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

Figures & data

Table 1 List of RT-PCR primers

Gene	Forward primer	Reverse primer
GAPDH	5′-TGACCACAGTCCATGCCATC-3′	5′-GACGGACACATTGGGGGTAG-3′
IL-1β	5′-GCAACTGTTCCTGAACTCAACT-3′	5′-ATCTTTTGGGGTCCGTCAACT-3′
TNF-α	5′-GACGTGGAACTGGCAGAAGAG-3′	5′-TTGGTGGTTTGTGAGTGTGAG-3′
iNOS	5′-GTTCTCAGCCCAACAATACAAGA-3′	5′-GTGGACGGGTCGATGTCAC-3′

Abbreviations: RT-PCR, reverse transcription polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IL-1β, interleukin 1β; TNF-α, tumor necrosis factor-alpha; iNOS, inducible nitric oxide synthase.

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.