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Anti-inflammatory and anti-invasive effects of methanol extract of Polyopes lancifolius by suppression of the NFκB pathway

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Abstract
Purpose: This study aimed to identify the anti-inflammatory and anti-invasive effects of a methanol extract of Polyopeslancifolius(MEPL)inlipopolysaccharide(LPS)-stimulatedBV2microgliacells.
Methods: We investigated the expression of mRNA and protein using RT-PCR and western blot analysis in LPSstimulated BV2 microglial cells. Level of nitric oxide (NO) production was analyzed using Griess reaction. The release of prostaglandin E2(PGE2)andtumornecrosisfactor-α(TNF-α)weredeterminedusingsandwichELISA.NF-κBactivationwasdetectedusingEMSAmethods.
Results: We found that MEPL significantly suppressed NO production in LPS-stimulated BV2 cells without any cytotoxicity. Our results also indicated that MEPL decreased the production of PGE2andTNF-αinLPS-stimulatedBV2cells.Furthermore,pretreatmentwithMEPLresultedinadownregulationofLPS-inducedmRNAandproteinexpressionofinducibleNOsynthase(iNOS),cyclooxygenase-2(COX-2),andTNF-α. Therefore, we investigated the effects of MEPL on nuclear factor-κB (NF-κB) activity, which is a potential transcriptional factor for regulating inflammatory genes such as iNOS, COX-2, and TNF-α. Our data showed that MEPL substantially inhibited the LPS-induced DNA-binding activity of NF-κB. MEPL also suppressed the LPS-induced degradation and phosphorylation of IκBα, and it consequently blocked p65 translocation from the cytosol to the nucleus.
Conclusion: These data show that MEPL may regulate LPS-induced NO, PGE2, andTNF αproductionbysuppressingNF-κB activity.
Author(s)
Matharage Gayani Dilshara
Issued Date
2013
Awarded Date
2013. 8
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000006462
Affiliation
제주대학교 대학원
Department
대학원 해양생명과학과
Advisor
Gi-Young Kim
Table Of Contents
Chapter I.
1. Introduction 3
2. Materials and method 5
2.1 Preparation of MEPL 5
2.2 Cell culture and sample treatment 5
2.3 Analysis of cell viability 6
2.4 Nitric oxide determination 6
2.5 Measurement of PGE2andTNF-α 6
2.6 Isolation of total RNA and RT-PCR 6
2.7 Western blot analysis 7
2.8 Electrophoretic mobility assay (EMSA) 7
2.9 Statistical analysis 8
3. Results
3.1 Effects of MEPL on cell viability 9
3.2 Effects of MEPL on LPS-induced NO and PGE2production 10
3.3 Effects of MEPL on LPS-induced iNOS and COX-2 protein and mRNA 12
3.4 Effects of MEPL on LPS-induced TNF-α production and mRNA Expression 14
3.5 Inhibitory effects of MEPL in LPS-induced NF-κB activity 16
4. Discussion 18
5. Conclusion 20
Chapter II.
1. Introduction 22
2. Materials and method 24
2.1 Preparation of MEPL 24
2.2 Cell culture and sample treatment 24
2.3 Cell viability assay 25
2.4 Isolation of total RNA and RT-PCR 25
2.5 Gelatin substrate gel zymography 25
2.6 Matrigel invasion assay 26
2.7 Western blot analysis 26
2.8 Electrophoretic mobility assay (EMSA) 27
2.9 Statistical analysis 27
3. Results
3.1 Effect of MEPL on cell viability 28
3.2 Suppression of MMP-9 gene transcription and its activity by MEPL 29
3.3 Inhibitory effect of MEPL on TNF-α-induced NF-κB activity 31
3.4 Reduction of invasion by MEPL in T24 bladder carcinoma cells 33
4. Discussion 35
5. Conclusion 37
Reference. 38
Degree
Master
Publisher
제주대학교 대학원
Citation
Matharage Gayani Dilshara. (2013). Anti-inflammatory and anti-invasive effects of methanol extract of Polyopes lancifolius by suppression of the NFκB pathway
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General Graduate School > Marine Life Sciences
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