Korean J Physiol Pharmacol.  2012 Apr;16(2):107-112. 10.4196/kjpp.2012.16.2.107.

3,4,5-Trihydroxycinnamic Acid Inhibits LPS-Induced iNOS Expression by Suppressing NF-kappaB Activation in BV2 Microglial Cells

• Lee JW
• Bae CJ
• Choi YJ
• Kim SI
• Kim NH
• Lee HJ
• Kim SS
• Kwon YS
• Chun W

• KMID: 2071755
• DOI: http://doi.org/10.4196/kjpp.2012.16.2.107

Abstract

Although various derivatives of caffeic acid have been reported to possess a wide variety of biological activities such as neuronal protection against excitotoxicity and anti-inflammatory property, the biological activity of 3,4,5-trihydroxycinnamic acid (THC), a derivative of hydroxycinnamic acids, has not been clearly examined. The objective of the present study is to evaluate the anti-inflammatory effects of THC on lipopolysaccharide (LPS)-stimulated BV2 microglial cells. THC significantly suppressed LPS-induced excessive production of nitric oxide (NO) and expression of iNOS, which is responsible for the production of iNOS. THC also suppressed LPS-induced overproduction of pro-inflammatory cytokines such as IL-1beta and TNF-alpha in BV2 microgilal cells. Furthermore, THC significantly suppressed LPS-induced degradation of IkappaB, which retains NF-kappaB in the cytoplasm. Therefore, THC attenuated nuclear translocation of NF-kappaB, a major pro-inflammatory transcription factor. Taken together, the present study for the first time demonstrates that THC exhibits anti-inflammatory activity through the suppression of NF-kappaB transcriptional activation in LPS-stimulated BV2 microglial cells.

Keyword

3,4,5-Trihydroxycinnamic acid (THC); BV2 microglial cells; Lipopolysaccharide; iNOS; NF-kappaB

MeSH Terms

Caffeic Acids
Coumaric Acids
Cytokines
Cytoplasm
Neurons
NF-kappa B
Nitric Oxide
Tetrahydrocannabinol
Transcription Factors
Transcriptional Activation
Tumor Necrosis Factor-alpha
Caffeic Acids
Coumaric Acids
Cytokines
NF-kappa B
Nitric Oxide
Tetrahydrocannabinol
Transcription Factors
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1 Chemical structure of 3,4,5-Trihydroxycinnamic acid (THC).

• 

  Fig. 2 THC inhibited NO production (A) and expression levels of iNOS protein (B) and mRNA (C) in LPS-stimulated BV2 microglial cells. (A) BV2 cells were pretreated with various concentrations of THC for 1 hr before incubation with LPS (200 ng/ml) for 24 hr. The amounts of nitrite in the supernatants were measured using Griess reagent. THC exhibited a significant suppression of LPS- induced NO production in a concentration-dependent manner. (B) The cell lysates were subjected to SDS-PAGE, and then protein levels of iNOS were determined by Western blot analysis. (C) After LPS treatment for 6 hr, total mRNA levels of iNOS were examined by RT-PCR. The β-actin was used as an internal control. Quantification of iNOS production was performed by densitometric analysis (lower). THC significantly suppressed LPS-induced iNOS expression in a concentration-dependent manner. The data are expressed as mean±S.D. (n=3), and are representative of three or more independent experiments. *p<0.05, **p<0.01 and ***p<0.001 indicate statistically significant differences from treatment with LPS alone.

• 

  Fig. 3 THC inhibited gene expression (A) and release (B) of TNF-α and IL-1β in LPS-stimulated BV2 microglial cells. (A) BV2 microglia cells were pretreated with the indicated concentrations of THC for 1 hr before LPS treatment (200 ng/ml), and total RNA was isolated at 6 hr after LPS treatment and mRNA levels were determined by RT-PCR analysis. THC significantly suppressed expression of TNF-α and IL-1β. Data from triplicate determination are shown (mean±SD). (B) Cell culture media were collected and subjected to TNF-α and IL-1β ELISA. Data represent three independent experiments in triplicate and are expressed as mean±SD. *p<0.05 and **p<0.01 indicate statistically significant differences from treatment with LPS alone. ##p<0.01 indicates statistically significant differences between indicated groups.

• 

  Fig. 4 Inhibitory effects of THC on LPS-induced IκB-α degration in BV2 microlial cells. Total cell lysates obtained 15 min after the LPS stimulation were subjected to Western blotting to assess the levels of IκB-α degradation (top). Quantification of IκB-α degradation was performed by densitometric analysis (lower). The β-actin was used as an internal control. Data from triplicate determination are shown (mean±S.D.). *p<0.05 and **p<0.01 indicate statistically significant differences from treatment with LPS alone.

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  Fig. 5 THC suppressed the nuclear localization of NF-κB in LPS-stimulated BV2 microglial cells. (A) BV2 microglia cells were pretreated with THC for 1 hr prior to stimulation with 200 ng/ml LPS for 1 hr. Localization of NF-κB p65 subunit was determined using an anti-p65 antibody and an Alexa 546-labeled goat anti-rabbit IgG antibody. Nuclei were visualized by Hoechst staining. Cells were visualized using confocal scanning microscopy. Scale bar=20 µm. (B) NF-κB localization was further examined by line scannin.

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