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Hydrangenol inhibits lipopolysaccharide-induced nitric oxide production in BV2 microglial cells by suppressing the NF-κB pathway and activating the Nrf2-mediated HO-1 pathway

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Abstract
We previously demonstrated the anti-inflammatory effects of water extract of Hydrangea macrophylla in lipopolysaccharide (LPS)-stimulated macrophage cells. Here, we investigated whether hydrangenol, one of bioactive component from H. macrophylla, attenuates the expression of nitric oxide (NO) and its regulatory gene, inducible NO synthase (iNOS), in LPS-stimulated BV2 microglial cells. Low dosages of hydrangenol inhibited LPS-stimulated NO release and iNOS expression without any accompanying cytotoxicity. Hydrangenol also suppressed LPS-induced nuclear translocation of the nuclear factor-κB (NF-κB) subunits by inhibiting IκBα phosphorylation and consequently inhibited DNA-binding activity of NF-κB. Additionally, the NF-κB inhibitors, pyrrolidine dithiocarbamate (PDTC) and proteasome inhibitor (PSI), diminish LPS-induced iNOS expression, indicating that hydrangenol-induced NF-κB inhibition might be a key regulator of iNOS expression. Furthermore, our data also showed that hydrangenol suppresses NO production by inducing heme oxygenase-1 (HO-1) and the presence of cobalt protoporphyrin (CoPP), a specific HO-1 inducer, potently suppressed LPS-induced NO. Additionally, hydrangenol promoted nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and subsequently increased its binding activity in specific ARE sites. Transient knockdown of Nrf2 remarkably downregulated hydrangenol-induced HO-1 expression, indicating that hydrangenol-induced Nrf2 is an upstream molecule of HO-1. Taken together, these data indicate that hydrangenol attenuates production of proinflammatory NO and iNOS in LPS-stimulated BV2 microglial cells by inhibiting NF-κB activation and stimulating the Nrf2/HO-1 signal pathway. Therefore, hydrangenol might be a good therapeutics in LPS-mediated inflammatory diseases.
Author(s)
김희주
Issued Date
2015
Awarded Date
2015. 8
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000007282
Alternative Author(s)
Kim, Hee Ju
Department
대학원 해양생명과학과
Table Of Contents
1. ABSTRACT 1
2. INTRODUCTION 3
3. MATERIALS AND METHODS 6
3.1. Reagents and antibodies 6
3.2. Cell culture and viability 6
3.3. Flow cytometric analysis 7
3.4. NO production 7
3.5. Isolation of total RNA and RT-PCR 8
3.6. Western blot analysis 8
3.7. Electrophoretic mobility assay (EMSA) 9
3.8. Transient knockdown of Nrf2 10
3.9. Statistical analysis 10

4. RESULTS 11
4.1. Hydrangenol has no influence on viability of BV2 microglial cells 11
4.2. Hydrangenol inhibits NO production and iNOS expression in LPS-stimulated BV2 microglial cells 13
4.3. Hydrangenol inhibits LPS-induced iNOS expression by suppressing NF-κB activation and nuclear translocation in BV2 microglial cells 15
4.4. Hydrangenol decrease NO release through expression of HO-1 in BV2 microglial cells17
4.5. Hydrangenol-induced Nrf2 is an important regulator for HO-1 induction 19

5. DISCUSSION 23
6. REFERENCES 26
Degree
Master
Publisher
제주대학교 대학원
Citation
김희주. (2015). Hydrangenol inhibits lipopolysaccharide-induced nitric oxide production in BV2 microglial cells by suppressing the NF-κB pathway and activating the Nrf2-mediated HO-1 pathway
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General Graduate School > Marine Life Sciences
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