抗氧化剂在梗阻性黄疸大鼠中枢神经系统氧化应激损伤中的应用研究
发布时间：2018-09-12 14:33
【摘要】：背景:梗阻性黄疸是指肝内胆管或肝外胆管等胆道系统发生机械性梗阻时,胆红素因胆汁向肠道的排泄受阻而返流入血所引起的黄疸。胆管受到阻塞时可有血清胆红素及总胆汁酸明显增高,而血清碱性磷酸酶(ALP)、谷氨酰转移酶(GGT)、谷丙转氨酶(ALT)等指标也可有显著增高。由于胆红素的亲脂性特点,其易与脂质含量多的组织结合而使组织细胞受损伤,而中枢神经系统又是机体除脂肪组织外脂质含量最丰富的器官,因此,胆红素对中枢神经系统损伤是最大的。胆红素浓度的升高可干扰正常脑细胞代谢,使细胞处于氧化应激状态,产生过多的氧自由基;并下调大脑中闭合蛋白的浓度,导致血脑屏障的破坏。于此同时大脑中caveolin-1(与脑血管屏障的损坏有关)和β-catenin的表达也可增加,以减轻胆红素对血脑屏障的损害。此外,胆红素对神经系统的损伤还产生大量的小分子物质,产生的氧自由基及脂质过氧化物可消耗大量的抗氧化酶,使体内沉积大量的高活性分子,氧化系统及抗氧化系统失去平衡,进而对组织及细胞产生氧化应激损伤。氧化应激损伤使生物膜损伤更重,当生物膜严重受损时,大量钙离子流入细胞内,可引起细胞内Ca2+超载,造成细胞功能障碍或丧失。在此基础上,小胶质细胞被激活,诱发炎症反应,最终导致神经营养因子的缺乏,甚至引起神经细胞的进行性变性及死亡。Chroni E等研究发现了梗阻性黄疸可以导致大鼠大脑中直接和间接氧化应激标记物的增加或降低,证实在梗阻性黄疸早期和晚期时的氧化应激状态与黄疸有关联,指出氧化应激是胆红素介导的脑病的一个重要机制。最近关于氧化应激反应的研究表明,Keap1-Nrf2-ARE抗氧化通路在机体抗氧化损伤的过程中起着重要的作用,它能维持细胞内氧化还原平衡,使细胞免受氧化性谷氨酸毒性和过氧化氢诱导的凋亡。NMDAR(N-methyl-D-aspartate receptor,N-甲基-D-天冬氨酸受体)是离子型谷氨酸受体一个亚型,主要分布于中枢神经系统的突触后膜。NMDAR主要参与兴奋性突触传递,具有配体电压双重门控的特性,对钙离子有较高的通透性。NMDAR的过度激活可引起兴奋性神经毒性,并通过一系列的反应,最终导致神经细胞死亡。而氧自由基也可以促进神经元和神经胶质细胞释放谷氨酸,同时又可以抑制谷氨酸的再摄取,谷氨酸激活NMDA受体通过Ca2+活化磷脂酶A又可产生氧自由基从而形成恶性循环。地卓西平马来酸盐(dizocilpinemaleate,MK-801)是一种非竞争性NMDA受体拮抗剂,它易于透过血脑屏障,对NMDAR通道进行变构调节,可有效阻滞谷氨酸(glutamic acid)与突触后膜上的受体结合,阻止NMDAR偶联的Ca2+通道激活,减少Ca2+内流,进而使NMDA受体的作用减弱,降低谷氨酸的毒性作用。大量研究结果证实MK-801对中毒、缺血缺氧、肝性脑病等多种情况下的神经元损伤具有保护作用。.白藜芦醇(resveratrol,Res)是在葡萄和红酒中大量存在的一种黄酮类多酚化合物,大量的体内和体外研究发现它有广泛的生物学效应和药学活性作用,如抵御活性自由基对机体细胞DNA的损伤并且防止细胞膜发生脂质过氧化反应等,并且已在心血管疾病、糖尿病、肾脏疾病等证实,发现白藜芦醇所产生的药理作用至少有部分是通过诱导HO-1的表达而发挥抗氧化应激损伤而进一步产生细胞保护作用的,但目前尚无在梗阻性黄疸发生时中枢神经系统中的应用研究。本实验首先建立梗阻性黄疸大鼠模型,再通过观察MK-801对梗阻性黄疸引起的大脑氧化应激反应中丙二醛(MDA),总抗氧化能力(T-AOC),超氧化物歧化酶(SOD),过氧化氢酶(CAT),核转录因子E2相关因子2(transcription factor NF-E2-related factor 2,Nrf2)蛋白,Nfr2 m RNA,血红素加氧酶-1(heineoxygenase-1,HO-1),NAD(P)H醌氧化还原酶1(NQO1,NAD(P)H quinone oxidoreductase 1)等指标的影响,进而深入探讨NMDA受体阻滞剂对梗阻性黄疸所致大脑氧化应激损伤是否具有保护作用及其具体机制。论文第三部分主要通过白藜芦醇对梗阻性黄疸大鼠中枢神经系统内MDA,SOD及HO-1研究其在氧化应激损伤的过程中的作用。第一部分MK-801对梗阻性黄疸大鼠中枢神经抗氧化应激系统的影响目的:本部分研究的目的在于成功建立梗阻性黄疸大鼠模型,并探讨MK-801对梗阻性黄疸大鼠中枢神经抗氧化应激系统的影响。方法:20只大鼠随机分为四组:I组(假手术组)、II组(对照组)、III组(低剂量处理组)和IV组(高剂量处理组),II,III,IV组为梗阻性黄疸模型组,III,IV组为MK-801组。第2天起III组腹腔注射MK-801,0.025mg/kg.d,IV组腹腔注射MK-801,0.25mg/kg.d;I组和II组以等容量生理盐水同时注射。四组大鼠术后3d采尾部静脉血以全自动生化仪检查血清总胆红素和直接胆红素浓度来确定模型制作是否成功,术后10天处死大鼠并取材(大脑皮质)按试剂盒要求行MDA及CAT、T-SOD、T-AOC含量或活性测定。结果:1假手术组实验鼠术后当日即恢复饮食及自由活动,且活动灵敏,小便淡黄。梗阻性黄疸模型组大鼠第2天开始出现尾尖及耳部等皮肤较薄的部位黄染,并逐渐出现全身黄染,尿液深黄,精神萎靡、反应迟缓。术后第三天,对照组、MK-801低剂量组、MK-801高剂量组大鼠大脑组织中直接胆红素的浓度和总胆汁酸含量(μmol/L)与假手术组相比明显增高,且有统计学差异(P0.01),考虑梗阻性黄疸模型制作成功。2对照组、MK-801低剂量组、MK-801高剂量组丙二醛的含量与假手术组相比升高,且有统计学差异(P0.05)。应用MK-801后丙二醛含量与对照组相比降低,且具有统计学意义,但MK-801高、低剂量组之间无统计学差异(P0.05)。3对照组氧化氢酶活性与假手术组相比降低,MK-801组过氧化氢酶活性比对照组明显增高,且有统计学差异(P0.05)。MK-801低剂量组、MK-801高剂量组之间过氧化氢酶活性无统计学差异(P0.05)4对照组总超氧化物歧化酶活性比假手术组降低,且有统计学意义,MK-801组总超氧化物岐化酶活性与对照组相比明显增高,且有统计学差异(P0.05)。MK-801高剂量组总超氧化物岐化酶活性与低剂量组相比无统计学差异。5对照组、MK-801低剂量组、MK-801高剂量组总抗氧化能力与假手术组相比明显增高,且有统计学意义(P0.05)。MK-801组比对照组总抗氧化能力升高,但高剂量组与对照组之间相比无统计学差异(P0.05)。结论1通过结扎实验动物胆总管可以成功建立梗阻性黄疸动物模型。2梗阻性黄疸时中枢神经系统机体存在氧化应激反应,机体通过增加过氧化氢酶等抗氧化应激损伤酶类的表达使抗氧化应激损伤能力增强,这与国内外目前研究一致。3 MK-801使梗阻性黄疸所致大鼠中枢神经系统氧化应激损伤的过程中脂质过氧化程度降低,并且可以提高SOD的活性及机体总抗氧化应激能力,高剂量的MK-801与低剂量的MK-801相比效果并未明显增加,反而使T-SOD活性比低剂量组降低,具体机制需要进一步研究。第二部分Keap1-Nrf2-ARE信号通路在梗阻性黄疸大鼠中枢神经系统氧化应激损伤中的作用探讨目的:研究Keap1-Nrf2-ARE信号通路在梗阻性黄疸大鼠中枢神经系统氧化应激损伤中的作用及在机体氧化应激反应过程中NMDA受体阻滞剂对Keap1-Nrf2-ARE信号通路的影响方法:四组大鼠分别为:I组(假手术组)、II组(对照组)、III组(低剂量处理组)和IV组(高剂量处理组),II,III,IV组为梗阻性黄疸模型组,第2天起III组腹腔注射MK-801,0.025mg/kg.d,IV组腹腔注射MK-801,0.25mg/kg.d,连续注射10d;I组和II组以等容量生理盐水同时注射。术后10天取材以Western bolt行HO-1,NQO1,Nrf2蛋白,以RT-PCR行Nrf2 m RNA测定。结果1与假手术组相比,OJ组Nrf2 m RNA表达均明显升高,且均有统计学差异,P0.05。MK-801组与对照组相比Nrf2 m RNA表达升高且有统计学意义,P0.05。高剂量组与对照组相比Nrf2 m RNA表达降低,但无统计学差异,P0.05。2与假手术组相比,OJ组Nrf2蛋白表达均增高,且有统计学意义,P0.05;MK-801组与对照组相比Nrf2蛋白表达增高,且有统计学意义,P0.05。不同的是高剂量组与低剂量组相比Nrf2蛋白表达减低,而且具有统计学意义,P0.05。3 OJ组与假手术组相比HO-1蛋白表达增高,且有统计学意义,P0.05。MK-801组与对照组相比HO-1蛋白表达增高,且有统计学意义,P0.05。高低剂量MK-801组比低剂量组相比,HO-1表达水平降低,且具有统计学差异P0.05。4OJ组与假手术组相比NQO1蛋白表达增高,且有统计学意义,P0.05。MK-801组与对照组相比NQO1蛋白表达增高,且有统计学意义,P0.05。高低剂量MK-801组比低剂量组相比,NQO1表达水平降低,且具有统计学差异P0.05。结论1再一次验证梗阻性黄疸时机体存在氧化应激反应。2机体通过Keap1-Nrf2-ARE信号通路增强了HO-1和NQO1及Nrf2蛋白的表达,增强了自身抗氧化应激能力。3 NMDA受体阻滞剂MK-801通过Keap1-Nrf2-ARE信号通路上调了II相解毒酶HO-1和NQO-1的蛋白表达,增加了机体抗氧化应激反应能力,从而发挥了它的神经保护作用。虽然高、低剂量的MK-801均能达到此作用,但高剂量的MK-801反而使Nrf2 m RNA、Nrf2蛋白、HO-1和NQO1蛋白的表达较低剂量组降低,可能与MK-801毒性作用有关。第三部分不同水平白藜芦醇对梗阻性黄疸大鼠中枢神经系统氧化应激目的:通过对研究梗阻性黄疸大鼠大脑皮质中氧化应激指标表达水平来探讨不同水平白藜芦醇对梗阻性黄疸大鼠中枢神经系统氧化应激损伤的影响及其氧化应激损伤中的保护作用和机制。方法:选择32只6周龄的雄性SD大鼠作为实验对象,将其随机分为四个组别,分笼饲养,每组8只,A组大鼠进行梗阻性黄疸假手术(只将胆总管游离出来,不做结扎),B、C、D组大鼠均进行手术制备梗阻性黄疸模型,术后每日C、D两组大鼠分别给予不同剂量的白藜芦醇溶液灌胃,A、B组则给予同等剂量生理盐水灌胃。分别在手术后的第14d采集血液标本行总胆红素、直接胆红素、谷丙转氨酶、谷草转氨酶测定,并采集大脑皮质标本,并对其中中丙二醛、超氧化物歧化酶活性、HO-1水平进行检测,观察不同组别的大鼠各项指标之间的差异。结果:术后TBIL及DBIL指标结果证明II、III、IV三组大鼠梗阻性黄疸模型制作成功。OJ组ALT、AST、MDA水平比假手术组升高,SOD、HO-1水平比假手术组降低。OJ组内反应肝功能损害指标ALT,AST及反应氧化应激损伤指标MDA的平均水平呈IIIIIIV的趋势,而氧化应激保护性指标SOD和HO-1平均值则呈IIIIIIV的趋势,且具有统计学意义。不同剂量的白藜芦醇组之间,指标相比也存在统计学差异(P0.05)。结论:白藜芦醇对梗阻性大鼠TBIL及DBIL影响不大,但可以保护其肝功能,并且可以能促进大脑皮质细胞组织中的丙二醛含量降低、SOD和HO-1活性升高,发挥抗氧化应激损伤的作用。
[Abstract]:BACKGROUND: Obstructive jaundice refers to jaundice caused by bilirubin reflux into the blood due to obstruction of excretion of bile from the intestinal tract when mechanical obstruction occurs in the intrahepatic or extrahepatic bile ducts. Alpha-aminotransferase (ALT) and other indicators can also be significantly increased. Because of the lipophilic characteristics of bilirubin, it is easy to combine with lipid-rich tissues and cause tissue and cell damage. The central nervous system is the most lipid-rich organ except adipose tissue. Therefore, bilirubin is the greatest damage to the central nervous system. The elevation of Caveolin-1 and beta-catenin in the brain can also increase the expression of caveolin-1 and beta-catenin in the brain to reduce the blood-brain barrier damage. In addition, bilirubin damage to the nervous system also produces a large number of small molecules, oxygen free radicals and lipid peroxides can consume a large number of antioxidant enzymes, so that a large number of highly active molecules deposited in the body, oxidative system and antioxidant system imbalance, and then produce oxidative stress damage to tissues and cells. Oxidative stress injury can aggravate the damage of biofilm. When the biofilm is severely damaged, a large amount of calcium ions flow into the cell, which can cause intracellular Ca2+ overload, resulting in cell dysfunction or loss. On this basis, microglia are activated, causing inflammation, eventually leading to the lack of neurotrophic factors, and even lead to the progress of nerve cells. Chroni E and other studies have found that obstructive jaundice can lead to the increase or decrease of direct and indirect oxidative stress markers in the brain of rats, confirming that oxidative stress in the early and late stages of obstructive jaundice is associated with jaundice, indicating that oxidative stress is an important mechanism of bilirubin-mediated encephalopathy. Studies on oxidative stress have shown that the Keap1-Nrf2-ARE antioxidant pathway plays an important role in the process of anti-oxidative damage in the body. It can maintain the balance of oxidation and reduction in cells, and protect cells from oxidative glutamate toxicity and hydrogen peroxide-induced apoptosis. NMDAR (N-methyl-D-aspartate receptor, N-methyl-D-aspartate receptor) receives NMDAR is a subtype of ionic glutamate receptor, mainly distributed in the postsynaptic membranes of the central nervous system. NMDAR is involved in excitatory synaptic transmission, has the characteristics of ligand voltage double-gated, and has a high permeability to calcium ions. Overactivation of NMDAR can cause excitatory neurotoxicity, and ultimately lead to a series of reactions. Neuronal cell death. Oxygen free radicals also promote the release of glutamate from neurons and glial cells, and inhibit glutamate reuptake. Glutamate-activated NMDA receptors produce oxygen free radicals through Ca2+ activated phospholipase A to form a vicious cycle. Dizocilpine maleate (MK-801) is a kind of dizocilpine maleate. Non-competitive NMDA receptor antagonists, which can easily regulate NMDAR channels through blood-brain barrier, can effectively block the binding of glutamic acid to receptors on postsynaptic membranes, prevent the activation of NMDAR-coupled Ca2+ channels, reduce Ca2+ influx, thereby weakening the role of NMDA receptors and reducing the toxicity of glutamate. Resveratrol (Res) is a kind of flavonoid polyphenols in grape and red wine. A large number of in vivo and in vitro studies have found that it has a wide range of biological effects and pharmacological activities, such as. Resveratrol has been shown to protect cells from DNA damage by reactive free radicals and lipid peroxidation. It has been demonstrated in cardiovascular diseases, diabetes mellitus and kidney diseases that the pharmacological effects of resveratrol are at least partially mediated by inducing the expression of HO-1 and exerting antioxidant stress damage to cells. In this study, we first established a rat model of obstructive jaundice, and then observed the effects of MK-801 on malondialdehyde (MDA), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), hydrogen peroxide (hydrogen peroxide) in oxidative stress of the brain induced by obstructive jaundice. Enzyme (CAT), transcription factor NF-E2-related factor 2 (Nrf2) protein, Nfr2 m RNA, heme oxygenase-1 (HO-1), NAD (P) H quinone oxidoreductase 1 (NQO1, NAD (P) and other indicators of the impact of NMDA receptor blockers on obstructive jaundice caused by large The third part of this paper mainly studies the effects of resveratrol on MDA, SOD and HO-1 in the central nervous system of rats with obstructive jaundice. Objective: To establish a successful model of obstructive jaundice in rats and investigate the effects of MK-801 on the central nervous system in rats with obstructive jaundice.Methods: Twenty rats were randomly divided into four groups: group I (sham operation group), group II (control group), group III (low dose treatment group) and group IV (high dose treatment group), group II, III, IV. Group II was injected intraperitoneally with MK-801,0.025mg/kg.d. Group IV was injected intraperitoneally with MK-801,0.25mg/kg.d. Group I and Group II were injected with normal saline of the same volume at the same time. The rats were sacrificed 10 days after operation and the contents or activities of MDA, CAT, T-SOD and T-AOC were determined according to the requirements of the kit. Results: 1. The rats in sham operation group resumed their diet and free movement on the day after operation, and their urine was light yellow. The rats in obstructive jaundice model group began to appear tail tips and tail tips on the second day. On the third day after operation, the concentration of direct bilirubin and total bile acid (micromol/L) in brain tissue of rats in control group, low dose MK-801 group and high dose MK-801 group were significantly higher than those in sham operation group (P The content of malondialdehyde in control group, MK-801 low-dose group and MK-801 high-dose group was significantly higher than that in sham-operation group (P 0.05). The content of malondialdehyde in MK-801 high-dose group was significantly lower than that in control group (P 0.05). The catalase activity of control group was lower than that of sham operation group, and the catalase activity of MK-801 group was significantly higher than that of control group (P 0.05). The catalase activity of low dose MK-801 group and high dose MK-801 group had no statistical difference (P 0.05). The total superoxide dismutase activity of control group was lower than that of sham operation group. The total superoxide dismutase activity of MK-801 group was significantly higher than that of the control group, and there was a statistical difference (P 0.05). The total superoxide dismutase activity of MK-801 high-dose group was not significantly different from that of low-dose group. The total antioxidant capacity of MK-801 group was higher than that of the control group, but there was no significant difference between the high-dose group and the control group (P 0.05). Conclusion 1 The animal model of obstructive jaundice can be successfully established by ligating the common bile duct of experimental animals. 2 There is oxidative stress in the central nervous system during obstructive jaundice. In response, the body enhances the ability of anti-oxidative stress injury by increasing the expression of catalase and other anti-oxidative stress injury enzymes, which is consistent with the current research at home and abroad. 3 MK-801 can reduce the degree of lipid peroxidation in the process of oxidative stress injury of the central nervous system induced by obstructive jaundice in rats, and can increase the activity of SOD and The total antioxidant stress ability of the body, the effect of high dose MK-801 and low dose MK-801 did not increase significantly, but decreased the activity of T-SOD compared with low dose group. The specific mechanism needs further study. Part II The role of Keap1-Nrf2-ARE signaling pathway in oxidative stress injury of central nervous system in obstructive jaundice rats AIM: To investigate the role of Keap 1-Nrf 2-ARE signaling pathway in oxidative stress injury of central nervous system in rats with obstructive jaundice and the effect of NMDA receptor blockers on Keap 1-Nrf 2-ARE signaling pathway during oxidative stress in vivo. Methods: Four groups of rats were divided into: group I (sham operation group), group II (control group), group III (low dose treatment group) and group IV. Group II, III and IV were treated with intraperitoneal injection of MK-801, 0.025mg/kg.d from day 2. Group IV was treated with intraperitoneal injection of MK-801, 0.25mg/kg.d for 10 consecutive days. Group I and II were injected with normal saline of equal volume at the same time. Results 1 Compared with the sham operation group, the expression of Nrf2 m RNA in OJ group was significantly higher, and there was statistical difference. Compared with the control group, the expression of Nrf2 m RNA in OJ group was significantly higher and statistically significant, P 0.05. The expression of Nrf2 protein in MK-801 group was significantly higher than that in the control group (P 0.05). The expression of HO-1 protein in high and low dose MK-801 group was lower than that in low dose MK-801 group. The expression of NQO1 protein in high and low dose MK-801 group was higher than that in sham operation group (P 0.05). The expression of NQO1 was significantly lower in MK-801 group than in low dose group (P 0.05). Conclusion1 Again, there was an oxidative stress response in the body during obstructive jaundice. 2 The expression of HO-1, NQO1 and Nrf2 proteins was enhanced by Keap1-Nrf2-ARE signaling pathway. 3 NMDA receptor blocker MK-801 regulates the expression of phase II detoxifying enzymes HO-1 and NQO-1 through the Keap1-Nrf2-ARE signaling pathway, increases the antioxidant stress response ability of the body, and thus exerts its neuroprotective effect. Although both high and low doses of MK-801 can achieve this effect, high doses of MK-801 can induce Nrf2 m RNA instead. The expression of Nrf2 protein, HO-1 and NQO1 protein decreased in the lower dose group, which may be related to the toxicity of MK-801. Part III Oxidative stress of central nervous system in rats with obstructive jaundice induced by resveratrol at different levels: To investigate the expression of oxidative stress in cerebral cortex of rats with obstructive jaundice. Methods: Thirty-two six-week-old male SD rats were randomly divided into four groups and fed in cages. Eight rats in each group were given sham operation for obstructive jaundice. Rats in group B, C and D were operated on to establish obstructive jaundice model. Rats in group C and D were separated daily after operation.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R575
本文编号：2239332
[Abstract]:BACKGROUND: Obstructive jaundice refers to jaundice caused by bilirubin reflux into the blood due to obstruction of excretion of bile from the intestinal tract when mechanical obstruction occurs in the intrahepatic or extrahepatic bile ducts. Alpha-aminotransferase (ALT) and other indicators can also be significantly increased. Because of the lipophilic characteristics of bilirubin, it is easy to combine with lipid-rich tissues and cause tissue and cell damage. The central nervous system is the most lipid-rich organ except adipose tissue. Therefore, bilirubin is the greatest damage to the central nervous system. The elevation of Caveolin-1 and beta-catenin in the brain can also increase the expression of caveolin-1 and beta-catenin in the brain to reduce the blood-brain barrier damage. In addition, bilirubin damage to the nervous system also produces a large number of small molecules, oxygen free radicals and lipid peroxides can consume a large number of antioxidant enzymes, so that a large number of highly active molecules deposited in the body, oxidative system and antioxidant system imbalance, and then produce oxidative stress damage to tissues and cells. Oxidative stress injury can aggravate the damage of biofilm. When the biofilm is severely damaged, a large amount of calcium ions flow into the cell, which can cause intracellular Ca2+ overload, resulting in cell dysfunction or loss. On this basis, microglia are activated, causing inflammation, eventually leading to the lack of neurotrophic factors, and even lead to the progress of nerve cells. Chroni E and other studies have found that obstructive jaundice can lead to the increase or decrease of direct and indirect oxidative stress markers in the brain of rats, confirming that oxidative stress in the early and late stages of obstructive jaundice is associated with jaundice, indicating that oxidative stress is an important mechanism of bilirubin-mediated encephalopathy. Studies on oxidative stress have shown that the Keap1-Nrf2-ARE antioxidant pathway plays an important role in the process of anti-oxidative damage in the body. It can maintain the balance of oxidation and reduction in cells, and protect cells from oxidative glutamate toxicity and hydrogen peroxide-induced apoptosis. NMDAR (N-methyl-D-aspartate receptor, N-methyl-D-aspartate receptor) receives NMDAR is a subtype of ionic glutamate receptor, mainly distributed in the postsynaptic membranes of the central nervous system. NMDAR is involved in excitatory synaptic transmission, has the characteristics of ligand voltage double-gated, and has a high permeability to calcium ions. Overactivation of NMDAR can cause excitatory neurotoxicity, and ultimately lead to a series of reactions. Neuronal cell death. Oxygen free radicals also promote the release of glutamate from neurons and glial cells, and inhibit glutamate reuptake. Glutamate-activated NMDA receptors produce oxygen free radicals through Ca2+ activated phospholipase A to form a vicious cycle. Dizocilpine maleate (MK-801) is a kind of dizocilpine maleate. Non-competitive NMDA receptor antagonists, which can easily regulate NMDAR channels through blood-brain barrier, can effectively block the binding of glutamic acid to receptors on postsynaptic membranes, prevent the activation of NMDAR-coupled Ca2+ channels, reduce Ca2+ influx, thereby weakening the role of NMDA receptors and reducing the toxicity of glutamate. Resveratrol (Res) is a kind of flavonoid polyphenols in grape and red wine. A large number of in vivo and in vitro studies have found that it has a wide range of biological effects and pharmacological activities, such as. Resveratrol has been shown to protect cells from DNA damage by reactive free radicals and lipid peroxidation. It has been demonstrated in cardiovascular diseases, diabetes mellitus and kidney diseases that the pharmacological effects of resveratrol are at least partially mediated by inducing the expression of HO-1 and exerting antioxidant stress damage to cells. In this study, we first established a rat model of obstructive jaundice, and then observed the effects of MK-801 on malondialdehyde (MDA), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), hydrogen peroxide (hydrogen peroxide) in oxidative stress of the brain induced by obstructive jaundice. Enzyme (CAT), transcription factor NF-E2-related factor 2 (Nrf2) protein, Nfr2 m RNA, heme oxygenase-1 (HO-1), NAD (P) H quinone oxidoreductase 1 (NQO1, NAD (P) and other indicators of the impact of NMDA receptor blockers on obstructive jaundice caused by large The third part of this paper mainly studies the effects of resveratrol on MDA, SOD and HO-1 in the central nervous system of rats with obstructive jaundice. Objective: To establish a successful model of obstructive jaundice in rats and investigate the effects of MK-801 on the central nervous system in rats with obstructive jaundice.Methods: Twenty rats were randomly divided into four groups: group I (sham operation group), group II (control group), group III (low dose treatment group) and group IV (high dose treatment group), group II, III, IV. Group II was injected intraperitoneally with MK-801,0.025mg/kg.d. Group IV was injected intraperitoneally with MK-801,0.25mg/kg.d. Group I and Group II were injected with normal saline of the same volume at the same time. The rats were sacrificed 10 days after operation and the contents or activities of MDA, CAT, T-SOD and T-AOC were determined according to the requirements of the kit. Results: 1. The rats in sham operation group resumed their diet and free movement on the day after operation, and their urine was light yellow. The rats in obstructive jaundice model group began to appear tail tips and tail tips on the second day. On the third day after operation, the concentration of direct bilirubin and total bile acid (micromol/L) in brain tissue of rats in control group, low dose MK-801 group and high dose MK-801 group were significantly higher than those in sham operation group (P The content of malondialdehyde in control group, MK-801 low-dose group and MK-801 high-dose group was significantly higher than that in sham-operation group (P 0.05). The content of malondialdehyde in MK-801 high-dose group was significantly lower than that in control group (P 0.05). The catalase activity of control group was lower than that of sham operation group, and the catalase activity of MK-801 group was significantly higher than that of control group (P 0.05). The catalase activity of low dose MK-801 group and high dose MK-801 group had no statistical difference (P 0.05). The total superoxide dismutase activity of control group was lower than that of sham operation group. The total superoxide dismutase activity of MK-801 group was significantly higher than that of the control group, and there was a statistical difference (P 0.05). The total superoxide dismutase activity of MK-801 high-dose group was not significantly different from that of low-dose group. The total antioxidant capacity of MK-801 group was higher than that of the control group, but there was no significant difference between the high-dose group and the control group (P 0.05). Conclusion 1 The animal model of obstructive jaundice can be successfully established by ligating the common bile duct of experimental animals. 2 There is oxidative stress in the central nervous system during obstructive jaundice. In response, the body enhances the ability of anti-oxidative stress injury by increasing the expression of catalase and other anti-oxidative stress injury enzymes, which is consistent with the current research at home and abroad. 3 MK-801 can reduce the degree of lipid peroxidation in the process of oxidative stress injury of the central nervous system induced by obstructive jaundice in rats, and can increase the activity of SOD and The total antioxidant stress ability of the body, the effect of high dose MK-801 and low dose MK-801 did not increase significantly, but decreased the activity of T-SOD compared with low dose group. The specific mechanism needs further study. Part II The role of Keap1-Nrf2-ARE signaling pathway in oxidative stress injury of central nervous system in obstructive jaundice rats AIM: To investigate the role of Keap 1-Nrf 2-ARE signaling pathway in oxidative stress injury of central nervous system in rats with obstructive jaundice and the effect of NMDA receptor blockers on Keap 1-Nrf 2-ARE signaling pathway during oxidative stress in vivo. Methods: Four groups of rats were divided into: group I (sham operation group), group II (control group), group III (low dose treatment group) and group IV. Group II, III and IV were treated with intraperitoneal injection of MK-801, 0.025mg/kg.d from day 2. Group IV was treated with intraperitoneal injection of MK-801, 0.25mg/kg.d for 10 consecutive days. Group I and II were injected with normal saline of equal volume at the same time. Results 1 Compared with the sham operation group, the expression of Nrf2 m RNA in OJ group was significantly higher, and there was statistical difference. Compared with the control group, the expression of Nrf2 m RNA in OJ group was significantly higher and statistically significant, P 0.05. The expression of Nrf2 protein in MK-801 group was significantly higher than that in the control group (P 0.05). The expression of HO-1 protein in high and low dose MK-801 group was lower than that in low dose MK-801 group. The expression of NQO1 protein in high and low dose MK-801 group was higher than that in sham operation group (P 0.05). The expression of NQO1 was significantly lower in MK-801 group than in low dose group (P 0.05). Conclusion1 Again, there was an oxidative stress response in the body during obstructive jaundice. 2 The expression of HO-1, NQO1 and Nrf2 proteins was enhanced by Keap1-Nrf2-ARE signaling pathway. 3 NMDA receptor blocker MK-801 regulates the expression of phase II detoxifying enzymes HO-1 and NQO-1 through the Keap1-Nrf2-ARE signaling pathway, increases the antioxidant stress response ability of the body, and thus exerts its neuroprotective effect. Although both high and low doses of MK-801 can achieve this effect, high doses of MK-801 can induce Nrf2 m RNA instead. The expression of Nrf2 protein, HO-1 and NQO1 protein decreased in the lower dose group, which may be related to the toxicity of MK-801. Part III Oxidative stress of central nervous system in rats with obstructive jaundice induced by resveratrol at different levels: To investigate the expression of oxidative stress in cerebral cortex of rats with obstructive jaundice. Methods: Thirty-two six-week-old male SD rats were randomly divided into four groups and fed in cages. Eight rats in each group were given sham operation for obstructive jaundice. Rats in group B, C and D were operated on to establish obstructive jaundice model. Rats in group C and D were separated daily after operation.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R575
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