硫氧还蛋白诱导物抵抗甲醛和吗啡毒性作用分子机制研究

发布时间：2018-05-15 03:22

本文选题：硫氧还蛋白-1 + 甲醛　；参考：《昆明理工大学》2012年博士论文

【摘要】：硫氧还蛋白1(Trx-1)是分子量为12KDa的多功能蛋白质,含有一个氧化还原的活性位点：-Cys-Gly-Pro-Cys-。硫氧还蛋白具有多种生物学功能：调节氧化还原、调节转录因子活性、抑制细胞凋亡以及调节炎症反应等。Trx-1能被许多应激诱导产生,如过氧化氢、有丝分裂原、多环芳烃、紫外线、病毒感染和缺血再灌注等。Trx-1具有神经营养因子样活性,在神经生长因子诱导的神经分化和突出生长其重要的作用。Trx-1高表达转基因小鼠与野生型小鼠相比,寿命显著地延长,而且对缺血再灌注损伤、糖尿病以及外源环境应激引起的毒害具有明显的抵抗作用。Trx-1与内质网应激密切相关,具有抵抗内质网应激损伤的作用。目前研究发现：替普瑞酮(GGA)、萝卜硫素、神经妥乐和泰莫普利等都可以通过诱导Trx-1的表达水平起细胞保护作用。其中,替普瑞酮的研究应用最为深入和透彻。替普瑞酮(GGA)是临床上广泛用于胃溃疡治疗的药物,能在多种细胞中诱导Trx-1的表达,对胃黏膜、肝脏和心脏等组织起保护作用。GGA还是一种脂溶性物质,能透过血脑屏障对神经组织起重要的保护作用。调控Trx-1的表达已成为一个新的疾病治疗策略,无毒性的Trx-1诱导物能促进神经再生,对氧化应激相关的神经系统疾病具有预防作用。因此,本论文拟从云南省的天然药物资源出发,寻找无毒性的Trx-1诱导物用于疾病的治疗研究。甲醛是一种常见的环境污染物,广泛存在于香烟烟雾、家具、汽车尾气、医疗和工业用品中。研究表明,室内甲醛浓度的增加是导致严重性建筑综合症的一个重要原因。甲醛对神经系统具有毒害作用,已被认为是一种潜在的“神经毒害物”。长期暴露于甲醛会造成神经毒害,引发神经退行性疾病,同时,甲醛能改变大鼠大脑的形态特征,导致行为异常和记忆功能障碍。因此,甲醛已经成为危害身体健康的一个重要因素。为了有效预防和治疗甲醛对人体的毒性,深入研究甲醛毒害的分子机制具有重要的意义。内质网是蛋白质合成和加工的主要场所,在维持细胞内钙离子浓度和蛋白糖基化具有重要的调节作用。当内质网功能紊乱以及未折叠/错误折叠蛋白过量累积时,会导致内质网应激。内质网中含有许多分子伴侣,当内质网应激发生时,糖调节蛋白78(GRP78)的表达水平会发生显著增加,以增强内质网的蛋白质折叠能力。内质网应激程度较低时,激活抗凋亡的分子伴侣；但当内质网应激过于严重和持久时,则激活JNK、CHOP和caspase-12,导致细胞凋亡,并且内质网应激与神经细胞死亡密切相关。甲醛是一种交联剂,能与巯基和氨基酸基团相互作用,导致蛋白质的多聚化,同时甲醛会导致蛋白质错误折叠和累积。甲醛与p淀粉样蛋白相互作用,产生具有神经毒性的淀粉样蛋白复合物。甲醛还会导致tau蛋白聚集,诱导人神经母细胞瘤和海马神经细胞的凋亡。然而,内质网应激介导的凋亡途径与甲醛所致神经细胞死亡的相关研究还未见报道。吗啡滥用已经成为全球严重的公共卫生和社会问题。长期反复使用吗啡会导致躯体依赖和精神依赖的发生,这限制了它的临床用途(治疗各种急性和慢性疼痛)。吗啡成瘾的主要表现是强迫性用药,即对药物的摄取和寻觅失去控制。成瘾的原因主要由于正性强化因素(欣快感和奖赏效应)和负性强化因素(减轻戒断症状)。中脑腹侧被盖区、伏隔核、前额叶皮层、杏仁核和海马等脑区与吗啡成瘾的过程相关。除了神经系统的表现外,吗啡在肝脏和肾脏中会导致氧化应激和细胞凋亡。长期使用阿片类药物会导致肝脏、肾脏损伤,表现有急性肝炎、慢性肝炎等肝脏损伤,以及肾病综合症、急性肾小球肾炎、间质性肾炎和肾功能衰竭。因此,在药物成瘾治疗的过程中需要注意阿片类药物所致的肝脏和肾脏损伤。寻找保护吗啡所致的肝脏和肾脏损伤的药物对于降低吗啡的副作用及更广泛地应用吗啡具有重要的意义。而且,目前还没有理想的药物和方法用于吗啡成瘾的治疗。甲醛和吗啡都可以导致氧化应激损伤、细胞凋亡和神经营养因子下调,但其作用方式各不相同；而Trx-1具有抗氧化、抗凋亡和神经营养因子活性,与甲醛和吗啡的毒害机制存在交叉。这就提出了两个问题：Trx-1诱导物是否可以抵抗甲醛的毒害作用；Trx-1诱导物是否可以抵抗吗啡成瘾和吗啡所致肝肾损伤。因此,本论文将研究这两方面的内容。主要的研究结果如下： (1)三七总皂甙(PNS)是三七的主要成分之一,以其为主要成分的药物被广泛应用于临床中。三七三醇组皂甙(PTS)是PNS的主要成分之一,PTS的主要成分是人参皂甙Rg1、R1和Re(占80%),而其中人参皂甙Rg1的含量高达60%以上。PTS以剂量依赖和时间依赖的方式增加PC12细胞内Trx-1的表达水平。体内动物实验结果：与对照组小鼠相比,PTS显著诱导小鼠大脑皮层、海马和纹状体区Trx-1的表达水平。人参皂甙Rg1是PTS的主要活性成分。我们研究发现：人参皂甙Rg1显著性地诱导PC12细胞内Trx-1的表达水平。 (2)内质网应激与神经细胞凋亡相关。在本论文中,我们研究发现：甲醛显著地诱导GRP78的表达水平；甲醛处理PC12细胞12小时后,能增加CHOP的表达；在甲醛作用24小时后pro-caspase-12的表达水平显著下降。这些结果表明：甲醛在一定程度上是通过内质网应激介导的凋亡途径导致神经毒害。 (3)氧化应激是甲醛导致细胞毒害的一个重要原因。Trx-1是一种重要的抗氧化物质,能被氧化应激诱导产生。本论文研究发现：甲醛作用PC12细胞12小时后,能诱导Trx-1的表达；但在甲醛作用24小时后会降低Trx-1的表达。采用siRNA干扰下调Trx-1的表达水平,则增加PC12细胞对甲醛的敏感性。这些结果表明：甲醛的神经毒害可能与其降低Trx-1的表达水平有关。 (4)Trx-1的高表达可以抵抗氧化应激和内质网应激所致的损伤。人参皂甙Rgl对PC12细胞的存活活力无影响,但是人参皂甙Rg1的预处理可以抵抗甲醛对PC12细胞的神经毒害,并抑制甲醛所致酪氨酸羟化酶表达水平的降低。人参皂甙Rgl可以恢复甲醛所致Trx-1表达水平的降低。而且,人参皂甙Rg1还可以抵抗甲醛所致的内质网应激：抑制甲醛所致GRP78和CHOP表达水平的增加,抑制甲醛所致pro-caspase-12表达水平的下降。这些研究结果表明：Trx-1诱导剂可以抵抗甲醛的神经毒害作用。 (5)吗啡反复作用会增加小鼠大脑皮层、海马、中脑腹侧被盖区和伏隔核内Trx-1和Hsp70的表达水平。这些结果表明：吗啡作用后细胞防御机制会被激活。本论文研究证实,GGA能诱导小鼠大脑内Trx-1和Hsp70的表达,减弱吗啡急性处理所致小鼠活动增强的效应,阻止吗啡引起的条件性位置偏爱,缓解吗啡戒断症状。GGA还可以抑制吗啡所致CREB活性的增强,以及ΔFosB和Cdk5表达的增加。在NAc区中,GGA能增强吗啡所致Trx-1和Hsp70表达的增加。这些结果表明：使用无细胞毒性的诱导物增强Trx-1的表达是吗啡成瘾治疗的新策略；GGA可用于吗啡成瘾的防治。 (6)GGA在肝脏和肾脏中能抑制吗啡所致MDA含量的增加,阻止吗啡所致caspase-9和caspase-3的激活。吗啡能通过线粒体介导的凋亡途径诱导肝脏和肾脏细胞的细胞凋亡,而不是通过内质网介导的途径。更重要的是,GGA还能增强吗啡对Trx-1和Hsp70的诱导作用。这些结果表明：GGA可用于吗啡所致肝肾损伤的防治。以上结果表明：内质网应激介导的凋亡途径与甲醛所致神经细胞凋亡存在相关性；人参皂甙Rg1是一种新的Trx-1诱导物,人参皂甙Rg1可以抵抗甲醛所致的神经毒害；Trx-1诱导物GGA可以抵抗吗啡成瘾和吗啡所致的肝肾损伤。这些研究结果可为甲醛和吗啡的毒害防治及其药物开发提供基础理论依据和新靶点。
[Abstract]:Thioredoxin 1 (Trx-1) is a multifunctional protein with molecular weight 12KDa and contains a redox active site: -Cys-Gly-Pro-Cys-. thioredoxin has a variety of biological functions: regulation of redox, regulation of transcription factor activity, inhibition of apoptosis, and regulation of inflammation, such as.Trx-1 can be induced by many stress, such as Hydrogen peroxide, mitogen, polycyclic aromatic hydrocarbons, ultraviolet, viral infection, and ischemia-reperfusion.Trx-1 have neurotrophic factor like activity, and the important role of neural differentiation and protruding growth induced by nerve growth factor is the important effect of.Trx-1 high expression transgenic mice. The toxic effects of injur, diabetes and exogenous environmental stress have obvious resistance to.Trx-1, which is closely related to endoplasmic reticulum stress and is resistant to endoplasmic reticulum stress damage. Current studies have found that tepreone (GGA), sulforaphane, neurotoxin and temopri can all play a role in inducing the expression level of Trx-1. The most widely used and thorough research of teprinone. Teprinone (GGA) is a clinically widely used drug for the treatment of gastric ulcers. It can induce the expression of Trx-1 in a variety of cells, protect the gastric mucosa, liver and heart and other tissues,.GGA or a fat soluble substance, which can penetrate the blood brain barrier to the nerve tissue. The regulation of the expression of Trx-1 has become a new treatment strategy for disease. Nontoxic Trx-1 inducers can promote nerve regeneration and have a preventive effect on nervous system diseases related to oxidative stress. Therefore, this paper is intended to develop from natural drug resources in Yunnan and seek non-toxic Trx-1 inducers for disease. The study of treatment.
Formaldehyde is a common environmental pollutant, widely found in cigarette smoke, furniture, automobile exhaust, medical and industrial supplies. Research shows that the increase in indoor formaldehyde concentration is an important cause of severe construction syndrome. Formaldehyde is toxic to the nervous system and has been considered as a potential "nerve poison". The long-term exposure to formaldehyde can cause nerve toxicity and cause neurodegenerative diseases. At the same time, formaldehyde can change the morphological characteristics of the brain and lead to abnormal behavior and memory dysfunction. Therefore, formaldehyde has become an important factor to harm the health of the body. In order to effectively prevent and treat the toxicity of formaldehyde to the human body, formaldehyde has been studied in depth. The molecular mechanism of toxicity is of great significance.
Endoplasmic reticulum (endoplasmic reticulum) is a major site for protein synthesis and processing. It plays an important regulatory role in maintaining intracellular calcium concentration and protein glycosylation. When endoplasmic reticulum dysfunction and excessive accumulation of unfolded / misfolded protein, endoplasmic reticulum stress is induced. The endoplasmic reticulum contains a number of molecular chaperones, when endoplasmic reticulum stress occurs, sugar The expression level of regulatory protein 78 (GRP78) increases significantly to enhance the protein folding ability of the endoplasmic reticulum. When the endoplasmic reticulum stress is low, it activates the anti apoptotic molecular chaperones; however, when endoplasmic reticulum stress is too severe and persistent, it activates JNK, CHOP and caspase-12, leading to cell apoptosis and endoplasmic reticulum stress and nerve cells. Death is closely related. Formaldehyde is a crosslinker, which can interact with the sulfhydryl and amino acid groups, resulting in protein polycondensation, and formaldehyde causes the error of protein folding and accumulation. Formaldehyde and P amyloid interact to produce a neurotoxic amyloid protein complex. Formaldehyde will also lead to the aggregation of tau protein. The apoptosis of human neuroblastoma and hippocampal neurons, however, the study of the relationship between endoplasmic reticulum stress mediated apoptosis pathway and formaldehyde induced neuronal death has not yet been reported.
Morphine abuse has become a serious public health and social problem worldwide. Long-term use of morphine can lead to the occurrence of body dependence and mental dependence, which restricts its clinical use (treatment of various acute and chronic pain). The main manifestation of morphine addiction is the compulsive drug use, that is, drug intake and loss of control. Addiction. The reasons are mainly due to positive intensifying factors (euphoria and reward effects) and negative intensifying factors (alleviating withdrawal symptoms). The ventral tegmental area of the midbrain, nucleus accumbens, prefrontal cortex, amygdala and hippocampus are related to the process of morphine addiction. In addition to the nervous system, morphine causes oxidative stress and finer in the liver and kidney. Apoptosis. Long-term use of opioids can lead to liver, kidney damage, acute hepatitis, chronic hepatitis and other liver injuries, as well as nephrotic syndrome, acute glomerulonephritis, interstitial nephritis and renal failure. Therefore, attention should be paid to the liver and kidney damage caused by opioid drugs in the process of drug addiction treatment. Drugs for the protection of morphine induced liver and kidney damage are important for reducing the side effects of morphine and more widely using morphine. Moreover, there are no ideal drugs and methods for the treatment of morphine addiction.
Both formaldehyde and morphine can cause oxidative stress damage, apoptosis and neurotrophic factors are down, but their ways of action are different, but Trx-1 has anti oxidation, anti apoptosis and neurotrophic factor activity, and the toxic mechanism of formaldehyde and morphine intersect. This puts forward two questions: whether Trx-1 inducer can resist formaldehyde Whether the Trx-1 inducers can resist morphine addiction or morphine induced liver and kidney injury, this paper will study these two aspects.
The main results are as follows:
(1) 37 total saponins (PNS) are one of the main components of 37, and their main components are widely used in clinical. 373 alcohol group saponins (PTS) are one of the main components of PNS. The main components of PTS are ginsenoside Rg1, R1 and Re (80%), and the content of the saponins Rg1 is above 60%.PTS in dose dependent and time dependent manner. The expression level of Trx-1 in PC12 cells was increased. In vivo animal experiment results: compared with the control group, PTS significantly induced the expression level of Trx-1 in the cerebral cortex, hippocampus and striatum of mice. Ginsenoside Rg1 was the main active component of PTS. We found that ginsenoside Rg1 significantly induces Trx-1 in PC12 cells. Expression level.
(2) endoplasmic reticulum stress is associated with neuronal apoptosis. In this paper, we found that formaldehyde significantly induces the expression level of GRP78; formaldehyde can increase the expression of CHOP after 12 hours of treatment with formaldehyde; the expression level of pro-caspase-12 decreases significantly after 24 hours of formaldehyde action. These results suggest that formaldehyde is to a certain extent. Neurotoxicity is caused by endoplasmic reticulum stress mediated apoptosis pathway.
(3) oxidative stress is an important cause of formaldehyde causing cell toxicity,.Trx-1 is an important antioxidant, which can be induced by oxidative stress. The study found that formaldehyde can induce the expression of Trx-1 after the action of PC12 cells for 12 hours, but the expression of Trx-1 will be reduced after the action of formaldehyde for 24 hours. SiRNA interference is used to reduce Trx-1. The expression level increased the sensitivity of PC12 cells to formaldehyde. These results indicate that the neurotoxicity of formaldehyde may be related to the reduction of Trx-1 expression level.
(4) the high expression of Trx-1 can resist the damage caused by oxidative stress and endoplasmic reticulum stress. Ginsenoside Rgl has no effect on the viability of PC12 cells, but the pretreatment of ginsenoside Rg1 can resist formaldehyde toxicity to PC12 cells and inhibit the decrease of tyrosine hydroxylase expression level induced by formaldehyde. Ginsenoside Rgl can be recovered. The expression level of Trx-1 is reduced by formaldehyde. Furthermore, ginsenoside Rg1 can also resist the endoplasmic reticulum stress induced by formaldehyde: inhibition of the increase of GRP78 and CHOP expression levels caused by formaldehyde and the decrease of pro-caspase-12 expression level caused by formaldehyde. These results suggest that Trx-1 inducers can resist the toxic effects of formaldehyde.
(5) repeated morphine can increase the expression level of Trx-1 and Hsp70 in the cerebral cortex, hippocampus, ventral tegmental area and nucleus accumbens in the mice. These results show that the cellular defense mechanism of morphine can be activated. This study proves that GGA can induce the expression of Trx-1 and Hsp70 in the brain of mice and reduce the activity of mice induced by morphine acute treatment. Dynamic enhancement, blocking morphine induced conditioned place preference, alleviating morphine withdrawal symptoms.GGA can also inhibit the enhancement of CREB activity induced by morphine, as well as the increase in the expression of delta FosB and Cdk5. In NAc region, GGA can enhance the expression of Trx-1 and Hsp70 induced by morphine. These results indicate that the use of cytotoxic inducers to enhance T Rx-1 expression is a new strategy for the treatment of morphine addiction, and GGA can be used for the prevention and treatment of morphine addiction.
(6) GGA can inhibit the increase of MDA content induced by morphine in the liver and kidney, and prevent the activation of caspase-9 and caspase-3 induced by morphine. Morphine can induce apoptosis in liver and kidney cells through mitochondrial mediated apoptosis pathway, rather than through endoplasmic reticulum mediated pathway. More importantly, GGA can also enhance morphine to Trx-1 and Hsp70 These results indicate that GGA can be used for the prevention and treatment of liver and kidney injury induced by morphine.
These results suggest that the apoptosis pathway mediated by endoplasmic reticulum stress is related to the neuronal apoptosis induced by formaldehyde; ginsenoside Rg1 is a new Trx-1 inducer, and ginsenoside Rg1 can resist formaldehyde induced neurotoxicity; Trx-1 inducer GGA can resist morphine addiction and morphine induced liver and kidney injury. The results provide theoretical basis and new targets for the prevention and control of formaldehyde and morphine.

【学位授予单位】：昆明理工大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R114;X503.1

【参考文献】