| 1BackgroundA 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.2ObjectiveThis 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.4ResultsThe 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. 5ConclusionsNatural 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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