Anti-Inflammatory Effects and Mechanisms of Action of Coussaric and Betulinic Acids Isolated from Diospyros kaki in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages

Abstract

Diospyros kaki Thunb. is widely distributed in East Asian countries, its leaves being mainly used for making tea. In this study, coussaric acid (CA) and betulinic acid (BA), both triterpenoid compounds, were obtained from D. kaki leaf extracts through bioassay-guided isolation. CA and BA showed anti-inflammatory effects via inhibition of the nuclear factor-κB (NF-κB) pathway, providing important information on their anti-inflammatory mechanism. Furthermore, they markedly inhibited nitric oxide (NO) and prostaglandin E₂ (PGE₂) production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages, and suppressed tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) levels. Furthermore, they decreased protein expression of inducible nitric oxide synthase and cyclooxygenase-2. Pre-treatment with CA and BA inhibited LPS-induced NF-κB. We further examined the effects of CA and BA on heme oxygenase (HO)-1 expression in RAW 264.7 macrophages: BA induced HO-1 protein expression in a dose-dependent manner, while CA had no effect. We also investigated whether BA treatment induced nuclear translocation of Nrf2. BA inhibited LPS-induced NF-κB-binding activity, as well as pro-inflammatory mediator and cytokine production (e.g., NO, PGE₂, TNF-α, IL-1β, IL-6), by partial reversal of this effect by SnPP, an inhibitor of HO-1. These findings further elucidate the anti-inflammatory mechanism of CA and BA isolated from D. kaki.

Keywords: Diospyros kaki Thunb.; anti-inflammation; betulinic acid (BA); coussaric acid (CA); heme oxygenase-1; nuclear factor-kappa B.

Figure 1

Structures of (A) coussaric acid (CA) and (B) betulinic acid (BA).

Figure 2

Effects of (A) CA and (B) BA on cell viability of RAW 264.7 macrophages stimulated with LPS. (A,B) Cells were incubated for 24 h with the indicated concentrations of CA and BA. Cell viability was determined as described in the Materials and Methods. Data shown represent the mean values of three experiments ±SD.

Figure 3

Effects of CA and BA on the production of (A) nitrite and (B) PGE₂ of RAW 264.7 macrophages stimulated with LPS. (A,B) The cells were pre-treated with indicated concentrations of CA and BA for 12 h, and then stimulated with LPS (1 μg/mL) for 18 h. The production of nitrite and PGE₂ was determined as described in the Materials and Methods. Data shown represent the mean values of three experiments ±SD. * p < 0.05 as compared to the group treated with LPS alone.

Figure 4

Effects of CA and BA on the production of (A,D) TNF-α; (B,E) IL-6; and (C,F) IL-1β in RAW 264.7 macrophages stimulated with LPS. (A–F) Cells were pre-treated with indicated concentrations of CA and BA for 3 h, and then stimulated with LPS (1 μg/mL) for 24 h. Production of TNF-α, IL-1β, and IL-6 was measured as described in the Materials and Methods. Data shown represent the mean values of three experiments ±SD. * p < 0.05 as compared with the group treated with LPS alone.

Figure 5

Effects of CA and BA on (A) iNOS and (B) COX-2 protein expression in RAW 264.7 macrophages stimulated with LPS. (A,B) Cells were pre-treated with indicated concentrations of CA and BA for 3 h, and then stimulated with LPS (1 μg/mL) for 24 h. Western blot analysis was performed as described in the Materials and Methods, and representative blots from three independent experiments that showed similar results were chosen. Data shown represent the mean values of three experiments ± SD. * p < 0.05 as compared with the group treated with LPS alone.

Figure 6

Effects of CA and BA on (A,B) NF-κB activation. (A,B) Cells were pre-treated with the indicated concentrations of CA and BA for 3 h, and then stimulated with LPS (1 μg/mL) for 30 min. Western blot analysis was performed as described in the Materials and Methods, and representative blots from three independent experiments that showed similar results were chosen. * p < 0.05 as compared with the control group. # p < 0.05 as compared with the group treated with LPS alone.

Figure 6

Effects of CA and BA on (A,B) NF-κB activation. (A,B) Cells were pre-treated with the indicated concentrations of CA and BA for 3 h, and then stimulated with LPS (1 μg/mL) for 30 min. Western blot analysis was performed as described in the Materials and Methods, and representative blots from three independent experiments that showed similar results were chosen. * p < 0.05 as compared with the control group. # p < 0.05 as compared with the group treated with LPS alone.

Figure 7

Effects of CA and BA on (A,B) HO-1 expression in RAW 264.7 macrophages. (A,B) Cells were incubated for 12 h with the indicated concentrations of CA, BA, and CoPP (20 μM), a HO-1 Inducer, was used as the positive control. Western blot analysis was performed as described in the Materials and Methods, and representative blots from three independent experiments that showed similar results were chosen. Data shown represent the mean values of three experiments ± SD. * p < 0.05 as compared with the control.

Figure 8

Effects of BA on the nuclear translocation of (A) Nrf2 and (B) Nrf2-mediated HO-1 in RAW 264.7 macrophages. (A) Cells were treated for the indicated periods with 10 μM BA. Nuclei were fractionated from the cytosol using PER-Mammalian Protein Extraction buffer, as described in the materials and methods; (B) RAW 264.7 macrophages were transiently transfected with Nrf2 siRNA and then treated with 10 μM BA for 12 h. Transfection and western blot analysis was performed as described in the Materials and Methods. Data shown represent the mean values of three experiments ±SD. * p < 0.05 as compared with the control.

Figure 9

Effects of SnPP on BA-mediated inhibition of (A–F) NF-κB activation and nitrite, PGE₂, TNF-α, IL-1β, and IL-6 production in LPS-stimulated RAW 264.7 macrophages. Cells were pre-treated with BA for 3 h in the presence or absence of SnPP (50 μM), and then stimulated with LPS (1 μg/mL) for (A) 30 min or (B–F) 24 h. Production of nitrite, PGE₂, TNF-α, IL-1β, and IL-6 and the degree of NF-κB binding were determined as described in Materials and Methods. Data shown represent the mean values of three experiments ± SD. * p < 0.05 compared with the group treated with LPS alone; # p < 0.05 compared with the group treated with BA and LPS.

Figure 9

Effects of SnPP on BA-mediated inhibition of (A–F) NF-κB activation and nitrite, PGE₂, TNF-α, IL-1β, and IL-6 production in LPS-stimulated RAW 264.7 macrophages. Cells were pre-treated with BA for 3 h in the presence or absence of SnPP (50 μM), and then stimulated with LPS (1 μg/mL) for (A) 30 min or (B–F) 24 h. Production of nitrite, PGE₂, TNF-α, IL-1β, and IL-6 and the degree of NF-κB binding were determined as described in Materials and Methods. Data shown represent the mean values of three experiments ± SD. * p < 0.05 compared with the group treated with LPS alone; # p < 0.05 compared with the group treated with BA and LPS.