Anti-inflammatory Effects of Curcumin in Microglial Cells

Abstract

Lipoteichoic acid (LTA) induces neuroinflammatory molecules, contributing to the pathogenesis of neurodegenerative diseases. Therefore, suppression of neuroinflammatory molecules could be developed as a therapeutic method. Although previous data supports an immune-modulating effect of curcumin, the underlying signaling pathways are largely unidentified. Here, we investigated curcumin's anti-neuroinflammatory properties in LTA-stimulated BV-2 microglial cells. Inflammatory cytokine tumor necrosis factor-α [TNF-α, prostaglandin E2 (PGE2), and Nitric Oxide (NO] secretion in LTA-induced microglial cells were inhibited by curcumin. Curcumin also inhibited LTA-induced inducible NO synthases (iNOS) and cyclooxygenase-2 (COX-2) expression. Subsequently, our mechanistic studies revealed that curcumin inhibited LTA-induced phosphorylation of mitogen-activated protein kinase (MAPK) including ERK, p38, Akt and translocation of NF-κB. Furthermore, curcumin induced hemeoxygenase (HO)-1HO-1 and nuclear factor erythroid 2-related factor 2 (Nrf-2) expression in microglial cells. Inhibition of HO-1 reversed the inhibition effect of HO-1 on inflammatory mediators release in LTA-stimulated microglial cells. Taken together, our results suggest that curcumin could be a potential therapeutic agent for the treatment of neurodegenerative disorders via suppressing neuroinflammatory responses.

Keywords: HO-1; TLR2; curcumin; microglial cells; neuroinflammation.

Figures

FIGURE 1

Effect of curcumin on BV-2 microglial cell viability. (A) BV2 cells were treated with various concentrations of curcumin (5, 10, and 20 μM) for 24 h. (B) BV2 cells were preincubated with curcumin (5, 10, and 20 μM) for 1 h, and then exposed to LTA (5 μg/ml) for 24 h. Cell viability was measured by MTT assay. Statistical significance was determined by one-way ANOVA. All data were mean ±SD of three experiments.

FIGURE 2

Curcumin inhibited neuroinflammatory mediators release from LTA-stimulated BV-2 microglial cells. Cells were treated with different concentrations of curcumin (5, 10, and 20 μM) for 1 h, then incubated with LTA (5 mg/ml) under serum-free conditions. (A) After 16 h of stimulation, nitrite content was measured using the Griess reaction. (B,C) The concentration of PGE2 and TNF-α, in the culture media was measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit. (D,E) Cells were treated with different concentrations of curcumin (5, 10, and 20 μM) for 1 h then incubated with LTA (5 μg/ml) under serum-free conditions. After 4 h of stimulation, the mRNA expression levels of iNOS and COX-2 were determined by qRT-PCR. Statistical significance was determined by one-way ANOVA. Each bar represented the mean (SD) from three independent experiments per group. #P < 0.01 vs. negative control, ∗P < 0.05, ∗∗P < 0.01 vs. the LTA-treated control.

FIGURE 3

Inhibitory effects of curcumin on LTA-induced activation of NF-κB in BV2 cells. BV-2 microglial cells were treated with curcumin followed by LTA (5 μg/ml) treatment for 0.5 h. Nuclear translocation of (NF-κB) p65 was confirmed by western blotting. The cytosolic extracts were analyzed by western blotting with anti-IκB-α and anti-p-IκB-α antibodies. For western blot detection of TBP, α-tubulin was used as a protein-loading control for each lane.

FIGURE 4

Curcumin inhibited LTA-induced phosphorylation of p38, ERK, and Akt in BV-2 microglial cells. (A) BV-2 microglial cells were treated with the indicated concentrations of curcumin for 1 h and then stimulated with LTA (5 μg/ml) for 1 h. An equal amount of cell extract was analyzed by western blotting with anti-p-ERK1/2, anti-p-c-Jun N-terminal kinase (JNK), anti-p-p38, and anti-p-Akt antibodies. ERK1/2, JNK, p38 and Akt bands indicated that the induction of total ERK1/2, JNK, p38, and Akt protein was not changed. BV-2 cells were treated with JNK inhibitor (JNK II, 10 mM), Akt inhibitor (Wor, 5 mM), ERK inhibitor (PD98059, 10 μM), or p38 inhibitor (SB230580, 10 μM) for 1 h, following treatment with LTA for 16 h. (B) Subsequently, the levels of NO production were determined. (C) The expression level of iNOS was also determined by qRT-PCR. Statistical significance was determined by Student’s t-test. Each bar represents the mean (SD) from three independent experiments per group. #P < 0.01 vs. negative control, ∗P < 0.05, and ∗∗P < 0.01 vs. the LTA-treated group.

FIGURE 5

Effects of HO-1 on curcumin-mediated anti-neuroinflammatory effects in LTA-stimulated microglial cells. (A,B) Cells were cultured with increasing concentrations of curcumin for 4 h or 20 μM of curcumin for the indicated times. mRNA expression level of HO-1 was determined by qRT-PCR. (C,D) Cells were cultured with increasing concentrations of curcumin for 8 h or 20 μM of curcumin for the indicated times. HO-1 protein expression was determined by western blot. (E,F) Cells were incubated with 20 μM curcumin for the indicated time or were incubated with the indicated concentration of curcumin for 1 h. Nuclear localization of Nrf2 was determined by western blot. TBP was used as a protein loading control for each lane. (G,H) The cells were incubated with curcumin for 1 h and then exposed to LTA with or without the HO-1 inhibitor SnPP (20 μM, HO-1 inhibitor) for 16 h. The secretion of NO and TNF-α were determined. Statistical significance was determined by Student’s t-test. Each bar represents the mean (SD) from three independent experiments per group. #P < 0.01 vs. negative control, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. the LTA-treated group.

FIGURE 6

Anti-inflammatory mechanism of curcumin in LTA-stimulated microglial cells. Curcumin had anti-inflammatory activity in LTA-stimulated microglial cells through inhibiting NF-κB and p38 MAPK activation, and induced the expression of Nrf2 and HO-1.