Anti-inflammatory effects of guggulsterone on murine macrophage by inhibiting LPS-induced inflammatory cytokines in NF-κB signaling pathway

Abstract

The present study was aimed to investigate the effects of guggulsterone (GS) on proinflammatory responses as well as the underlying molecular mechanisms in macrophage upon lipopolysaccharide (LPS) stimulation. Effects of GS on viability of Raw264.7 cells were examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Real-time polymerase chain reaction (PCR) was employed to examine the mRNA expression of cytokines, including interleukin 1β (IL-1β), tumor necrosis factor-alpha (TNF-α), and inducible nitric oxide synthase (iNOS). Phosphorylations of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinases (p38), and inhibitor of nuclear factor kappaB (IκB) were determined using immunoblotting. The results revealed that GS was not toxic to Raw264.7 cells at designated concentrations. We demonstrated that GS significantly suppressed the elevated mRNA expression of proinflammatory cytokines, including IL-1β, TNF-α, and iNOS in a dose-dependent manner. GS treatment reduced the level of IκB phosphorylation in LPS-stimulated macrophages in a dose-dependent manner. Use of BAY 11-7082, an inhibitor of nuclear factor-kappaB (NF-κB), led to significantly suppressing effects on IL-1β and TNF-α expression similar as that of GS-treated cells. Our findings suggest that GS possesses anti-inflammatory activity, which may be attributed to downregulation of iNOS and inhibition of NF-κB activity in LPS-stimulated Raw264.7 cells.

Keywords: Anti-inflammatory effects; IL-1β; NF-κB; TNF-α; guggulsterone; lipopolysaccharides.

Figure 1

GS suppressed mRNA expression of proinflammatory cytokines IL-1β, TNF-α, and iNOS in LPS-stimulated Raw264.7 cells. Notes: Cells were pretreated with GS at indicated concentrations (1, 2.5, 5, 10, 12.5, and 25 μM) for 2 hours, then cells were challenged with 1 μg/mL LPS for 2 hours. After treatments, the cells were lysed for mRNA extraction and gene expression level was analyzed by real-time PCR. mRNA expression of (A) IL-1β, (B)TNF-α, and (C) iNOS is presented. Data are shown as relative fold change after normalization to GAPDH and expressed as mean ± SD of the three independent experiments. *P<0.05 as compared to LPS alone. **P<0.01 as compared to LPS alone. ***P<0.001 as compared to LPS alone. Abbreviations: GS, guggulsterone; IL-1β, interleukin 1β; TNF-α, tumor necrosis factor-alpha; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; PCR, polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SD, standard deviation.

Figure 2

GS inhibited LPS-induced NF-κB pathway in Raw264.7 cells. Notes: (A) Cells were incubated with GS at indicated concentrations (1, 2.5, 5, 10, 12.5, and 25 μM) for 4 hours and then stimulated with 1 μg/mL LPS for 30 minutes. After treatments, phosphorylation of IκB was demonstrated by immunoblot. Level of GAPDH was used as control. mRNA expression of (B) IL-1β and (C) TNF-α relative to GAPDH is presented. GS and BAY 11-7082 inhibited proinflammatory cytokines mRNA expression in LPS-stimulated Raw264.7 cells through interfering with NF-κB activation. Data are shown as relative fold change after normalization to GAPDH and expressed as mean ± SD of the three independent experiments. *P<0.05 as compared to LPS alone. Abbreviations: GS, guggulsterone; NF-κB, nuclear factor-kappaB; LPS, lipopolysaccharide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IL-1β, interleukin 1β; TNF-α, tumor necrosis factor-alpha; SD, standard deviation.

Figure 3

Effect of GS on phosphorylation of ERK1/2, JNK, and p38 in LPS-stimulated Raw264.7 cells. Notes: Cells were incubated with GS at indicated concentrations (1, 2.5, 5, 10, 12.5, and 25 μM) for 4 hours and then stimulated with 1 μg/mL LPS for 30 minutes. After treatments, the cells were lysed for protein extraction. Phosphorylation of indicated kinases was demonstrated by immunoblot using specific antibodies and chemiluminescence development. Level of GAPDH was used as control. Abbreviations: GS, guggulsterone; ERK, extracellular signal-related kinase; JNK, c-Jun N-terminal kinase; p38, p38 mitogen-activated protein kinase; LPS, lipopolysaccharide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.