Anti-osteoclastogenetic activities of dearylheptanoids through the regulation of TNF receptor associated factor 6 signal pathways in in-vitro model
- 주제(키워드) osteoclast
- 발행기관 강릉원주대학교
- 지도교수 주성수
- 발행년도 2018
- 학위수여년월 2018. 2
- 학위명 석사
- 학과 및 전공 일반대학원 해양분자생명과학과
- 본문언어 영어
- 저작권 강릉원주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
The bone is made up of new bone by the osteocyte, and the old bone is absorbed by the osteoclast to maintain healthy bone. Bone tissue is determined by the balance between osteoclast bone formation rate and osteoclast resorption rate. Among the essential components of osteoclast formation, receptor activators of NF-κB ligand (RANKL) binds to RANK receptors in osteoclast precursors and mature osteoclasts and stimulates bone resorption by concentrating RANKL. Therefore, in this study, primarily aimed to induce osteoclasts using sRANKL in pre-osteoclastic cell line (RAW 264.7). Secondly, diarylheptanoid hirsutenone (HTN) was chosen as a modulator of the degradation of tumor necrosis factor receptor related factor 6 (TRAF6) and IkappaB (IκB) using western blotting analysis. In addition, lipopolysaccharide (LPS)-induced pro-inflammatory cytokines were screened in bone marrow cells to assess whether HTN plays a critical role in bone resorption environments. Results demonstrated that HTN effectively inhibited the pro-inflammatory repertoires, such as inducible nitric oxide synthase (iNOS) and interferon (IFN)γ in macrophages-depleted bone marrow cells. Also found that HTN inhibited TRAF6 and phosphor-IκB in sRANKL-treated. Coincidently, HTN significantly inhibited p-IκB in LPS-treated mouse spleen cells. Taken together, conclude that HTN plays a crucial role in directly or indirectly inhibiting osteoclast formation by promoting TRAF6 degradation and inhibiting NF-κB signaling. These results suggest that HTN may inhibit osteoclast triggering processes and thus a promising new candidate for the diseases initiating from bone loss.
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ABSTRACT ................................................................. 1
초록................................................................................... 2
LIST OF FIGURES .................................................... 3
LIST OF TABLES ...................................................... 4
1 INTRODUCTION................................................ 5
2 MATERIALS AND METHODS........................ 7
2.1 Reagents........................................................................ 7
2.2 Cell culture.................................................................... 7
2.2.1 Pre-osteoclastic cell lines......................................... 7
2.2.2 Osteoclasts differentiation...................................... 8
2.2.3 Isolation of splenocytes from C57BL/6 spleen…... 9
2.2.4 Interferon gamma overexpression in splenocytes stimulated by monoclonal anti-CD3....................... 9
2.3 Immunocytochemistry (ICC)...................................... 10
2.4 Nitric oxide (NO) production....................................... 10
2.5 Western blot analysis................................................... 11
2.6 4',6-diamidino-2-phenylindole (DAPI) staining........ 11
2.7 Lactate dehydrogenase (LDH) assay.......................... 12
2.8 Tartrate-resistant acid phosphatase (TRAP) positive cell staining...................................................... 12
2.9 mRNA profiles of osteoclastogenesis-related system repertoires..................................................................... 13
2.10 Luciferase reporter gene assay.................................... 14
2.11 Cell viability assay........................................................ 14
2.12 Mouse Interferon-γ enzyme-linked immunosorbent assay (ELISA) assay..................................................... 15
2.13 Polymerase chain reaction (PCR) .............................. 15
2.14 Statistical analysis........................................................ 15
3 RESULTS............................................................. 17
3.1 Construction of osteoclastic cell model....................... 17
3.2 Cell viability by Hirsutenone (HTN) in RAW 264.7 cells................................................................................ 19
3.3 RANKL-dependent signal pathway analysis............. 20
3.4 DAPI staining for multinucleasted RAW 264.7 cells. 22
3.5 Immunocytochemistry analysis of osteoclast-associated receptor (OSCAR) in RAW 264.7 cell....... 24
3.6 Induction of osteoclastic cells in bone marrow monocytes and anti-inflammatory effects of HTN..... 26
3.7 NF-κB luciferase assay in RAW 264.7 cells................ 28
3.8 Osteoclastogenesis-related mRNA gene expression in mouse splenocytes.................................................... 29
3.9 Inhibition of IκB phosphorylation by HTN in mouse splenocytes.................................................................... 31
3.10 Cytotoxicity and Profiles of mRNAs in macrophage-depleted bone marrow cells (MDBMC)...................... 33
4 DISCUSSION....................................................... 35
5 REFERENCES..................................................... 39
