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Neuroprotection of Active Principles from the Leaves of Laurus nobilis and Mori Cortex Radicis in Neuronal Cell Culture and Rodent Models of Parkinsons Disease: Regulatory Effect on the Ubiquitin-Proteasome

DC Field Value Language
dc.contributor.advisor마응천-
dc.contributor.author함아롬-
dc.date.accessioned2019-07-02T15:45:10Z-
dc.date.available2019-07-02T15:45:10Z-
dc.date.issued2012-02-
dc.identifier.other000000000649-
dc.identifier.urihttps://hdl.handle.net/10371/156650-
dc.identifier.urihttp://dcollection.snu.ac.kr:80/jsp/common/DcLoOrgPer.jsp?sItemId=000000000649ko_KR
dc.description.abstractPD is characterized by selective loss of dopaminergic (DAergic) neurons in the substantia nigra (SN) and the presence of Lewy bodies, the pathological hallmarks of PD composed of aggregated a-synuclein (ASYN) occurring inside neurons. The modification of ASYN may initiate the accumulation of abnormal proteins by impairment of the ubiquitin-proteasome proteolytic (UPP) pathway. A failure of the UPP pathway, which degrades intracellular proteins, has also been thought to play an important role in the pathogenesis of PD. The aims of this study were to assess the neuroprotective effect of the active principles from the Leaves of Laurus nobilis, Mori Cortex radicis, and Cudrania tricuspidata against dopamine (DA)-induced neuronal toxicity by regulation of UPP and ASYN-mediated pathways in both in vitro and in vivo models of PD. We observed a marked increase in apoptosis and the generation of ROS and decreased cell viability by DA. Pretreatment of the cells for 24 h with each of cstunolide, dehydrocostuslactone, and spirafolide from Laurus nobilis before DA exposure notably increased the cell survival and lowered the intracellular ROS level. Moreover costunolide had inductive effects on the expression of Nurr1 (nuclear receptor related-1), DA transporter (DAT), and vesicular monoamine transporter type 2 (VMAT2) in DA treated human DAergic SH-SY5Y cells, and a reciprocal reduction in ASYN was observed at both the transcriptional and translational levels in vitro. E6-associated protein (E6-AP) has been known to promote the degradation of ASYN. Flow cytometric and western blotting analyses revealed that reynosin significantly protected against DA-induced cell death in both SH-SY5Y cells and 6-hydroxydopamine (6-OHDA)-induced tyrosine hydroxylase (TH)-positive cell loss in the SN region of rat brain. In addition, reynosin concomitantly up-regulated E6-AP protein expression and down-regulated over-expression of the ASYN protein in both DA-treated cells and a rodent PD model system. And the treatment with moracenin D isolated from Mori Cortex radicis resulted in an up-regulation of Nurr1 mRNA levels and a down-regulation of ASYN mRNA levels. Additionally, the ASYN protein expression was decreased in accordance with an increase in Nurr1 protein expression. The protective effect of moracenin D was presumably due to the correlative effects on the up-regulation of nurr1 and down-regulation of ASYN expressions against DA induction. Iisozaluzanin C, magnolialide, santamarine, lucentolide, 1β-hydroxyarbusculin A, and methyl 1β, 2β, 6-tryhydroxy-5, 7H-eudesma-4(15), 11(13)-dien-12-oate (METO) were isolated from the chloroform extract of Laurus nobilis. Incubation of DA alone increased in fluorescence, as the protein aggregation. However co-incubation of magnolialide or 1β-hydroxyarbusculin A with DA significantly inhibited protein aggregation, as determined by thioflavin S fluorescence. Further magnolialide, isozaluzanin C, santamarine, and lucentolide co-treated with DA increased proteasome activity significantly in the DA-induced markedly decreased proteasome activity. Parkin has E3 ubiquitin ligase activity and is mediated UPP pathway. Therefore the mutants were designed point mutation of E3 ligase activity associated RING region. After cell lysates of the over-expressed Parkin were prepared and conducted E3 ligase activity by autoubiquitination assay. The C289G missense mutation is in functional domains in the Parkin protein; altered decreasing the E3 ligase activity in both cell lysates of immunoprecipitated C289G mutant and C289G mutant with 6-OHDA. 5, 7-Dihydroxychromone significantly increased the decreasing E3 ubiquitin ligase activity of Parkin by 6-OHDA-induced in C289G mutant cells.
Taken together, the present results indicated that the neuroprotective effects of isolated compounds from natural plants on DA-induced cytotoxicity and mutation of Parkin in models of PD may be, at least in part, attributable to the its potent regulation of UPP or ASYN-mediated pathways. Therefore these active principles could be considered as a candidate therapy for the treatment of PD.
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dc.description.tableofcontentsⅠ. Introduction 3 _x000D_
Ⅱ. Materials and methods 6 _x000D_
2.1. Plant materials 6 _x000D_
2.2. Reagents 7 _x000D_
2.3. Cell culture 7 _x000D_
2.4. Cell viability by FACS 8 _x000D_
2.5. Detection of cell apoptosis by Annexin V-FITC staining 8 _x000D_
2.6. Detection of intracellular ROS 8 _x000D_
2.7. Reverse transcription polymerase chain reaction (RT-PCR) 9 _x000D_
2.8. Primary cortical neurons culture 10 _x000D_
2.9. 4', 6-diamidino-2-phenylindole staining of neuron apoptotic cells 11 _x000D_
2.10. Animal care and maintenance 11 _x000D_
2.11. 6-OHDA lesions surgery of rat brain and treatment procedure 12 _x000D_
2.12. Tissue collection and preparation 13 _x000D_
2.13. Immunohistochemistry 13 _x000D_
2.14. Image capture and analysis 14 _x000D_
2.15. Western blot analysis 14 _x000D_
2.16. Quantification of protein aggregation 15 _x000D_
2.17. Proteasome activity 15 _x000D_
2.18. Construction of the expression vectors 16 _x000D_
2.19. Plasmid 16 _x000D_
2.20. Transient transfection assay 17 _x000D_
2.21. Immunocytochemistry 17_x000D_
2.22. Immunoprecipitation 18_x000D_
2.23. E3 ubiquitin ligase activity 18_x000D_
2.24. Statistical analysis 18_x000D_
Ⅲ. Results 19_x000D_
Part 1. 19_x000D_
1.1. HPLC analysis of HFL 19_x000D_
1.2. Cytotoxic effect of sesquiterpene lactones of HFL on SH-SY5Y cells 19 _x000D_
1.3. Anti-apoptotic effect of sesquiterpene lactones on DA-induced cells 20_x000D_
1.4. Effects of sesquiterpene lactones against H2O2-induced a cell death 20_x000D_
1.5. Effects of sesquiterpene lactones of HFL on intracellular ROS levels 21_x000D_
Part 2. 21_x000D_
2.1 Moracenin D from Mori Cortex Radicis protects SH-SY5Y cells against DA-induced cell death by regulating Nurr1 and ASYN expression 21_x000D_
2.1.1. Protection from DA-induced cell death by Moracenin D 21_x000D_
2.1.2. mRNA and Protein expressions of Nurr1 and ASYN by moracenin D 22_x000D_
2.2. Regulatory effects of costunolide on dopamine metabolism-associated genes inhibit dopamine-induced apoptosis in human dopaminergic SH-SY5Y cells 23_x000D_
2.2.1. Protective effect of costunolide on the DA-induced apoptosis in cells 23_x000D_
2.2.2. Effect of costunolide on mRNA of DA metabolism associated genes 23_x000D_
2.2.3.Effect of costunolide on proteins of DA metabolism associated genes 23_x000D_
Part 3. 24 _x000D_
3.1. Protection from DA-induced cell death by reynosin 24 _x000D_
3.2. Effects of reynosin on E6-AP and ASYN protein expression in cells 25 _x000D_
3.3. Neuroprotective effect of reynosin on the DA-induced apoptosis of primary cultured neurons of rat 26 _x000D_
3.4. Inhibitory effect of HFL against the loss of DAergic neurons in rat models of PD 27 _x000D_
3.5. Identification of ASYN over-expression by HFL 27 _x000D_
3.6. Effects of reynosin on TH-positive DAergic neurons in the SN in 6-OHDA lesions rats 27 _x000D_
3.7. Effect of reynosin on E6-AP and ASYN protein expression in rats with lesions 28 _x000D_
Part 4. 29 _x000D_
4.1. Effect of moracenin D from Mori Cortex radicis on protein aggregation _x000D_
and proteasome dysfunction in vitro 29 _x000D_
4.1.1. Effect of moracenin D and moracin P on protein aggregation 29 _x000D_
4.1.2. Effect of moracenin D and moracin P on ubiquitin conjugates 29 _x000D_
4.1.3. Effect of moracenin D on proteasome activity 29 _x000D_
4.2. Novel sesquiterpenes from the leaves of Laurus nobilis (Lauraceae) on protein aggregation and proteasome activity in dopamine-induced SH-SY5Y cells 30 _x000D_
4.2.1. Effect of sesquiterpenes on cell death induced by DA 30 _x000D_
4.2.2. Inhibition of Magnolialide and METO on protein aggregation 30 _x000D_
4.2.3. Sesquiterpenes inhibit proteasome dysfunction in vitro 31 _x000D_
Part 5. 31 _x000D_
5.1. Generation of transiently transfected Parkin-expressing SH-SY5Y cells 31 _x000D_
5.2. Parkin mutations and 6-OHDA reduced its E3 ubiquitin ligase activity 32 _x000D_
5.3. 7-Hydroxycoumarin and 5, 7-Dihydroxychromone from Cudrania tricuspidata attenuate 6-OHDA-induced cell death in C289G mutant transfected cells 32 _x000D_
5.4. 5, 7-Dihydroxychromone regulates Parkin E3 ligase activity 33 _x000D_
Ⅳ. Discussion 33 _x000D_
Ⅴ. References 48 _x000D_
Abstract in Korean 99
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dc.format.extent120-
dc.language.isoeng-
dc.publisher서울대학교 대학원-
dc.subject.ddc615-
dc.titleNeuroprotection of Active Principles from the Leaves of Laurus nobilis and Mori Cortex Radicis in Neuronal Cell Culture and Rodent Models of Parkinsons Disease: Regulatory Effect on the Ubiquitin-Proteasome-
dc.typeThesis-
dc.typeDissertation-
dc.contributor.AlternativeAuthorAhrom Ham-
dc.description.degreeDoctor-
dc.contributor.affiliation제약학과-
dc.date.awarded2012-02-
dc.contributor.major천연물과학전공-
dc.identifier.holdings000000000006▲000000000011▲000000000649▲-
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