of the hepatic interintestinal oxidative stress, which preliminarily indicates that the intestinal tract may be a new treatment field for the liver ROS; it is confirmed through the acupoint The feasibility of interposition potential monitoring and the oxidative stress state of the interconnected organs of the meridians revealed that food in addition to providing nutrition and energy also has the role of regulating the system of human oxidative stress treatment, providing new perspectives and directions for digestive tract and intestinal diseases, and raising the understanding of the interaction between food and human body to a new level.
【学位授予单位】：浙江工商大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R151
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