天然产物白首乌二苯酮的神经保护作用和两面针碱的免疫调控作用机制研究
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摘要
一、背景
天然产物来源广泛，结构多样，药理作用独特，大多数都经过长期的临床应用，其疗效确切，使用安全性高。目前有50%的临床应用药物来自天然产物及其衍生物，并且许多结构新颖的天然产物及其衍生物正处于临床和临床前研究阶段。因此，从天然产物中寻找高效低毒易得的先导化合物成为药物研究的热点，天然产物在今后仍将是新药的重要来源。
随着各种分离、分析和结构测定方法以及色谱、波谱联用技术的飞速发展，化学分离和结构鉴定已经不是天然药物研究的瓶颈。目前天然产物研究面临的一个问题就在于其药理活性和作用机制的阐述较少或研究不深，尤其是结构新颖的天然产物。针对合理阐释其作用机制和结合靶标这一难点，本文第一章综述了基因组学、蛋白质组学和计算机辅助的方法在天然产物靶标发现和机制研究中的应用，希望能够为活性天然产物靶标发现和机制研究的方法及相关技术提供一个比较全面的认识，然后进一步将第一章的方法应用到天然产物cynandione A和氯化两面针碱（nitidine chloride，后文所用两面针碱均指氯化两面针碱）的机制研究中，来阐明这两个天然产物如何发挥相应的药理活性。
脑卒中是一种死亡率高、致残率高的常见病、多发病，并且近几年来发病率呈上升趋势。缺血性脑卒中约占所有脑卒中（stroke）的80%，是指局部脑组织因血液循环障碍，缺血、缺氧而发生的软化坏死。缺血性脑卒中的病因主要是由于供应脑部血液的动脉出现粥样硬化和血栓形成，使管腔狭窄甚至闭塞，导致局灶性急性脑供血不足而发病；也有因异常物体（固体、液体、气体）沿血液循环进入脑动脉或供应脑血液循环的颈部动脉，造成血流阻断或血流量骤减而产生相应支配区域脑组织软化坏死。目前临床治疗脑卒中的药物由于其价格、抗药性以及对出血、胃肠道、神经系统等毒副作用，应用受到了一定的限制，因此，寻找高效低毒易得的分子来防治脑卒中尤为必要。
第二章介绍的天然产物cynandione A是从白首乌中分离得到的苯乙酮类化合物，近年来文献报导cynandione A具有抗氧化、抗谷氨酸兴奋毒和胃保护等药理活性。但目前对其作用机制尚无文献报道，值得进一步的深入研究。
多发性硬化症（multiple sclerosis）至今仍是整个医学界未解的难题之一。该病是一种慢性、自身免疫性疾病，通过破坏脑、脊髓以及视神经的神经纤维保护层髓磷脂，影响正常的神经传递而导致身体残疾。由于该病具有极高的复发率和致残率，已成为常见的、重要的神经系统疾病之一。由于该病的发病高峰在30岁左右，因此，严重影响青壮年人口的工作和生活质量，也给国家和政府造成极大负担，制约社会和经济发展。
目前临床上对该病尚无特效治疗方法，缺乏令人满意的治疗药物。急性期主要给予糖皮质激素，复发-缓解期主要治疗药物包括：干扰素-γ，醋酸格拉替雷和单克隆抗体那他珠单抗。重度患者还可以考虑自体干细胞移植。西医的激素疗法虽然能够缓解急性期症状，但毒副作用明显，故不推荐长期使用;免疫调节剂干扰素、格尔德霉素和那他珠单抗等不仅费用昂贵，且长期使用针对单一靶点药物容易出现耐受。且上述药物均需经静脉、或注射给药，不能有效改善多发性硬化症复发和髓鞘脱失等问题。因此，研制能够有效控制多发性硬化症的复发、改善髓鞘脱失的药物具有重大意义。传统中医药具有多靶点、系统调节的作用特点，因此，针对防治多发性硬化症这一复杂性、难治性疾病具有一定优势。
第三章介绍的天然产物两面针碱是从芸香科植物两面针[Zanthoxylum nitidum(Roxb.) DC]的根中分离到的具有多种药理活性的苯骈菲啶类生物碱，是两面针的主要有效成分，是两面针及其制剂的主要质量控制指标成分。近年来文献报导氯化两面针碱在抗炎、镇痛、抗真菌、抗疟以及抗肿瘤等方面具有广泛的药理活性、开发利用价值和应用前景。基于上述研究进展，并且目前对两面针碱的免疫调控作用尚未进行充分报道，本章首先探讨了两面针碱潜在的治疗多发性硬化症的药理活性，进而在体外细胞模型上研究两面针碱对不同种类和活化状态的免疫细胞的调控作用，最后采用亲和层析的方法寻找与两面针碱存在相互作用的蛋白质以解释其作用靶标，更深一层地阐明两面针碱的作用机制。
2、目的
采用文献综述的方法进一步研究活性天然产物cynandione A和两面针碱的作用机制，为合理开发利用天然产物cynandione A和两面针碱提供重要的科学依据，并为寻找及发现新的神经保护和免疫调控药物提供化学小分子模板。
3、方法
（1）通过清除自由基、抗氧化和抗谷氨酸兴奋毒的活性筛选，发现活性先导物cynandione A，然后，采用二维电泳和LC-MS的蛋白质组学方法研究cynandione A神经保护作用的机制。
（2）体内实验采用大鼠大脑中动脉缺血再灌注（缺血性脑卒中）模型，探讨cynandioneA神经保护作用及其体内抗缺血性脑卒中损伤的机制。SD大鼠经10%水合氯醛（350mg/kg）腹腔麻醉，分离右侧颈总动脉（common carotid artery，CCA）、颈内动脉（internalcarotid artery，ICA）、颈外动脉（external carotid artery，ECA）。结扎CCA近心端、ECA起始端后，在CCA处剪“V”形小口，沿CCA插入线栓（长40mm，直径0.26mm，并于20mm长处标记），经ICA向颅内插至大脑中动脉分叉处，插入深度为18-20mm。再灌注时外拉线使球端回至ECA，拔除插线，结扎ECA。假手术组除不插线外，其余步骤同手术组。大脑中动脉栓塞后2h恢复血供。通过神经功能缺失体征评分、大鼠存活率和四氮唑法染色评价cynandione A抗脑缺血损伤的神经保护作用。通过大鼠脑组织HE染色和DPYSL2、HMGB1免疫组织化学分析探讨cynandione A神经保护作用。
（3）实验性自身免疫性脑脊髓膜炎（experimental autoimmune encephalomyelitis，EAE）是一种得到普遍认可和应用的多发性硬化症实验动物模型。造模方法是皮下注射MOG33-55并辅以CFA，通过树突状细胞激活体内CD4+T细胞，产生Th17细胞，并使之穿过血脑屏障，攻击自身神经髓鞘，从而导致中枢神经白质脱髓鞘，引起EAE。免疫当天记为第0天，于第0天和第2天给予各组小鼠腹腔注射400ng/只百日咳毒素（PTX）。通过神经功能评分、脊髓组织HE染色、髓鞘碱性蛋白的免疫组织化学以及血浆和组织细胞因子的水平来评价两面针碱防治实验性自身免疫性脑脊髓膜炎的作用。
（4）进一步作用机制的研究是采用基因芯片、二维电泳和计算机辅助的方法推断两面针碱可能的作用通路。首先确定合理的给药浓度、刺激剂剂量、给药时间和不同种类的细胞模型。分别考察两面针碱对活化状态下的骨髓细胞、骨髓来源的树突状细胞、小鼠腹腔巨噬细胞、RAW264.7巨噬细胞、脾细胞和磁珠分选的CD4+T细胞的影响。然后验证三种方法推断的两面针碱调控的作用通路。
（5）两面针碱作用靶标的研究是采用生物素标记的亲和层析方法。生物素-抗生物素蛋白（biotin-avidin）系统常被用来寻找天然产物或活性小分子的靶蛋白。这种方法首先需要将小分子通过连接链（spacer）与生物素相连，然后通过生物素与亲和柱的亲和力将小分子固定在亲和柱上，接着在此亲和柱上加入细胞提取物，与小分子有作用的蛋白就会被保留。最后，改变流动相将靶蛋白洗脱并进行功能和结构确定。基于生物素-抗生物素蛋白系统可以寻找靶标蛋白的性质，我们希望选择合适的两面针碱衍生物，通过嫁接连接臂与生物素相连获得生物素标记的两面针碱衍生物，将来用于寻找与两面针碱作用的细胞内靶标蛋白。通过pulldown实验和western blot实验证明两面针碱与质谱鉴定到的蛋白的相互作用，再通过SPR和ITC等实验表明此蛋白与两面针碱确实存在相互作用，从而对下游通路产生影响，进一步产生防治EAE的作用。
4、结果
第二章介绍了天然产物cynandione A的神经保护作用机制研究。首先通过细胞水平清除自由基、抗氧化和抗谷氨酸兴奋毒的活性筛选，发现了神经保护活性先导化合物cynandione A，然后进一步采用二维电泳和LC-MS的蛋白质组学方法研究其调控的蛋白质，结果分析cynandione A能够下调DPYSL2和HMGB1蛋白的表达，并且能抑制RAF-MEK-ERK1/2通路而起到抗谷氨酸兴奋毒的保护作用。体内药效评价发现该天然产物能够减轻大鼠脑缺血再灌注后的神经功能缺失的评分，延长大鼠存活率，改善大鼠脑缺血再灌注的脑组织病理学，显示出一定的抗脑缺血损伤的保护作用。并且组织分布证明cynandione A能够透过血脑屏障。进一步的作用机制研究表明cynandione A是通过下调DPYSL2蛋白的表达和抑制HMGB1蛋白的截留来发挥抗脑缺血的保护作用。
第三章研究了天然产物两面针碱的免疫调控的作用机制和靶标。首先在整体动物水平发现两面针碱腹腔内注射能够显著推迟EAE发病时间并且减轻EAE的行为学评分。组织病理学考察发现两面针碱能够减少单核炎性细胞浸润和髓鞘脱失。另外，两面针碱显著减少血清和组织炎性因子IL-1β，IL-6和TNF-α水平，而提高抑炎性因子IL-10水平。以上结果显示两面针碱具有抑制小鼠实验性自身免疫性脑脊髓炎的药理活性。在其体内有效的基础上，我们筛选了脑脊髓炎发病过程中起到关键作用的树突状细胞、巨噬细胞和CD4~+T细胞三类细胞，进一步研究两面针碱对其的调控作用。通过活性筛选发现两面针碱能够促进活化的树突状细胞和巨噬细胞分泌IL-10，然后采用基因组学、蛋白质组学和计算机辅助手段鉴定了两面针碱的作用通路MAPK和NF-κB，并通过实验证明两面针碱能够增强活化的MAPK和NF-κB通路而促进IL-10分泌。进一步研究发现IL-10对NF-κB通路存在负反馈调节作用，从而减少炎性因子IL-1β，IL-6和TNF-α的分泌。另外，我们还通过采用IL-10的抗体、外源性的IL-10以及IL-10敲除实验证明两面针碱是通过IL-10来缓解EAE的发病。
在天然产物两面针碱的免疫调控的作用机制基础上，实验进一步采用亲和层析的方法寻找与两面针碱存在相互作用的蛋白质以解释其作用靶标，更深一层地阐明两面针碱的作用机制。通过pulldown实验和western blot实验证明两面针碱可与HSP90相互作用，SPR和ITC等实验表明HSP90与两面针碱确实存在相互作用。两面针碱通过与HSP90结合，调控HSP90与某伴侣蛋白的相互作用，从而诱导下游通路提高IL-10的分泌，并且进一步产生防治EAE的作用。
5、结论
天然产物cynandione A能够下调DPYSL2和HMGB1蛋白的表达，并且能抑制RAF-MEK-ERK1/2通路而起到抗谷氨酸兴奋毒的保护作用。体内药效评价发现cynandione A是通过下调DPYSL2蛋白的表达和抑制HMGB1蛋白的截留来发挥抗脑缺血的保护作用。
天然产物两面针碱能够增强活化的MAPK和NF-κB通路而促进IL-10分泌。进一步研究发现IL-10对NF-κB通路存在负反馈调节作用，从而减少炎性因子IL-1β，IL-6和TNF-α的分泌。另外，我们还通过采用IL-10的抗体、外源性的IL-10以及IL-10敲除实验证明两面针碱是通过IL-10来缓解EAE的发病。进一步的作用靶标研究发现两面针碱与HSP90存在相互作用，并且两面针碱通过与HSP90结合，调控HSP90与某伴侣蛋白的相互作用，从而诱导下游通路提高IL-10的分泌，并且进一步产生防治EAE的作用。
这些发现拓展了天然产物cynandione A和两面针碱在神经保护和免疫调控作用的认识，进一步揭示了它们在防治缺血性脑卒中和多发性硬化症方面的应用潜力，为寻找及发现新的神经保护和免疫调控药物提供化学小分子模板。
1Background
A natural product is a chemical compound or substance produced by a livingorganism (found in nature) that usually has a pharmacological or biological activity for usein pharmaceutical drug discovery and drug design. Natural products may be extracted fromtissues of terrestrial plants, marine organisms or microorganism fermentation broths. Acrude extract from any one of these sources typically contains novel, structurally diversechemical compounds, of which the natural environment is a rich source. Drugs derivedfrom natural products have been used for control of major diseases like cardiovasculardiseases, tumors and infectious diseases. Currently, about50%clinical drugs are derivedfrom natural products, and more natural products and their derivatives are in clinical orpre-clinical trials. Therefore, the research and development of natural products is a hottopic in drug development, and they remain an important source of new drugs.
As the rapid development of various chromatographic separation, analysis, structuredetermination approaches, chemical separation and structural identification are not thebottleneck of natural products research. However, a problem of natural products research isthat their pharmacological activities and mechanisms of action are not well clarilied andunderstood, especially the novel natural products. Therefore, the first chapter reviewsgenomics, proteomics, as well as computer-aided strategies to appropriate thesemechanisms and the implications for target discovery of natural products. Furthermore, weapply these approaches to discover the target genes or proteins of natural productscynandione A and nitidine chloride and clarify the mechanisms of their actions.
Stroke is the leading cause of adult disability and the third most common cause ofdeath in industrialized nations. Ischemic stroke occurs when there is an acute blockage ofarterial blood flow to the brain. Neurons contain low levels of endogenous antioxidants;consequently, the brain is very much vulnerable to the injurious effects of reactive oxygenspecies (ROS). Indeed, it has been shown that oxidative stress is a contributory factor inthe determination of the fate of cells during cerebral ischemia and in the outcome of acuteischemic stroke. To date, thrombolytic therapy with tissue plasminogen activators is theonly approved therapy in the United States and Canada for the treatment of acute ischemicstroke. Thrombolytic therapy with tissue plasminogen activator, however, is limited by a very narrow time window. Part of the reason for this limited3-hour window of efficacy isthe generation of free radicals associated with delayed reperfusion that causes oxidativedamage to the brain, which could increase the risk of cerebral hemorrhage and edema.Though numerous potential neuroprotective agents targeting different injurious factors inthe ischemic cascade including oxidative stress have been investigated, almost all of themexcept edaravone have proven inefficacious in humans in studies with a vigorous trialdesign.
Cynandione A and28other derivative compounds were identified from the roots ofCynanchum (C.) wilfordii and C. auriculatum by liquid chromatography electrosprayionization tandem mass spectrometry. Furthermore, cynandione A was obtained duringactivity-guided isolation of the active component of the methanolic extract from dried rootsof C. wilfordii and was found to mitigate neurotoxicity induced by a variety of neurotoxicagents such as H2O2, the excitotoxic neurotransmitter, L-glutamate and kainate in vitro. Itwas further shown that cynandione A could protect against CCl4-mediated hepatotoxicityin vitro, probably due to its radical scavenging properties. In the second chapter, we soughtto further characterize the neuroprotective effects of cynandione A and otheracetophenones extracted from the roots of C. auriculatum and the underlying mechanismusing pheochromocytoma tumor cell line PC12and to investigate whether cynandione Aprotected against ischemic injuries in rats with experimentally induced cerebral ischemia.
Multiple sclerosis remains one of the unsolved problems in the medical community.This chronic autoimmune disease is able to destroy the brain, spinal cord, and nerve fiberlayer of the myelin and affects normal nerve, then causes physical disabilities. Because ofhigh recurrence rate and morbidity, chronic course in young people, multiple sclerosis hasbecome a common disease of the nervous system and makes a great burden to the socialand economic development. Current medications in the treatment of multiple sclerosis,including glucocorticoids, relapse-remission treatment and stem cell transplantation, arelimited by a number of well-characterized clinical side-effects, such as hepatotoxicity,blood dyscrasias, and gastrointestinal and cardiac toxic effects. Therefore, the explorationof new anti-multiple sclerosis drugs with high efficacy and less toxicity is eagerly needed.Traditional Chinese medicine (TCM), a unique medical system with the significantcharacteristic of the use of multi-component drugs, can hit multiple targets with itscomponents, improve therapeutic efficacy, reduce drug-related side effects and may alsobe an effective way of decreasing drug resistance. Natural products have aroused much interest recently due to its superiority in the treatment of complex multi-factor diseases,such as multiple sclerosis.
Nitidine chloride, a benzo[c]phenanthridine alkaloids isolated from Zanthoxylumnitidium (Roxb.) DC., was reported to have significant anti-tumor, anti-fungal,anti-malarial, and anti-inflammatory activities. In our previous study, nitidine chlorideexhibited analgesic and anti-inflammatory activities, however, the precise molecular targetand underlying mechanisms of its anti-inflammatory and immunomodulatory efficacy arepoorly clarified to date. In the third chapter, we explored genomic, proteomic, andcomputational approaches to investigate its possible mechanisms that contributed tonitidine mediated signal transduction pathways in the regulation of IL-10secretion in DCsand macrophages. Moreover, nitidine chloride effectively inhibited pro-inflammatorycytokines secretion, significantly enhanced IL-10secretion, and mitigated the occurrenceof EAE in vivo. Therefore, our findings indicated that nitidine chloride is a promisingcandidate compound that can be further optimized to be a therapeutic agent for multiplesclerosis.
2Objective
This subject will reveal the molecular mechanism of action of bioactive naturalproducts cynandione A (to protect against ischemic injuries in rats with experimentallyinduced cerebral ischemia) and nitidine (to promote IL-10secretion in EAE treatment formultiple sclerosis) and will provide important scientific foundation and information fordrug design and development to study novel structure and mechanism neuroprotective,anti-inflammatory and immune-modulating agents.
3Methods
(1) Cellular level and cerebral ischemia animal model identified cynandione A as aneuroprotective lead, and using two-dimensional electrophoresis and LC-MS proteomicsapproaches to study its neuroprotective effect.
(2) Using rat middle cerebral artery ischemia reperfusion model to explorecynandione A neuroprotective effect and its mechanism in vivo. Adult maleSprague-Dawley rats (250-300g) were injected intraperitoneally with5or30mg/kgcynandione A or8mg/kg nimodipine for3days. Rats were anesthetized by peritonealinjection of chloral hydrate at350mg/kg, and the middle cerebral artery was occluded for 2h with a silicone rubber-coated nylon monofilament (40mm in length and0.26mm indiameter). The occluding filament was withdrawn after2h to allow reperfusion. Rats wereinjected intraperitoneally with5or30mg/kg cynandione A or8mg/kg nimodipine for3days following occlusion of the middle cerebral artery. In sham-operated rats, the leftcommon carotid artery was exposed and external carotid artery was opened withoutintroducing the filament into the internal carotid artery. Neurological deficit score of eachrat was obtained at24and72h after occlusion of the middle cerebral artery. Rats were alsosacrificed3days after reperfusion, and rat brains were dissected coronally into2-mm brainslices using a metallic brain matrix. Slices were immediately stained by immersion in1%2,3,5-triphenyl tetrazolium chloride and then in4%paraformaldehyde for preservation.After brain tissues preservation, the areas of the infarcted regions and of both hemisphereswere calculated for each coronal slice by image analysis software ImageJ. Also, HEstaining and immunohistochemical analysis were performed to explore neuroprotectiveeffect of cynandione A.
(3) Experimental autoimmune encephalomyelitis (EAE), an inflammatorydemyelinating disease of the central nervous system (CNS), is the prime model for humanmultiple sclerosis. Female C57BL/6mice were intraperitoneally injected with nitidine atthe dose of10mg/kg day which is lower than the LD50for nitidine for a week beforeMOG sensitization, and the nitidine treatment continued through all the experiment. Thecontrol groups were injected with DMSO or PBS of equal volume with the former. Thenthe mice were immunized by subcutaneous injection at three sites of dorsal with300μg ofMOG35-55peptide emulsified in complete Freund’s adjuvant supplemented with0.5mgMycobacterium tuber-culosis strain H37RA. Furthermore, mice received intraperitonealinjections with400ng of pertussis toxin at the time of immunization and48h later.Clinical signs of disease were observed usually between EAE induction and assessed dailyexactly according to the following scoring criteria:0, no detectable signs of EAE;1,flaccid tail;2, hind limb weakness or abnormal gait;3, complete hind limb paralysis;4,paralysis of fore and hind limbs; and5, moribund or death. Intermediate scores (0.5) wereassigned if the neurological signs exhibited a lower severity than typically observed.Finally, blood serum and spinal cord from mice suffering EAE induction for21days wereobtained and analyzed.
(4) Further mechanism research is to explore gene chip, two-dimensionalelectrophoresis and computer aided approaches to investigate the possible mechanism of action of nitidine. To determine reasonable dosage concentration, stimulants dose, dosingtime and different kinds of cell model. Bone marrow cells, bone marrow-derived dendriticcells, peritoneal macrophages, RAW264.7macrophages, spleen cells and CD4+T cellsseparated by magnetic beads were obtained and analyzed by ELISA, respectively. Thenverify its regulatory pathways.
(5) In order to further understand its biological mechanisms and to identify its targetproteins, a biotinylated derivative of nitidine was synthesized, using a peptide-bond as thelinkage between artemisinin and D-biotin. This approach starts with immobilizing abioactive compound on a matrix in a way that does not interfere with its activity. The smallmolecule ligand is modified by introducing an appropriate functional group (referred to asa linker), through which it can be immobilized by attachment to the affinity matrix——astep that is important for later phase separation. There are various commercially availableactivated resins that allow for the attachment of specific chemical groups (for example,sulfhydryl, amino, hydroxyl or carboxyl groups). After the immobilized small moleculeligand has been incubated with protein extracts and any unbound proteins havesubsequently been removed in a series of washing steps, specifically bound proteins areseparated by solid-phase elution, using buffer conditions that disrupt the interactionbetween the target protein and the immobilized small molecule ligand. Finally, the proteinis typically identified by MS. Predicting or inferring the protein targets need to bevalidated in the biological method. Through the pulldown and western blot experiments,SPR, and ITC experiments showed that nitidine is able to bind and interact with the proteinin macrophages, thus influence on the downstream pathways, further promotes IL-10secretion in EAE treatment for MS.
4Results
The second chapter described neuroprotective effects of natural product cynandione A.Viability assays using the WST-8method and LDH release assays showed that cynandioneA dose-dependently attenuated glutamate-induced cytotoxicity. Comparative proteomicanalysis by two-dimensional gel electrophoresis and MALDI-TOF MS/MS of PC12cellstreated with cynandione A showed10μM cynandione A caused broad changes in proteinexpression in PC12cells including downregulation of high mobility group box1(HMGB1)and dihydropyrimidinase-like2(DPYSL2) and inhibit RAF-MEK-ERK1/2pathway.Immunoblotting studies showed that10μM cynandione A aborted glutamate-induced increase in DPYSL2and HMGB1levels in PC12cells and30mg/kg cynandione A alsoattenuated the rise in HMGB1levels and mitigated DPYSL2cleavage in brain tissues ofrats with cerebral ischemia. Furthermore, rats with cerebral ischemia treated with30mg/kgcynandione A exhibited markedly improved neurological deficit scores at24and72hcompared with control and a7.2%reduction in cerebral infarction size at72h (P <0.05vs.control). Our findings demonstrated that cynandione A mitigated ischemic injuries andshould be further explored as a neuroprotective agent for ischemic stroke.
The third chapter demonstrated anti-inflammatory and immune-modulating effects ofnatural product nitidine. Nitidine is a clinically efficient ingredient from extracts ofzanthoxylum nitidum, an indigenous plant in Southern China that shows anti-inflammatoryand analgesic properties. Here, we found that this molecule was able to interfere withmaturation of DCs and their ability to present antigens to T cells, and nitidine-treatedBMDCs could secrete more IL-10and less IL-1β，IL-6and TNF-α in response tolipopolysaccharide (LPS) in vitro. Up-regulation of MAPK and NF-κB pathwaysactivation was shown to be responsible for IL-10preferential production. These resultswere consistent with the observation that IL-10concentration of serum fromnitidine-treated mice suffered from EAE induction was higher compared with controlgroup. In addition, nitidine was able to interfere with the onset of EAE and decreased EAEclinicopathological features in mice by inhibiting immune response to MOG35-55peptides.Further study found that IL-10is able to negative feedback regulate the NF-κB pathway,thus reducing IL-1β，IL-6and TNF-α secretion. In addition, through IL-10antibody,exogenous IL-10and IL-10knockout experiments showed that nitidine could alleviate theincidence of EAE by IL-10. Our results suggest that nitidine has powerfulanti-inflammatory and immune-modulating function in vitro, as well as in vivo, and wouldbe regarded as a new anti-inflammatory and immune-modulating drug with greatpotentiality for the treatment of autoimmune diseases in the future.
In order to further understand its biological mechanisms, affinity chromatography wasadopted to identify target proteins of nitidine. Through the pulldown and western blotexperiments, SPR, and ITC experiments showed that nitidine is able to bind and interactwith the protein in macrophages, thus influence on the downstream pathways, furtherpromotes IL-10secretion in EAE treatment for multiple sclerosis.
5Conclusions
Natural product cynandione A caused downregulation of HMGB1and DPYSL2andinhibit RAF-MEK-ERK1/2pathway to attenuate glutamate-induced cytotoxicity in vitro.Furthermore, cynandione A protected against ischemic injuries in rats with experimentallyinduced cerebral ischemia. Our findings demonstrated that cynandione A mitigatedischemic injuries and should be further explored as a neuroprotective agent for ischemicstroke.
Our present study found that nitidine could mitigate EAE and secrete more IL-10andless IL-1β，IL-6and TNF-α in response to LPS in vitro. Further mechanism study foundthat nitidine could promote the activated dendritic cells and macrophages to secrete IL-10.Affinity chromatography results suggested that nitidine is able to bind and interact withHSP90in dendritic cells and macrophages. This subject will reveal the molecularmechanism of action of nitidine to promote IL-10secretion in EAE treatment for multiplesclerosis and will provide important scientific foundation and information for drug designand development to study novel structure and mechanism anti-inflammatory andimmune-modulating agents.
天然产物来源广泛，结构多样，药理作用独特，大多数都经过长期的临床应用，其疗效确切，使用安全性高。目前有50%的临床应用药物来自天然产物及其衍生物，并且许多结构新颖的天然产物及其衍生物正处于临床和临床前研究阶段。因此，从天然产物中寻找高效低毒易得的先导化合物成为药物研究的热点，天然产物在今后仍将是新药的重要来源。
随着各种分离、分析和结构测定方法以及色谱、波谱联用技术的飞速发展，化学分离和结构鉴定已经不是天然药物研究的瓶颈。目前天然产物研究面临的一个问题就在于其药理活性和作用机制的阐述较少或研究不深，尤其是结构新颖的天然产物。针对合理阐释其作用机制和结合靶标这一难点，本文第一章综述了基因组学、蛋白质组学和计算机辅助的方法在天然产物靶标发现和机制研究中的应用，希望能够为活性天然产物靶标发现和机制研究的方法及相关技术提供一个比较全面的认识，然后进一步将第一章的方法应用到天然产物cynandione A和氯化两面针碱（nitidine chloride，后文所用两面针碱均指氯化两面针碱）的机制研究中，来阐明这两个天然产物如何发挥相应的药理活性。
脑卒中是一种死亡率高、致残率高的常见病、多发病，并且近几年来发病率呈上升趋势。缺血性脑卒中约占所有脑卒中（stroke）的80%，是指局部脑组织因血液循环障碍，缺血、缺氧而发生的软化坏死。缺血性脑卒中的病因主要是由于供应脑部血液的动脉出现粥样硬化和血栓形成，使管腔狭窄甚至闭塞，导致局灶性急性脑供血不足而发病；也有因异常物体（固体、液体、气体）沿血液循环进入脑动脉或供应脑血液循环的颈部动脉，造成血流阻断或血流量骤减而产生相应支配区域脑组织软化坏死。目前临床治疗脑卒中的药物由于其价格、抗药性以及对出血、胃肠道、神经系统等毒副作用，应用受到了一定的限制，因此，寻找高效低毒易得的分子来防治脑卒中尤为必要。
第二章介绍的天然产物cynandione A是从白首乌中分离得到的苯乙酮类化合物，近年来文献报导cynandione A具有抗氧化、抗谷氨酸兴奋毒和胃保护等药理活性。但目前对其作用机制尚无文献报道，值得进一步的深入研究。
多发性硬化症（multiple sclerosis）至今仍是整个医学界未解的难题之一。该病是一种慢性、自身免疫性疾病，通过破坏脑、脊髓以及视神经的神经纤维保护层髓磷脂，影响正常的神经传递而导致身体残疾。由于该病具有极高的复发率和致残率，已成为常见的、重要的神经系统疾病之一。由于该病的发病高峰在30岁左右，因此，严重影响青壮年人口的工作和生活质量，也给国家和政府造成极大负担，制约社会和经济发展。
目前临床上对该病尚无特效治疗方法，缺乏令人满意的治疗药物。急性期主要给予糖皮质激素，复发-缓解期主要治疗药物包括：干扰素-γ，醋酸格拉替雷和单克隆抗体那他珠单抗。重度患者还可以考虑自体干细胞移植。西医的激素疗法虽然能够缓解急性期症状，但毒副作用明显，故不推荐长期使用;免疫调节剂干扰素、格尔德霉素和那他珠单抗等不仅费用昂贵，且长期使用针对单一靶点药物容易出现耐受。且上述药物均需经静脉、或注射给药，不能有效改善多发性硬化症复发和髓鞘脱失等问题。因此，研制能够有效控制多发性硬化症的复发、改善髓鞘脱失的药物具有重大意义。传统中医药具有多靶点、系统调节的作用特点，因此，针对防治多发性硬化症这一复杂性、难治性疾病具有一定优势。
第三章介绍的天然产物两面针碱是从芸香科植物两面针[Zanthoxylum nitidum(Roxb.) DC]的根中分离到的具有多种药理活性的苯骈菲啶类生物碱，是两面针的主要有效成分，是两面针及其制剂的主要质量控制指标成分。近年来文献报导氯化两面针碱在抗炎、镇痛、抗真菌、抗疟以及抗肿瘤等方面具有广泛的药理活性、开发利用价值和应用前景。基于上述研究进展，并且目前对两面针碱的免疫调控作用尚未进行充分报道，本章首先探讨了两面针碱潜在的治疗多发性硬化症的药理活性，进而在体外细胞模型上研究两面针碱对不同种类和活化状态的免疫细胞的调控作用，最后采用亲和层析的方法寻找与两面针碱存在相互作用的蛋白质以解释其作用靶标，更深一层地阐明两面针碱的作用机制。
2、目的
采用文献综述的方法进一步研究活性天然产物cynandione A和两面针碱的作用机制，为合理开发利用天然产物cynandione A和两面针碱提供重要的科学依据，并为寻找及发现新的神经保护和免疫调控药物提供化学小分子模板。
3、方法
（1）通过清除自由基、抗氧化和抗谷氨酸兴奋毒的活性筛选，发现活性先导物cynandione A，然后，采用二维电泳和LC-MS的蛋白质组学方法研究cynandione A神经保护作用的机制。
（2）体内实验采用大鼠大脑中动脉缺血再灌注（缺血性脑卒中）模型，探讨cynandioneA神经保护作用及其体内抗缺血性脑卒中损伤的机制。SD大鼠经10%水合氯醛（350mg/kg）腹腔麻醉，分离右侧颈总动脉（common carotid artery，CCA）、颈内动脉（internalcarotid artery，ICA）、颈外动脉（external carotid artery，ECA）。结扎CCA近心端、ECA起始端后，在CCA处剪“V”形小口，沿CCA插入线栓（长40mm，直径0.26mm，并于20mm长处标记），经ICA向颅内插至大脑中动脉分叉处，插入深度为18-20mm。再灌注时外拉线使球端回至ECA，拔除插线，结扎ECA。假手术组除不插线外，其余步骤同手术组。大脑中动脉栓塞后2h恢复血供。通过神经功能缺失体征评分、大鼠存活率和四氮唑法染色评价cynandione A抗脑缺血损伤的神经保护作用。通过大鼠脑组织HE染色和DPYSL2、HMGB1免疫组织化学分析探讨cynandione A神经保护作用。
（3）实验性自身免疫性脑脊髓膜炎（experimental autoimmune encephalomyelitis，EAE）是一种得到普遍认可和应用的多发性硬化症实验动物模型。造模方法是皮下注射MOG33-55并辅以CFA，通过树突状细胞激活体内CD4+T细胞，产生Th17细胞，并使之穿过血脑屏障，攻击自身神经髓鞘，从而导致中枢神经白质脱髓鞘，引起EAE。免疫当天记为第0天，于第0天和第2天给予各组小鼠腹腔注射400ng/只百日咳毒素（PTX）。通过神经功能评分、脊髓组织HE染色、髓鞘碱性蛋白的免疫组织化学以及血浆和组织细胞因子的水平来评价两面针碱防治实验性自身免疫性脑脊髓膜炎的作用。
（4）进一步作用机制的研究是采用基因芯片、二维电泳和计算机辅助的方法推断两面针碱可能的作用通路。首先确定合理的给药浓度、刺激剂剂量、给药时间和不同种类的细胞模型。分别考察两面针碱对活化状态下的骨髓细胞、骨髓来源的树突状细胞、小鼠腹腔巨噬细胞、RAW264.7巨噬细胞、脾细胞和磁珠分选的CD4+T细胞的影响。然后验证三种方法推断的两面针碱调控的作用通路。
（5）两面针碱作用靶标的研究是采用生物素标记的亲和层析方法。生物素-抗生物素蛋白（biotin-avidin）系统常被用来寻找天然产物或活性小分子的靶蛋白。这种方法首先需要将小分子通过连接链（spacer）与生物素相连，然后通过生物素与亲和柱的亲和力将小分子固定在亲和柱上，接着在此亲和柱上加入细胞提取物，与小分子有作用的蛋白就会被保留。最后，改变流动相将靶蛋白洗脱并进行功能和结构确定。基于生物素-抗生物素蛋白系统可以寻找靶标蛋白的性质，我们希望选择合适的两面针碱衍生物，通过嫁接连接臂与生物素相连获得生物素标记的两面针碱衍生物，将来用于寻找与两面针碱作用的细胞内靶标蛋白。通过pulldown实验和western blot实验证明两面针碱与质谱鉴定到的蛋白的相互作用，再通过SPR和ITC等实验表明此蛋白与两面针碱确实存在相互作用，从而对下游通路产生影响，进一步产生防治EAE的作用。
4、结果
第二章介绍了天然产物cynandione A的神经保护作用机制研究。首先通过细胞水平清除自由基、抗氧化和抗谷氨酸兴奋毒的活性筛选，发现了神经保护活性先导化合物cynandione A，然后进一步采用二维电泳和LC-MS的蛋白质组学方法研究其调控的蛋白质，结果分析cynandione A能够下调DPYSL2和HMGB1蛋白的表达，并且能抑制RAF-MEK-ERK1/2通路而起到抗谷氨酸兴奋毒的保护作用。体内药效评价发现该天然产物能够减轻大鼠脑缺血再灌注后的神经功能缺失的评分，延长大鼠存活率，改善大鼠脑缺血再灌注的脑组织病理学，显示出一定的抗脑缺血损伤的保护作用。并且组织分布证明cynandione A能够透过血脑屏障。进一步的作用机制研究表明cynandione A是通过下调DPYSL2蛋白的表达和抑制HMGB1蛋白的截留来发挥抗脑缺血的保护作用。
第三章研究了天然产物两面针碱的免疫调控的作用机制和靶标。首先在整体动物水平发现两面针碱腹腔内注射能够显著推迟EAE发病时间并且减轻EAE的行为学评分。组织病理学考察发现两面针碱能够减少单核炎性细胞浸润和髓鞘脱失。另外，两面针碱显著减少血清和组织炎性因子IL-1β，IL-6和TNF-α水平，而提高抑炎性因子IL-10水平。以上结果显示两面针碱具有抑制小鼠实验性自身免疫性脑脊髓炎的药理活性。在其体内有效的基础上，我们筛选了脑脊髓炎发病过程中起到关键作用的树突状细胞、巨噬细胞和CD4~+T细胞三类细胞，进一步研究两面针碱对其的调控作用。通过活性筛选发现两面针碱能够促进活化的树突状细胞和巨噬细胞分泌IL-10，然后采用基因组学、蛋白质组学和计算机辅助手段鉴定了两面针碱的作用通路MAPK和NF-κB，并通过实验证明两面针碱能够增强活化的MAPK和NF-κB通路而促进IL-10分泌。进一步研究发现IL-10对NF-κB通路存在负反馈调节作用，从而减少炎性因子IL-1β，IL-6和TNF-α的分泌。另外，我们还通过采用IL-10的抗体、外源性的IL-10以及IL-10敲除实验证明两面针碱是通过IL-10来缓解EAE的发病。
在天然产物两面针碱的免疫调控的作用机制基础上，实验进一步采用亲和层析的方法寻找与两面针碱存在相互作用的蛋白质以解释其作用靶标，更深一层地阐明两面针碱的作用机制。通过pulldown实验和western blot实验证明两面针碱可与HSP90相互作用，SPR和ITC等实验表明HSP90与两面针碱确实存在相互作用。两面针碱通过与HSP90结合，调控HSP90与某伴侣蛋白的相互作用，从而诱导下游通路提高IL-10的分泌，并且进一步产生防治EAE的作用。
5、结论
天然产物cynandione A能够下调DPYSL2和HMGB1蛋白的表达，并且能抑制RAF-MEK-ERK1/2通路而起到抗谷氨酸兴奋毒的保护作用。体内药效评价发现cynandione A是通过下调DPYSL2蛋白的表达和抑制HMGB1蛋白的截留来发挥抗脑缺血的保护作用。
天然产物两面针碱能够增强活化的MAPK和NF-κB通路而促进IL-10分泌。进一步研究发现IL-10对NF-κB通路存在负反馈调节作用，从而减少炎性因子IL-1β，IL-6和TNF-α的分泌。另外，我们还通过采用IL-10的抗体、外源性的IL-10以及IL-10敲除实验证明两面针碱是通过IL-10来缓解EAE的发病。进一步的作用靶标研究发现两面针碱与HSP90存在相互作用，并且两面针碱通过与HSP90结合，调控HSP90与某伴侣蛋白的相互作用，从而诱导下游通路提高IL-10的分泌，并且进一步产生防治EAE的作用。
这些发现拓展了天然产物cynandione A和两面针碱在神经保护和免疫调控作用的认识，进一步揭示了它们在防治缺血性脑卒中和多发性硬化症方面的应用潜力，为寻找及发现新的神经保护和免疫调控药物提供化学小分子模板。
1Background
A natural product is a chemical compound or substance produced by a livingorganism (found in nature) that usually has a pharmacological or biological activity for usein pharmaceutical drug discovery and drug design. Natural products may be extracted fromtissues of terrestrial plants, marine organisms or microorganism fermentation broths. Acrude extract from any one of these sources typically contains novel, structurally diversechemical compounds, of which the natural environment is a rich source. Drugs derivedfrom natural products have been used for control of major diseases like cardiovasculardiseases, tumors and infectious diseases. Currently, about50%clinical drugs are derivedfrom natural products, and more natural products and their derivatives are in clinical orpre-clinical trials. Therefore, the research and development of natural products is a hottopic in drug development, and they remain an important source of new drugs.
As the rapid development of various chromatographic separation, analysis, structuredetermination approaches, chemical separation and structural identification are not thebottleneck of natural products research. However, a problem of natural products research isthat their pharmacological activities and mechanisms of action are not well clarilied andunderstood, especially the novel natural products. Therefore, the first chapter reviewsgenomics, proteomics, as well as computer-aided strategies to appropriate thesemechanisms and the implications for target discovery of natural products. Furthermore, weapply these approaches to discover the target genes or proteins of natural productscynandione A and nitidine chloride and clarify the mechanisms of their actions.
Stroke is the leading cause of adult disability and the third most common cause ofdeath in industrialized nations. Ischemic stroke occurs when there is an acute blockage ofarterial blood flow to the brain. Neurons contain low levels of endogenous antioxidants;consequently, the brain is very much vulnerable to the injurious effects of reactive oxygenspecies (ROS). Indeed, it has been shown that oxidative stress is a contributory factor inthe determination of the fate of cells during cerebral ischemia and in the outcome of acuteischemic stroke. To date, thrombolytic therapy with tissue plasminogen activators is theonly approved therapy in the United States and Canada for the treatment of acute ischemicstroke. Thrombolytic therapy with tissue plasminogen activator, however, is limited by a very narrow time window. Part of the reason for this limited3-hour window of efficacy isthe generation of free radicals associated with delayed reperfusion that causes oxidativedamage to the brain, which could increase the risk of cerebral hemorrhage and edema.Though numerous potential neuroprotective agents targeting different injurious factors inthe ischemic cascade including oxidative stress have been investigated, almost all of themexcept edaravone have proven inefficacious in humans in studies with a vigorous trialdesign.
Cynandione A and28other derivative compounds were identified from the roots ofCynanchum (C.) wilfordii and C. auriculatum by liquid chromatography electrosprayionization tandem mass spectrometry. Furthermore, cynandione A was obtained duringactivity-guided isolation of the active component of the methanolic extract from dried rootsof C. wilfordii and was found to mitigate neurotoxicity induced by a variety of neurotoxicagents such as H2O2, the excitotoxic neurotransmitter, L-glutamate and kainate in vitro. Itwas further shown that cynandione A could protect against CCl4-mediated hepatotoxicityin vitro, probably due to its radical scavenging properties. In the second chapter, we soughtto further characterize the neuroprotective effects of cynandione A and otheracetophenones extracted from the roots of C. auriculatum and the underlying mechanismusing pheochromocytoma tumor cell line PC12and to investigate whether cynandione Aprotected against ischemic injuries in rats with experimentally induced cerebral ischemia.
Multiple sclerosis remains one of the unsolved problems in the medical community.This chronic autoimmune disease is able to destroy the brain, spinal cord, and nerve fiberlayer of the myelin and affects normal nerve, then causes physical disabilities. Because ofhigh recurrence rate and morbidity, chronic course in young people, multiple sclerosis hasbecome a common disease of the nervous system and makes a great burden to the socialand economic development. Current medications in the treatment of multiple sclerosis,including glucocorticoids, relapse-remission treatment and stem cell transplantation, arelimited by a number of well-characterized clinical side-effects, such as hepatotoxicity,blood dyscrasias, and gastrointestinal and cardiac toxic effects. Therefore, the explorationof new anti-multiple sclerosis drugs with high efficacy and less toxicity is eagerly needed.Traditional Chinese medicine (TCM), a unique medical system with the significantcharacteristic of the use of multi-component drugs, can hit multiple targets with itscomponents, improve therapeutic efficacy, reduce drug-related side effects and may alsobe an effective way of decreasing drug resistance. Natural products have aroused much interest recently due to its superiority in the treatment of complex multi-factor diseases,such as multiple sclerosis.
Nitidine chloride, a benzo[c]phenanthridine alkaloids isolated from Zanthoxylumnitidium (Roxb.) DC., was reported to have significant anti-tumor, anti-fungal,anti-malarial, and anti-inflammatory activities. In our previous study, nitidine chlorideexhibited analgesic and anti-inflammatory activities, however, the precise molecular targetand underlying mechanisms of its anti-inflammatory and immunomodulatory efficacy arepoorly clarified to date. In the third chapter, we explored genomic, proteomic, andcomputational approaches to investigate its possible mechanisms that contributed tonitidine mediated signal transduction pathways in the regulation of IL-10secretion in DCsand macrophages. Moreover, nitidine chloride effectively inhibited pro-inflammatorycytokines secretion, significantly enhanced IL-10secretion, and mitigated the occurrenceof EAE in vivo. Therefore, our findings indicated that nitidine chloride is a promisingcandidate compound that can be further optimized to be a therapeutic agent for multiplesclerosis.
2Objective
This subject will reveal the molecular mechanism of action of bioactive naturalproducts cynandione A (to protect against ischemic injuries in rats with experimentallyinduced cerebral ischemia) and nitidine (to promote IL-10secretion in EAE treatment formultiple sclerosis) and will provide important scientific foundation and information fordrug design and development to study novel structure and mechanism neuroprotective,anti-inflammatory and immune-modulating agents.
3Methods
(1) Cellular level and cerebral ischemia animal model identified cynandione A as aneuroprotective lead, and using two-dimensional electrophoresis and LC-MS proteomicsapproaches to study its neuroprotective effect.
(2) Using rat middle cerebral artery ischemia reperfusion model to explorecynandione A neuroprotective effect and its mechanism in vivo. Adult maleSprague-Dawley rats (250-300g) were injected intraperitoneally with5or30mg/kgcynandione A or8mg/kg nimodipine for3days. Rats were anesthetized by peritonealinjection of chloral hydrate at350mg/kg, and the middle cerebral artery was occluded for 2h with a silicone rubber-coated nylon monofilament (40mm in length and0.26mm indiameter). The occluding filament was withdrawn after2h to allow reperfusion. Rats wereinjected intraperitoneally with5or30mg/kg cynandione A or8mg/kg nimodipine for3days following occlusion of the middle cerebral artery. In sham-operated rats, the leftcommon carotid artery was exposed and external carotid artery was opened withoutintroducing the filament into the internal carotid artery. Neurological deficit score of eachrat was obtained at24and72h after occlusion of the middle cerebral artery. Rats were alsosacrificed3days after reperfusion, and rat brains were dissected coronally into2-mm brainslices using a metallic brain matrix. Slices were immediately stained by immersion in1%2,3,5-triphenyl tetrazolium chloride and then in4%paraformaldehyde for preservation.After brain tissues preservation, the areas of the infarcted regions and of both hemisphereswere calculated for each coronal slice by image analysis software ImageJ. Also, HEstaining and immunohistochemical analysis were performed to explore neuroprotectiveeffect of cynandione A.
(3) Experimental autoimmune encephalomyelitis (EAE), an inflammatorydemyelinating disease of the central nervous system (CNS), is the prime model for humanmultiple sclerosis. Female C57BL/6mice were intraperitoneally injected with nitidine atthe dose of10mg/kg day which is lower than the LD50for nitidine for a week beforeMOG sensitization, and the nitidine treatment continued through all the experiment. Thecontrol groups were injected with DMSO or PBS of equal volume with the former. Thenthe mice were immunized by subcutaneous injection at three sites of dorsal with300μg ofMOG35-55peptide emulsified in complete Freund’s adjuvant supplemented with0.5mgMycobacterium tuber-culosis strain H37RA. Furthermore, mice received intraperitonealinjections with400ng of pertussis toxin at the time of immunization and48h later.Clinical signs of disease were observed usually between EAE induction and assessed dailyexactly according to the following scoring criteria:0, no detectable signs of EAE;1,flaccid tail;2, hind limb weakness or abnormal gait;3, complete hind limb paralysis;4,paralysis of fore and hind limbs; and5, moribund or death. Intermediate scores (0.5) wereassigned if the neurological signs exhibited a lower severity than typically observed.Finally, blood serum and spinal cord from mice suffering EAE induction for21days wereobtained and analyzed.
(4) Further mechanism research is to explore gene chip, two-dimensionalelectrophoresis and computer aided approaches to investigate the possible mechanism of action of nitidine. To determine reasonable dosage concentration, stimulants dose, dosingtime and different kinds of cell model. Bone marrow cells, bone marrow-derived dendriticcells, peritoneal macrophages, RAW264.7macrophages, spleen cells and CD4+T cellsseparated by magnetic beads were obtained and analyzed by ELISA, respectively. Thenverify its regulatory pathways.
(5) In order to further understand its biological mechanisms and to identify its targetproteins, a biotinylated derivative of nitidine was synthesized, using a peptide-bond as thelinkage between artemisinin and D-biotin. This approach starts with immobilizing abioactive compound on a matrix in a way that does not interfere with its activity. The smallmolecule ligand is modified by introducing an appropriate functional group (referred to asa linker), through which it can be immobilized by attachment to the affinity matrix——astep that is important for later phase separation. There are various commercially availableactivated resins that allow for the attachment of specific chemical groups (for example,sulfhydryl, amino, hydroxyl or carboxyl groups). After the immobilized small moleculeligand has been incubated with protein extracts and any unbound proteins havesubsequently been removed in a series of washing steps, specifically bound proteins areseparated by solid-phase elution, using buffer conditions that disrupt the interactionbetween the target protein and the immobilized small molecule ligand. Finally, the proteinis typically identified by MS. Predicting or inferring the protein targets need to bevalidated in the biological method. Through the pulldown and western blot experiments,SPR, and ITC experiments showed that nitidine is able to bind and interact with the proteinin macrophages, thus influence on the downstream pathways, further promotes IL-10secretion in EAE treatment for MS.
4Results
The second chapter described neuroprotective effects of natural product cynandione A.Viability assays using the WST-8method and LDH release assays showed that cynandioneA dose-dependently attenuated glutamate-induced cytotoxicity. Comparative proteomicanalysis by two-dimensional gel electrophoresis and MALDI-TOF MS/MS of PC12cellstreated with cynandione A showed10μM cynandione A caused broad changes in proteinexpression in PC12cells including downregulation of high mobility group box1(HMGB1)and dihydropyrimidinase-like2(DPYSL2) and inhibit RAF-MEK-ERK1/2pathway.Immunoblotting studies showed that10μM cynandione A aborted glutamate-induced increase in DPYSL2and HMGB1levels in PC12cells and30mg/kg cynandione A alsoattenuated the rise in HMGB1levels and mitigated DPYSL2cleavage in brain tissues ofrats with cerebral ischemia. Furthermore, rats with cerebral ischemia treated with30mg/kgcynandione A exhibited markedly improved neurological deficit scores at24and72hcompared with control and a7.2%reduction in cerebral infarction size at72h (P <0.05vs.control). Our findings demonstrated that cynandione A mitigated ischemic injuries andshould be further explored as a neuroprotective agent for ischemic stroke.
The third chapter demonstrated anti-inflammatory and immune-modulating effects ofnatural product nitidine. Nitidine is a clinically efficient ingredient from extracts ofzanthoxylum nitidum, an indigenous plant in Southern China that shows anti-inflammatoryand analgesic properties. Here, we found that this molecule was able to interfere withmaturation of DCs and their ability to present antigens to T cells, and nitidine-treatedBMDCs could secrete more IL-10and less IL-1β，IL-6and TNF-α in response tolipopolysaccharide (LPS) in vitro. Up-regulation of MAPK and NF-κB pathwaysactivation was shown to be responsible for IL-10preferential production. These resultswere consistent with the observation that IL-10concentration of serum fromnitidine-treated mice suffered from EAE induction was higher compared with controlgroup. In addition, nitidine was able to interfere with the onset of EAE and decreased EAEclinicopathological features in mice by inhibiting immune response to MOG35-55peptides.Further study found that IL-10is able to negative feedback regulate the NF-κB pathway,thus reducing IL-1β，IL-6and TNF-α secretion. In addition, through IL-10antibody,exogenous IL-10and IL-10knockout experiments showed that nitidine could alleviate theincidence of EAE by IL-10. Our results suggest that nitidine has powerfulanti-inflammatory and immune-modulating function in vitro, as well as in vivo, and wouldbe regarded as a new anti-inflammatory and immune-modulating drug with greatpotentiality for the treatment of autoimmune diseases in the future.
In order to further understand its biological mechanisms, affinity chromatography wasadopted to identify target proteins of nitidine. Through the pulldown and western blotexperiments, SPR, and ITC experiments showed that nitidine is able to bind and interactwith the protein in macrophages, thus influence on the downstream pathways, furtherpromotes IL-10secretion in EAE treatment for multiple sclerosis.
5Conclusions
Natural product cynandione A caused downregulation of HMGB1and DPYSL2andinhibit RAF-MEK-ERK1/2pathway to attenuate glutamate-induced cytotoxicity in vitro.Furthermore, cynandione A protected against ischemic injuries in rats with experimentallyinduced cerebral ischemia. Our findings demonstrated that cynandione A mitigatedischemic injuries and should be further explored as a neuroprotective agent for ischemicstroke.
Our present study found that nitidine could mitigate EAE and secrete more IL-10andless IL-1β，IL-6and TNF-α in response to LPS in vitro. Further mechanism study foundthat nitidine could promote the activated dendritic cells and macrophages to secrete IL-10.Affinity chromatography results suggested that nitidine is able to bind and interact withHSP90in dendritic cells and macrophages. This subject will reveal the molecularmechanism of action of nitidine to promote IL-10secretion in EAE treatment for multiplesclerosis and will provide important scientific foundation and information for drug designand development to study novel structure and mechanism anti-inflammatory andimmune-modulating agents.
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