百可利对帕金森病模型震颤症状抑制作用及机制研究

发布时间：2018-05-16 17:00

本文选题：百可利 + 帕金森病震颤　；参考：《沈阳药科大学》2012年博士论文

【摘要】：百可利(Baicalein),化学名称为5, 6, 7 -三羟基黄酮,是最早从唇形科(Labiatae)黄芩属(Scutellaria)多年生草本中药植物黄芩(Scutellaria baicalensis Georgi)的干燥根中提取得到的黄酮类化合物,是黄芩的最主要活性成分之一。百可利具有多种药理作用,如:抗菌、抗病毒、抗炎、抗变态反应、抗氧化、清除氧自由基、抗肿瘤、抗凝等。百可利对多种疾病包括炎症、肿瘤、纤维化性疾病、心脑血管性疾病具有良好的防治作用。最近研究表明百可利可以保护局部缺血的神经元免受损伤,减轻炎症介导的多巴胺能神经元变性。这些研究结果提示百可利可能是一个有效的治疗神经退行性疾病的化合物。2007年本实验室经过高通量筛选证明百可利对多巴胺和6-羟基多巴胺(6-OHDA)引起的多巴胺能神经元损伤具有选择性神经保护活性,后经多种筛选模型评价,证明其可靠的生物活性,并经动物模型评价,显示出一定的药理作用,初步定为先导化合物,编号为DL0705。然而,百可利溶解性较差,在水中几乎不溶,为此,课题组对其化学成分进行了晶型研究,发现和证明其存在有多晶型现象。通过稳定性实验与生物学试验证明:晶β型稳定性好,生物利用度高,属优势药物晶型。临床前研究结果表明:百可利能够减轻帕金森病震颤症状及其所导致的神经症候、日常生活运动障碍等,有望成为作用独特的治疗帕金森病的药物,具有良好的药用价值和新药开发前景。在前期研究的基础上,本论文在确证百可利治疗帕金森病震颤有效的基础上,对其作用机制进行了探讨。第一章百可利防治中枢神经系统退行性疾病研究现状本章首先在查阅了大量文献的基础上,对近几十年的百可利的药理学研究进展作一综述。该综述包括七个方面:抗菌抗病毒作用、抗炎及抗变态反应作用、抗氧化作用、抗肿瘤作用、抗纤维化作用、心脑血管保护作用以及神经保护及促细胞转化与分化作用。然后在第二节探讨了百可利与神经系统退行性疾病的关系,从抑制神经细胞退行性改变的启动因子、阻断神经细胞退行性改变的信号传导过程以及激活内源性神经保护机制等方面探讨百可利用于防治神经系统退行性疾病的科学性。第三节是关于百可利与帕金森病的讨论。本章最后对百可利防治帕金森病进行了展望。第二章百可利对6-OHDA致帕金森病大鼠震颤症状抑制作用及机制研究本章采用6-OHDA前脑内侧束(MFB)定位注射建立偏侧大鼠帕金森病动物模型,通过检测行为学,神经递质和多巴胺能神经元数目来评价百可利在体内的活性和药效。然后通过检测酪氨酸羟化酶(TH),多巴胺转运体(DAT),囊泡单胺转运体2 (VMAT2),胶质源性纤维蛋白(GFAP),小胶质细胞特异性蛋白(0X42),谷氨酰胺合成酶(GS),GABA转运体(GABA-T),谷氨酸脱羧酶67 (GAD67),谷氨酸脱氢酶(GDH),细胞色素氧化酶亚单位Ⅰ (C0I),腺苷A2A受体(A2AR),D1受体(D1R),D2受体(D2R)和乙酰胆碱受体(AchR)等多种相关指标来探讨百可利可能的抗帕金森病作用机制。实验结果进一步肯定了本实验室的前期研究工作成果,并得出以下结论:1.百可利可剂量和时间依赖性的减轻帕金森病(PD)大鼠的肌肉震颤频率和震颤幅度,在给药后1Omin即可出现药效,30min达到最高,药效可持续5个小时。其抗震颤作用的强度可优于多巴胺递质补充剂美多芭。2.通过比较震颤型PD动物和旋转型PD动物病理生理上的区别,发现以震颤症状为主的PD大鼠损伤主要累及黑质致密部(SNc),且损伤程度较轻,功能紊乱主要出现在丘脑底核(STN)和外侧苍白球(GPe);而以旋转症状为主的PD大鼠损伤主要累及黑质内侧部(SNM)和黑质外侧部(SNL),且受损相对严重,功能紊乱主要出现在纹状体。3.百可利可平衡PD大鼠基底神经节区多巴胺(DA)、谷氨酸(Glu)和γ-氨基丁酸(GABA)的神经递质的紊乱,增加GPe的抑制性输出,抑制STN的兴奋性输出而拮抗基底神经节过度的抑制性输出,恢复丘脑皮层的正常兴奋状态。同时发现百可利对DA的作用明显比Glu和GABA弱,可说明百可利治疗PD震颤症状效果好,而对强直-运动不能症状(大鼠的旋转症状)效果差。4.百可利对神经递质的调节作用与其增加DAT和GS的蛋白表达,降低GABA-T的蛋白表达有明显相关性。5.百可利可通过上调D1和D2受体,下调A2A受体而发挥其抗PD作用。6.百可利可拮抗PD状态下星型胶质细胞和小胶质细胞的过度激活而发挥对DA神经的保护作用。第三章百可利对MPTP帕金森病食蟹猴模型的治疗作用及机制研究为进一步确证百可利的抗PD震颤作用,本章采用了症状及病理、生化改变方面均酷似人类PD,而且稳定可靠的1-甲基-4-苯基-1,2, 3, 6-四氢吡啶(MPTP)致帕金森病食蟹猴模型,进一步观察百可利对帕金森病动物行为学的影响,并深入探讨其作用机制。结果表明,百可利能改善MPTP致帕金森病食蟹猴模型的行为学异常,对上肢精细运动障碍、僵冻、运动减缓和静止震颤等PD症状均有不同程度的缓解。在行为学实验基础上,本章通过realtime RT-PCR实验,在基因水平上探讨了与PD发病机制相关的几种酶学的改变,结果发现百可利可上调PD猴THmRNA表达,验证了百可利的神经保护作用。此外,百可利可下调纹状体儿茶酚氧位甲基转移酶(COMT),单胺氧化酶B(MAO-B )和GABA-T mRNA的表达,上调STN的GS mRNA的表达,进一步在基因水平上解释了百可利调节基底神经节DA ,Glu和GABA递质平衡的作用。COI、GAD67和GDHmRNA检测结果表明,百可利可明显增加PD猴GPe神经元的活性,抑制GPi和STN的神经元活性。这些结果进一步证明了百可利通过平衡PD动物基底神经节神经递质的紊乱,使GPi、GPe和STN的活动恢复常态,从而发挥其抗PD震颤的作用。第四章百可利抗Glu诱导的中脑DA能神经元毒性作用及机制研究随着对帕金森病的深入研究,人们逐渐认识到Glu在PD的发病中扮演了重要角色。由于DA的减少,Glu 一方面通过氧化性神经毒性和兴奋性神经毒性诱导DA神经元变性坏死;另一方面,DA的减少可增加STN的谷氨酸能神经元的兴奋性使Glu释放增多,导致基底神经节运动环路功能紊乱而参与PD运动症状的产生。在第二、三章实验中发现百可利对Glu有明显的拮抗作用,在此工作基础上,本章通过体外培养中脑DA能神经元,重点研究百可利对Glu诱导的氧化性神经毒性和兴奋性神经毒性的拮抗作用,并对Glu介导的下游的几个功能性蛋白进行研究。结果发现,百可利可明显抑制Glu诱导的氧化性神经毒性,表现在增加神经元的存活率,降低细胞内活性氧簇(ROS)含量,增加线粒体膜电位,抑制细胞凋亡率等。在采用荧光探针Fura-2/AM和镉还原法对Glu诱导的细胞内钙离子增多和一氧化氮(NO)释放增加的实验中发现,百可利可明显拮抗Glu介导的细胞内钙的增多和NO释放增加,说明百可利可拮抗Glu介导的兴奋性神经毒作用,同时也间接反映了百可利可通过抑制Glu介导的钙内流而降低STN神经元的兴奋性,进而控制PD的震颤症状。随后我们观察了百可利对Glu致大鼠原代中脑DA能神经元损伤后神经元型一氧化氮合酶(nNOS)、FOS和钙/钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)蛋白表达的影响。免疫印记实验结果显示百可利可明显拮抗Glu诱导的nNOS的表达,说明百可利的抗氧化和神经元保护作用与抑制nNOS活性有关。FOS蛋白结果表明,百可利通过下调该蛋白的活性而对PD的震颤症状起到一个长期调节作用,而百可利对CaMKⅡ蛋白表达无影响。T型钙通道是近年来新发现的抗帕金森病震颤的一个新靶点。帕金森病患者基底神经节运动环路功能紊乱而导致丘脑网状核(NRT)产生抑制性突触后电位(IPSP),随后激活T型钙通道而产生低阈值的钙棘波(LTS),LTS可诱发NRT神经元产生节律性的放电,这种节律性放电可诱导丘脑-皮层的节律性运动和外周的肌肉震颤活动。这一过程被认为是PD震颤的最后通路。基于此理论,本章探讨了百可利对T型钙通道蛋白表达的影响,结果发现百可利可明显下调T型钙通道CAV3. 1和CAV3. 3蛋白表达,提示这可能是百可利抗PD震颤的一个新靶点。第五章黄酮类化合物及其组合抗帕金森病震颤作用比较本章对百可利与相关的黄酮类化合物及其组合抗帕金森病震颤作用进行了比较。实验结果表明,与特发性震颤抑制剂普萘洛尔和经典的抗PD震颤药美多芭相比,黄酮类的抗PD震颤作用有明显的优势。同时通过合用药物的药效来看,黄酮类药物之间可能有相同或相似的作用机制。因此对黄酮类化合物抗PD作用机制做深一步的研究,寻找对神经系统具有活性的先导化合物,从而发现潜在的高效低毒神经药物,对于帕金森病的治疗具有非常深远的意义。综合上述各章研究结果,可以认为,百可利抗帕金森病震颤的作用主要是通过:1.上调DAT, GS蛋白表达,下调GABA-T蛋白表达,抑制COMT和MAO-B的mRNA的表达;2.平衡PD大鼠基底神经节区DA、Glu和GABA神经递质的紊乱;3.上调D1和D2受体,下调A2A受体;4.抑制ROS含量,增加线粒体膜电位水平,抑制细胞凋亡率,拮抗Glu氧化性神经毒性;5.抑制钙内流、nNOS表达和NO释放而拮抗Glu兴奋性神经毒性;6.下调Glu诱导的FOS蛋白的表达;7.下调T型钙通道CAV3.1和CAV3. 3蛋白表达。
[Abstract]:Baicalein, the chemical name of 5, 6, 7 - three hydroxy flavones, is the first flavonoids extracted from the dry root of Labiatae baicalensis Georgi (Scutellaria) perennial herb (Scutellaria baicalensis Georgi) in the family of Huang Qinshu (Scutellaria). It is one of the most important active ingredients of Scutellaria. Use, such as antibacterial, antiviral, anti-inflammatory, antiallergic, antioxidant, scavenging oxygen free radicals, anti-tumor, anticoagulant, and so on. Cisco has a good prevention and control effect on many diseases including inflammation, tumor, fibrotic disease, cardiovascular and cerebrovascular diseases. These findings suggest that curco may be an effective compound for the treatment of neurodegenerative diseases in.2007, a high throughput screening test in the laboratory to demonstrate selective neuroprotective activity of curyl on dopaminergic neuron damage induced by dopamine and 6- hydroxy dopamine (6-OHDA). After evaluation of a variety of screening models, it proved its reliable biological activity, and was evaluated by animal model. It showed a certain pharmacological effect. It was preliminarily designated as a pilot compound, which was numbered DL0705., but the solubility of 100 Keli was poor and almost insoluble in water. Therefore, the research group has studied its chemical composition and found and proved its existence. The phenomenon of polycrystalline form. Through the stability test and biological test, it is proved that the stability of crystal beta type is good, the bioavailability is high, which belongs to the dominant drug type. The results of pre clinical study show that it can reduce the symptoms of Parkinson's disease and the symptoms of the nerve, the disturbance of daily life and so on, and it is expected to become a unique therapeutic Parkinson. The medicine of the disease has good medicinal value and the prospect of the development of the new medicine. On the basis of the earlier study, this paper has discussed the mechanism of its action on the basis of confirming the effectiveness of the treatment of the tremor of Parkinson's disease. On the basis of this review, the progress of pharmacology in the last few decades is reviewed. This review includes seven aspects: antiviral and antiviral action, anti-inflammatory and antiallergic effect, antioxidation, antitumor, anti fibrosis, cardio cerebral vascular protection, neuroprotection and cell differentiation and differentiation. The two section discusses the relationship between BALCO and neurodegenerative diseases, the scientific nature of the third section of the treatment of the degenerative diseases of the nervous system, from the starting factors that inhibit the degeneration of the nerve cells, the signal transduction process of blocking the degeneration of the nerve cells, and the activation of the endogenous neuroprotective mechanism. A discussion about the prevention and treatment of Parkinson's disease. In this chapter, a prospect of the prevention and treatment of Parkinson's disease in the present chapter. Second a study on the inhibition of the tremor symptoms of 6-OHDA induced Parkinson's disease in rats and the mechanism study on the establishment of an animal model of partial rat Parkinson's disease by 6-OHDA anterior medial fasciculus injection (MFB). The number of neurotransmitters and dopaminergic neurons to evaluate the activity and efficacy of cytosine in the body. Then by detecting tyrosine hydroxylase (TH), dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT2), glial derived fibrin (GFAP), microglia specific protein (0X42), glutamine synthetase (GS), GABA transporter (GABA-T) ) glutamic acid decarboxylase 67 (GAD67), glutamic dehydrogenase (GDH), cytochrome oxidase subunit I (C0I), adenosine A2A receptor (A2AR), D1 receptor (D1R), D2 receptor (D2R) and acetylcholine receptor (AchR) were used to explore the mechanism of the anti Parkinson action of BCO, and the experimental results further affirmed the early stage of this laboratory. The results of the work are studied and the following conclusions are drawn: 100 the frequency of muscle tremor and the amplitude of tremor of Parkinson's disease (PD) rats can be reduced by dose and time dependence. The efficacy of 1Omin can be found after the administration, and the maximum of 30min is reached and the efficacy of the drug is sustainable for 5 hours. The strength of its anti tremor effect is superior to the dopamine supplement mdopa.2. By comparing the pathophysiological differences between the tremor type PD animals and the rotating PD animals, it is found that the PD rats with the main tremor symptoms mainly involve the dense part of the substantia nigra (SNc), and the damage degree is lighter. The dysfunction mainly occurs in the nucleus of the subthalamic nucleus (STN) and the lateral globus pallidus (GPe), and the damage of the PD rats mainly with rotation symptoms is mainly involved in the black. The medial part of the mass (SNM) and the lateral part of the substantia nigra (SNL) and the damage are relatively serious. The dysfunction mainly occurs in the Basilar Ganglion of the striatum, which can balance the dopamine (DA), the glutamic acid (Glu) and the gamma aminobutyric acid (GABA) neurotransmitter, adding the inhibitory output of GPe and inhibiting the excitatory output of STN and antagonizing the basilar nerve. It was found that the effect of coryl on DA was significantly lower than that of Glu and GABA, and that the effect of Corp on the symptoms of PD tremor was better, but the effect of the tetanic motion (rat's rotation symptoms) on the effect of.4. on the neurotransmitters was worse than that of DAT and GS. The protein expression of GABA-T has a significant correlation with the expression of GABA-T protein,.5. 100 can protect the DA nerve by up regulation of D1 and D2 receptor, down regulation of A2A receptor and its anti PD action,.6. 100 can antagonize the overactivation of astrocytes and microglia in PD state, and the third cap on MPTP Parkinson's disease The therapeutic effect and mechanism of cynomolgus monkey model to further confirm the anti PD tremor effect of cynoli, this chapter adopts the symptoms and pathology, and the biochemical changes resemble human PD, and the stable and reliable 1- methyl -4- phenyl -1,2, 3, 6- four hydropyridine (MPTP) induced Parkinson's disease cynomolgus monkey model, further observe the action of cork to Parkinson's disease The effect of physical behavior is discussed and the mechanism of action is discussed in depth. The results show that cynoli can improve the behavioral abnormality of MPTP induced Parkinson's disease cynomolgus monkey model. The PD symptoms such as fine motor obstacle, freeze, movement slowing down and static tremor are alleviated in different degrees. On the basis of the study, this chapter passes the realtime RT-PCR experiment. The changes in several enzymes related to the pathogenesis of PD were discussed at the gene level. The results showed that 100 could up regulate the expression of THmRNA in PD monkey and verify the neuroprotective effect of clonolis. In addition, Bai can downregulate the expression of corpus striatum catechol oxygen methyltransferase (COMT), monoamine oxidase B (MAO-B) and GABA-T mRNA, up regulation of STN GS M The expression of RNA further explains the role of cloni at the gene level to regulate the balance of DA, Glu and GABA transmitters in the basal ganglia,.COI, GAD67 and GDHmRNA detection results show that BCO can significantly increase the activity of GPe neurons in PD monkeys and inhibit the activity of GPi and STN neurons. These results further demonstrate that the BCO is balanced by the balance of PD animals. The disorder of neurotransmitters in the basal ganglia, making the activities of GPi, GPe and STN restored to normal, and thus exerting their anti PD tremors. Fourth the toxicity and mechanism of the Glu induced DA energy neurons in Glu, with the in-depth study of the disease, people gradually realized that Glu plays an important role in the pathogenesis of PD. Glu, on the one hand, induces degeneration and necrosis of DA neurons by oxidative neurotoxicity and excitatory neurotoxicity; on the other hand, the decrease of DA can increase the excitability of STN glutamate neurons and increase the release of Glu, leading to the dysfunction of the basal ganglia movement loop and the production of PD movement symptoms. In the second, third chapter, the experiment was conducted. It was found that BCO has obvious antagonism to Glu. On the basis of this work, this chapter focuses on the study of the antagonism of clori on the oxidative neurotoxicity induced by Glu and excitatory neurotoxicity induced by Glu in vitro by culture of DA energy neurons in the midbrain in vitro, and studies several active proteins in the downstream of Glu. The oxidative neurotoxicity induced by Glu was inhibited by increasing the survival rate of neurons, reducing the content of intracellular reactive oxygen species (ROS), increasing the mitochondrial membrane potential and inhibiting the rate of cell apoptosis. In the experiment using fluorescence probe Fura-2/AM and cadmium reduction method, the increase of intracellular calcium ion increase and nitric oxide (NO) release induced by Glu was increased. Now, 100 can obviously antagonize the increase of intracellular calcium and the increase of NO release mediated by Glu, indicating that 100 can antagonize the excitatory neurotoxicity mediated by Glu, and also indirectly reflect the inhibition of the excitatory properties of STN neurons by inhibiting the Glu mediated calcium influx, and controlling the tremor symptoms of PD. The effect of Glu on the expression of neuronal nitric oxide synthase (nNOS), FOS and calcium / calmodulin dependent protein kinase II (CaMK II) protein after DA neurons injury in the primary mesencephalon of rats. The results of immuno imprint experiment showed that 100 can obviously antagonize the expression of Glu induced nNOS, indicating the antioxidative and neuronal protection of centico The results of.FOS protein related to inhibition of nNOS activity showed that by down-regulation of the activity of the protein, cloni had a long-term regulatory effect on the tremor symptoms of PD, and the no influence of the.T type calcium channel on the expression of CaMK II protein was a new target for the recent discovery of the tremor of Parkinson's disease. The basal ganglia of Parkinson's disease patients The dysfunction of the motor loop causes the inhibitory postsynaptic potential (IPSP) of the thalamus reticular nucleus (NRT), and then activates the T calcium channel to produce a low threshold calcium spinous wave (LTS). LTS induces a rhythmic discharge of NRT neurons. This rhythmic discharge can induce the rhythmic movement of the thalamus cortex and the muscle tremor activity of the peripheral muscles. The process is considered to be the final pathway of PD tremor. Based on this theory, this chapter explores the effect of calcis on the expression of T type calcium channel protein. It is found that BCO can obviously reduce the expression of T type calcium channel CAV3. 1 and CAV3. 3 protein, suggesting that this may be a new target for the anti PD tremor of BCP. The fifth chapter flavonoids and their combination resistance. Comparison of the tremor effects of pranolol and related flavonoids and their combination against Parkinson's disease in Parkinson's disease. Experimental results show that the anti PD tremor effect of flavonoids is obviously superior to that of the idiopathic tremor inhibitor propranolol and the classic anti PD tremor. According to the drug effect, the flavonoids may have the same or similar mechanism of action. Therefore, a deep study on the anti PD mechanism of flavonoids is made to find the precursor compounds with active nerve system, and the potential highly effective and low toxic neurodrugs can be found. It has a profound meaning for the treatment of Parkinson's disease. According to the results of the above chapters, it is believed that the effect of tremule on Parkinson's disease is mainly through: 1. up regulation of DAT, GS protein expression, downregulation of GABA-T protein expression, inhibition of mRNA expression of COMT and MAO-B; 2. balance of DA in the basal ganglia, Glu and GABA neurotransmitters in PD rats; 3. up D1 and D2 receptors, down regulated receptors; 4 Inhibiting the ROS content, increasing the mitochondrial membrane potential, inhibiting the apoptosis rate and antagonizing the oxidative neurotoxicity of Glu; 5. inhibition of calcium influx, nNOS expression and NO release against Glu excitotoxicity; 6. down regulation of Glu induced FOS protein expression; 7. down regulation of T type calcium channel CAV3.1 and CAV3. 3 protein expression.

【学位授予单位】：沈阳药科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R285.5;R-332

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