A mechanism study on the regulation of neurodegenerative disease using the Lycopus maackianus extracts, compounds isolated from Lindera erythrocarpa, and ramalin-synthetic derivative compounds = 애기쉽싸리 추출물, 비목나무 유래 화합물 및 ramalin 합성유도체 화합물을 이용한 퇴행성뇌질환 조절 기전 연구

목차 (Table of Contents)

• Chapter 1. General Information 1
• Ⅰ. Lycopus maackianus 2
• Ⅱ. Lindera erythrocarpa 4
• Ⅲ. Ramalin isolated from the Antarctic lichen (Ramalina terebrata) 6
• Chapter 2. Lycopus maackianus Makino MeOH Extract Exhibits Antioxidant and Anti Neuroinflammatory Effects in vitro and in vivo 7
• Ⅰ. Introduction 8
• Ⅱ. Materials and Methods 11
• 1. Preparation of extract from L. maackianus 11
• 2. Materials 11
• 3. Metabolite profiling analysis using 11
• 4. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity 12
• 5. 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity 13
• 6. Cell culture and viability assay 13
• 7. Determination of nitrite levels 14
• 8. Determination of PGE2 levels 14
• 9. Determination of IL-6 and TNF-α levels 14
• 10. Western blot analysis 15
• 11. Preparation of cytosolic and nuclear fractions 15
• 12. NF-κB localization and immunofluorescence 15
• 13. Origin and maintenance of parent zebrafish 15
• 14. Determination of ROS levels in HT22 cells 16
• 15. LPS and H2O2 treatment of zebrafish embryos 16
• 16. Cell death measurement and image analysis in zebrafish embryos 17
• 17. Determination of PGE2 levels 17
• 18. Determination of NO levels and image analysis in zebrafish embryos 17
• 19. Statistical analyses 17
• Ⅲ. Results 19
• 1. UHPLC-TOF-HRMS analysis of L. maackianus MeOH extract 19
• 2. Effect of L. maackianus extract on DPPH and ABTS radical scavenging activity 21
• 3. Effects of L. maackianus extract on the levels of pro-inflammatory mediators and cytokines in LPS-induced BV2 cells 23
• 4. Effects of L. maackianus extract on NF-κB activation in LPS-induced BV2 cells 26
• 5. Effects of L. maackianus extract on glutamate-induced HT22 cells 28
• 6. Effects of L. maackianus extract on HO-1 expression and Nrf2 nuclear translocation 30
• 7. Antioxidant effect of L. maackianus extract in zebrafish model stimulated with H2O2 33
• 8. Anti-inflammatory effect of L. maackianus extract in zebrafish model stimulated with LPS 36
• Chapter 3. Linderone and Dihydropashanone isolated from Lindera erythrocarpa plays Neuroprotective Effects via the Regulating NF-κB and Nrf2 Pathway in BV2 and HT22 Cells 47
• Ⅰ. Introduction 48
• Ⅱ. Materials and Methods 50
• 1. Materials 50
• 2. Extraction and isolation 50
• 3. Cell culture and cell viability assays 53
• 4. Nitrite assays 53
• 5. PGE2 assay 53
• 6. Assays for IL-6 and TNF-α 53
• 7. Western blotting analysis 53
• 8. p65 Localization 54
• 9. Determination of ROS levels in HT22 cells 54
• 10. Statistical analysis 54
• Ⅲ. Results 55
• 1. Structural Elucidation of Isolated Compounds 55
• 2. Effects of compounds isolated from L. erythrocarpa on cell viability 62
• 3. Effects of isolated compounds from of L. erythrocarpa on the BV2, RAW264.7 and HT22 Cells 66
• 4. Effects of compound 7 and 13 on the cell viability of BV2 and HT22 Cells 70
• 5. Effects of linderone and dihydropashanone on inflammatory factors in BV2 cells 72
• 6. Effects of linderone and dihydropashanone on NF-κB activation in BV2 cells 75
• 7. Effects of linderone and dihydropashanone on glutamate-induced oxidative stress in HT22 cells 77
• 8. Effects of linderone and dihydropashanone on Nrf2/HO-1 pathway in HT22 and BV2 cells 79
• Ⅳ. Discussion 83
• Ⅴ. Conclusion 86
• Chapter 4. Ramalin-synthetic derivative compounds play Neuroprotective Effects in BV2 and HT22 Cells 87
• Ⅰ. Introduction 88
• Ⅱ. Materials and Methods 90
• 1. Chemicals and reagents 90
• 2. Cell culture and cytotoxicity assay 90
• 3. Nitrite assay 90
• 4. Inflammatory factors assays 91
• 5. Western blot analysis 91
• 6. NF-κB (p65) immunofluorescence 92
• 7. BV2-HT22 cell co-culture 92
• 8. Terminal dexynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay 92
• 9. Terminal dexynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay 93
• 10. Statistical analysis 93
• Ⅲ. Results 94
• 1. The structure of RA derivatives 94
• 2. Inhibitory effect of RA derivatives on nitrite production 96
• 4. Protective effect of RA derivatives on HT22 cell death 105
• 5. Effects of RA-10 on inflammation in BV2 cell 114
• 6. Effects of RA-10 on NF-kappa B activation in BV2 cell 117
• 7. Effects of RA-10 on HT22 cell co-cultured with BV2 cell 119
• 8. The effect of NF-κB activation in HT22 apoptosis induced by neuro-inflammation 125
• Ⅳ. Discussion 127
• Ⅴ. Conclusion 131
• References 132