Anti-Inflammatory Effect of Garcinol Extracted from Garcinia dulcis via Modulating NF-κB Signaling Pathway

Abstract

Garcinia is a significant medicinal plant with many beneficial phytoconstituents, including garcinol. This study investigated the anti-inflammatory effect of garcinol isolated from Garcinia dulcis fruit in LPS-activated THP-1 and Raw 264.7 macrophages. The results demonstrated that the low concentration of garcinol did not alter cell viability. Furthermore, co-incubation of garcinol with LPS inhibited the production of pro-inflammatory cytokines, including TNF-α, IL-8, IL-6, IL-1β, and pro-inflammatory mediators, including iNOS and COX-2 at the mRNA and protein expression levels. Garcinol also decreased the secretion of TNF-α, IL-6, IL-1β, PGE2, and NO. Moreover, the anti-inflammatory effects involved an alteration in the NF-κB signaling pathway. Downregulation of pIKKα/β, pIκBα, and pNF-κB was observed, hence reducing the translocation of pNF-κB from the cytosol into the nucleus, which subsequently decreased the production of pro-inflammatory molecules. Therefore, garcinol isolated from Garcinia dulcis is a potential candidate as an anti-inflammatory agent for inflammation-related disease treatment.

Keywords: Garcinia dulcis; NF-κB; RAW 264.7; THP-1; garcinol; inflammation.

Figure 1

Chemical structure of garcinol (A) and Garcinia dulcis fruit (B).

Figure 2

The effect of various concentrations of garcinol on the cell viability of THP-1 cells and RAW264.7 was determined (A). The cell viability of another experiment that performed the co-incubation of garcinol or 5 µM of dexamethasone (dexa) with LPS (500 ng/mL) was evaluated (B). The results of each group are presented as the relative expression to control. The data are presented as mean ± SD from six replicates examination in three independent experiments. For statistical analyses, One-way ANOVA followed by Dunnett’s test was used. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Effects of garcinol (10, 20, and 30 µM) on the mRNA expression level of a pro-inflammatory-related molecule, including TNF-α, COX-2, iNOS, IL-8, IL-6, and IL -1β in THP-1 cells (A,B) and RAW 264.7 cells (C,D) activated with LPS 500 ng/mL. 5 µM of dexamethasone (dexa) was used as a positive control. The gene expression was determined by using qRT-PCR. For normalization, the GAPDH gene was used. The results of qRT-PCR of mentioned molecules are presented as a relative fold change of the control. The data are presented in three independent experiments as the mean ± SD of triplicate examination. For statistical analyses, One-way ANOVA and Dunnett’s test were used. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

Effects of garcinol (10, 20, and 30 µM) on the protein expression level of a pro-inflammatory-related molecule including TNF-α, iNOS, COX-2, IL-8 IL-6, and IL-1β in THP-1 cells (A–C) and RAW264.7 cells (D–F) activated with 500 ng/mL of LPS. Western blot analysis was used to determine the protein expression, and β-actin was used for normalization. The data are presented in the three independent experiments’ mean ± SD of triplicate examination (n = 9). For statistical analyses, One-way ANOVA and Dunnett’s test were used. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 5

Effects of garcinol (10, 20, and 30 µM) on the secretion of pro-inflammatory cytokines composed of TNF-α, IL-6, and IL-1β were determined in THP-1 cells (A) and RAW264.7 cells (B) activated with LPS. The supernatants from the culture media of each group were determined for the secretion level of each cytokine using a sandwich ELISA test kit for each cytokine. The data are presented as the mean ± SD of duplicate examination in three independent experiments (n = 6). For statistical analyses, One-way ANOVA and Dunnett’s test were used. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 6

Effects of garcinol (10, 20, and 30 µM) on the expression of related-molecule in the NF-κB signaling pathway in LPS-treated THP-1 cells (A–C) and RAW264.7 cells (D–F) were determined by using western blot analysis. β-actin was used for normalization. The data are presented as the mean ± SD of triplicate examination in three independent experiments (n = 9). For statistical analyses, One-way ANOVA and Dunnett’s test were used. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 7

Effects of garcinol (10, 20, and 30 µM) on NO (A) and PGE2 (B) production were determined in LPS-activated THP-1 cells and RAW264.7 cells. The supernatant from the culture media of each group was determined for the NO and PGE2 secretion level by using a Griess test and ELISA, respectively. The data are presented as the mean ± SD of six replicates, examining three independent experiments. For statistical analyses, One-way ANOVA and Dunnett’s test were used. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 8

Proposed model for the anti-inflammatory activity of garcinol in macrophages. Black lines represent the standard response for LPS activation; red lines represent garcinol activity found in this present study; and red dashed lines represent the hypothesized activities of garcinol.