Anti-inflammatory Activity of 1-docosanoyl Cafferate Isolated from Rhus verniciflua in LPS-stimulated BV2 Microglial Cells

Jae-Won Lee; Il-Young Cheong; Hae-Sung Kim; Jae Jun Lee; Yong-Suk Lee; Yong-Soo Kwon; Myong-Jo Kim; Hee Jae Lee; Sung-Soo Kim; Wanjoo Chun

doi:10.4196/kjpp.2011.15.1.9

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Lee, Cheong, Kim, Lee, Lee, Kwon, Kim, Lee, Kim, and Chun: Anti-inflammatory Activity of 1-docosanoyl Cafferate Isolated from Rhus verniciflua in LPS-stimulated BV2 Microglial Cells

Original Article

Korean J Physiol Pharmacol 2011;15(1):9-15.

Published online: 18 February 2011

DOI: https://doi.org/10.4196/kjpp.2011.15.1.9

Anti-inflammatory Activity of 1-docosanoyl Cafferate Isolated from Rhus verniciflua in LPS-stimulated BV2 Microglial Cells

Jae-Won Lee^1,^#, Il-Young Cheong^2,^#, Hae-Sung Kim¹, Jae Jun Lee¹, Yong-Suk Lee^2,⁵, Yong-Soo Kwon³, Myong-Jo Kim⁴, Hee Jae Lee¹, Sung-Soo Kim¹, Wanjoo Chun^1,

¹Department of Pharmacology, Kangwon National University, Chuncheon 200-701, Korea

²Department of Anesthesiology, College of Medicine, Kangwon National University, Chuncheon 200-701, Korea

³Department of College of Pharmacy, Kangwon National University, Chuncheon 200-701, Korea

⁴Division of Bio-resources Technology, Kangwon National University, Chuncheon 200-701, Korea

⁵Korea Institute of Radiological and Medical Sciences, Seoul 139-706, Korea

Corresponding to: Wanjoo Chun, Department of Pharmacology, College of Medicine, Kangwon National University, Hyoja 2-dong, Chuncheon 200-701, Korea. (Tel) 82-33-250-8853, (Fax) 82-33-255-8809, (E-mail) wchun@kangwon.ac.kr

^# These authors contributed equally to this work and should be considered co-first authors.

Received 19 November 2010 Revised 17 January 2011 Accepted 17 January 2011

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Although various derivatives of caffeic acid have been reported to possess a wide variety of biological activities such as protection of neuronal cells against excitotoxicity, the biological activity of 1-docosanoyl cafferate (DC) has not been examined. The objective of the present study was to evaluate the anti-inflammatory effects of DC, isolated from the stem bark of Rhus verniciflua, on lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Pretreatment of cells with DC significantly attenuated LPS-induced NO production, and mRNA and protein expression of iNOS in a concentration-dependent manner. DC also significantly suppressed LPS-induced release of cytokines such as TNF-α and IL-1β. Consistent with the decrease in cytokine release, DC dose-dependently and significantly attenuated LPS-induced mRNA expression of these cytokines. Furthermore, DC significantly suppressed LPS-induced degradation of IKB, which retains NF-kB in the cytoplasm. Therefore, nuclear translocation of NF-kB induced by LPS stimulation was significantly suppressed with DC pretreatment. Taken together, the present study suggests that DC exerts its anti-inflammatory activity through the suppression of NF-kB translocation to the nucleus.

Keywords: 1-Docosanoyl cafferate (DC), BV2 microglial cells, Lipopolysaccharide, Cytokines, iNOS, NF-kB

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Fig. 1.

Chemical structure of 1-docosanoyl cafferate (DC).

Fig. 2.

Effects of DC on NO production and iNOS expression in LPS-stimulated BV2 microglial cells. (A) Effect of DC on the viability of BV2 microglial cells. No noticeable cell death was observed up to 20 μM. (B) Concentration-dependent suppression of LPS-induced NO production by DC and CA (caffeic acid). (C) Inhibitory effect of DC on LPS-induced upregulation of iNOS mRNA expression. (D) Suppression of LPS-induced iNOS protein expression by DC: top, quantitative analysis of immunoblots; bottom, representative immunoblot of iNOS. β-Actin was used as an internal control. Quantitative data represent three independent experiments and are expressed as mean±SD. ^∗p<0.05, ^∗∗p<0.01, and ^∗∗∗p< 0.001 indicate statistically significant differences compared to LPS alone. ^#p<0.05 indicate statistically significant differences between the indicated groups.

Fig. 3.

Inhibitory effects of DC on LPS-induced release of TNF-α (A) and IL-1β (B) in BV2 microglial cells. BV2 microglia cells were incubated with 200 ng/ml of LPS in the presence or absence of the indicated concentrations of DC for 24 hr. Cell culture media were collected and subjected to TNF-α and IL-1β sandwich ELISAs. Data represent three independent experiments, each run in triplicate, and are expressed as mean±SD. ^∗p<0.05 and ^∗∗p<0.01 indicate statistically significant differences compared to LPS alone. ^##p<0.01 indicates a statistically significant difference between the indicated groups.

Fig. 4.

Effects of DC on the gene expression of TNF-α (A) and IL-1β (B) in LPS-stimulated BV2 microglial cells. Cells were incubated with DC for 1 hr prior to exposure to 200 ng/ml LPS. Total RNA was isolated 6 hr after LPS treatment. TNF-α and IL-1β mRNA levels were determined by real time PCR. Data represent three independent experiments, each done in triplicate, and are expressed as mean±SD. ^∗p<0.05 indicates a statistically significant difference compared to LPS alone.

Fig. 5.

Inhibitory effects of DC on LPS-induced IkB-α degradation in BV2 microglial cells. The intracellular level of IKB-α was determined using immunoblotting analysis: top, quantitative analysis of immunoblots; bottom, representative immunoblot of IKB-α. β-Actin was used as an internal control. Quantitative data represent three independent experiments and are expressed as mean±SD. ^∗p <0.05 and ^∗∗p<0.01 indicate statistically significant differences compared to LPS alone. ^##p<0.01 indicates a statistically significant difference between the indicated groups.

Fig. 6.

Blockade by DC of nuclear translocation of the p65 subunit of NF-kB in LPS-stimulated BV2 microglial cells. (A) Localization of the NF-kB p65 subunit was determined using a p65 antibody and an Alexa 546-labeled goat anti-rabbit IgG antibody. Nuclei were visualized by Hoechst staining (Hoechst 33258). In basal conditions, immunostaining of p65 subunit was diffuse throughout the cytoplasm. LPS stimulation resulted in the translocation of p65 subunits into the nucleus. Pretreatment with DC attenuates LPS-induced nuclear translocation of the p65 subunit. (B) Line scanning analysis of confocal images further visualizes the intracellular localization of the p65. Scale bar, 20μm.

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