3,4,5-Trihydroxycinnamic Acid Inhibits Lipopolysaccharide-Induced Inflammatory Response through the Activation of Nrf2 Pathway in BV2 Microglial Cells

Abstract

3,4,5-Trihydroxycinnamic acid (THC) is a derivative of hydroxycinnamic acids, which have been reported to possess a variety of biological properties such as anti-inflammatory, anti-tumor, and neuroprotective activities. However, biological activity of THC has not been extensively examined. Recently, we reported that THC possesses anti-inflammatory activity in LPS-stimulated BV2 microglial cells. However, its precise mechanism by which THC exerts anti-inflammatory action has not been clearly identified. Therefore, the present study was carried out to understand the anti-inflammatory mechanism of THC in BV2 microglial cells. THC effectively suppressed the LPS-induced induction of pro-inflammatory mediators such as NO, TNF-α, and IL-1β. THC also suppressed expression of MCP-1, which plays a key role in the migration of activated microglia. To understand the underlying mechanism by which THC exerts these anti-inflammatory properties, involvement of Nrf2, which is a cytoprotective transcription factor, was examined. THC resulted in increased phosphorylation of Nrf2 with consequent expression of HO-1 in a concentration-dependent manner. THC-induced phosphorylation of Nrf2 was blocked with SB203580, a p38 MAPK inhibitor, indicating that p38 MAPK is the responsible kinase for the phosphorylation of Nrf2. Taken together, the present study for the first time demonstrates that THC exerts anti-inflammatory properties through the activation of Nrf2 in BV2 microglial cells, suggesting that THC might be a valuable therapeutic adjuvant for the treatment of inflammation-related disorders in the CNS.

Keywords: 3; 4; 5-trihydroxycinnamic acid; Heme oxygenase-1; Neuro-inflammation; Nrf2.

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

Fig. 2.. Inhibitory effects of THC on LPS-induced inflammatory mediators in BV2 microglial cells. Cells were stimulated with LPS in presence or absence of THC 24 hr. (A) The amount of nitrite in culture medium was measured by using the Griess reagents. The level of TNF-α (B) and IL-1β (C) mRNA were determined by real-time PCR. THC significantly suppressed expression of TNF-α and IL-1β. Data from triplicate determination are shown (mean ± SD). *p<0.05 and **p<0.01 indicate statistically significant differences from treatment with LPS alone.

Fig. 3.. Effect of THC on MCP-1 protein and mRNA expression in LPS-stimulated BV2 microglia cells. BV2 microglial cells were pretreated with THC (10, 50 and 100 μM) for 1 hr and then treated with LPS (200 ng/ml). (A) Cell lysates were subjected to SDS-PAGE, and then protein levels of MCP-1 were determined by Western blot analysis (top). Quantitative analysis was performed by densitometric analysis (bottom). (B) After 6 hr of LPS stimulation, representative image (top) and quantitative analysis (bottom) of MCP-1 mRNA expression were obtained using RT-PCR. THC significantly inhibited LPS-induced MCP-1 expression in both mRNA and protein levels. β-Actin was used as an internal control. Images are representative of three independent experiments that shows similar results. *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.. Effects of THC on Nrf2 activation and HO-1 expression. (A&B) Phosphorylation and nuclear translocation of Nrf2. BV2 microglia cells were treated with THC (10-100 μM), and then Western blot analysis was performed. Total level of phosphorylated Nrf2 was determined (A) and nuclear translocation of Nrf2 was examined in nuclear and cytosol fractions (B). (C, D) HO-1 protein and mRNA expression. HO-1 protein level (C) was determined by immunoblotting after THC treatment. After 6 hr of THC treatment, the total RNA was isolated, HO-1 mRNA level (D) was determined by RT-PCR. β-Actin was used as an internal control. Quantitative analysis was carried out using densitometric analysis. Images are representative of three independent experiments that shows similar results. *p<0.05 and **p<0.01 indicate statistically significant differences from treatment with LPS alone.

Fig. 5.. THC-induced Nrf2 phosphorylation was mediated by p-38 MAPK. BV2 microglial cells were stimulated with 200 ng/ml LPS in the absence or presence of THC. (A) Western blot analysis was then performed to evaluate the activation of MAP kinases signaling pathways (top: representative image, bottom: quantitative analysis). Phosphorylation of p38 was increased in a concentration-dependent manner, but phosphorylation of ERK and JNK was decreased with THC treatment, suggesting that p-38 might play an important role in the THC-induced Nrf2 phosphorylation. (B) BV2 microglia cells were pretreated with the p38 inhibitor, SB203580 (5 μM), for 1 hr prior to THC treatment. THC-induced Nrf2 phosphorylation was blocked via p38 inhibition (top: representative image, bottom: quantitative analysis), indicating that p38 is responsible for the THC-induced phosphorylation of Nrf2. β-Actin was used as an internal control. Images are representative of three independent experiments that shows similar results. Quantitative analysis was carried out using densitometric analysis. *p<0.05 and **p<0.01 indicate statistically significant differences from treatment with LPS alone.