Anti-inflammatory effects of Hwang-Heuk-San, a traditional Korean herbal formulation, on lipopolysaccharide-stimulated murine macrophages

Abstract

Background: Hwang-Heuk-San (HHS), a Korean traditional herbal formula comprising four medicinal herbs, has been used to treat patients with inflammation syndromes and digestive tract cancer for hundreds of years; however, its anti-inflammatory potential is poorly understood. The aim of the present study was to investigate the anti-inflammatory effects of HHS using a lipopolysaccharide (LPS)-activated RAW 264.7 macrophage model.

Methods: The inhibitory effects of HHS on LPS-induced nitric oxide (NO), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) production were examined using Griess reagent and enzyme-linked immunosorbent assay (ELISA) detection kits. The effects of HHS on the expression of inducible NO synthase (iNOS), IL-1β and TNF-α, their upstream signal proteins, including nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and activator protein (AP-1), were also investigated.

Results: A noncytotoxic concentration of HHS significantly reduced the production of NO, IL-1β and TNF-α in LPS-stimulated RAW 264.7 cells, which was correlated with reduced expression of iNOS, IL-1β and TNF-α at the mRNA and protein levels. HHS efficiently blocked the phosphorylation of MAPKs, especially that of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) but not that of the p38 MAPK. The reduced production of inflammatory molecules by HHS was followed by decreased activity of NF-κB and AP-1.

Conclusions: These results suggest that HHS may offer therapeutic potential for treating inflammatory diseases accompanied by macrophage activation.

Fig. 1

Inhibition of NO production and iNOS expression by HHS in LPS-stimulated RAW 264.7 macrophages. The cells were pretreated with various concentrations of HHS for 1 h and then incubated with LPS (100 ng/ml) for 24 h. a The amount of NO production in the medium was measured using the Griess reaction. Statistical significance was determined by a one-way ANOVA (*, p < 0.05 vs. untreated control; ^#, p < 0.05 vs. LPS-treated group). b Total cellular proteins (30 μg) were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and detected with anti-iNOS antibody and an ECL detection system. c After LPS treatment for 6 h, total RNA was prepared for RT-PCR analysis of iNOS gene expression. The amplified PCR products were run in 1 % agarose gel and visualized by EtBr staining. Actin and GAPDH were used as internal controls for the Western blot and RT-PCR assays, respectively

Fig. 2

Inhibition of IL-1β production and its expression by HHS in LPS-stimulated RAW 264.7 macrophages. The cells were pretreated with various concentrations of HHS for 1 h and then incubated with LPS (100 ng/ml) for 24 h. a The amounts of IL-1β were measured in culture media using a commercial ELISA kit. Statistical significance was determined by a one-way ANOVA (*, p < 0.05 vs. untreated control; ^#, p < 0.05 vs. LPS-treated group). b Total cellular proteins (30 μg) were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and detected with anti-IL-1β antibody and an ECL detection system. c After LPS treatment for 6 h, total RNA was prepared for RT-PCR analysis of IL-1β gene expression. The amplified PCR products were run in 1 % agarose gel and visualized by EtBr staining. Actin and GAPDH were used as internal controls for the Western blot and RT-PCR assays, respectively

Fig. 3

Inhibition of TNF-α production and its expression by HHS in LPS-stimulated RAW 264.7 macrophages. The cells were pretreated with various concentrations of HHS for 1 h and then incubated with LPS (100 ng/ml) for 24 h. a The amounts of TNF-α were measured in culture media using a commercial ELISA kit. Statistical significance was determined by a one-way ANOVA (*, p < 0.05 vs. untreated control; ^#, p < 0.05 vs. LPS-treated group). b Total cellular proteins (30 μg) were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and detected with anti-TNF-α antibody and an ECL detection system. c After LPS treatment for 6 h, total RNA was prepared for RT-PCR analysis of TNF-α gene expression. The amplified PCR products were run in 1 % agarose gel and visualized by EtBr staining. Actin and GAPDH were used as internal controls for the Western blot and RT-PCR assays, respectively

Fig. 4

Attenuation of LPS-induced NF-κB activation by HHS in RAW 264.7 macrophages. a The cells were treated with the indicated concentrations of HHS for 1 h before LPS treatment (100 ng/ml) for 30 min. Nuclear and cytosolic proteins were resolved on 10 % SDS-polyacrylamide gels, followed by Western blotting using anti-NF-κB p65 and anti-IκBα antibodies. Lamin B and actin were used as internal controls for the nuclear and cytosolic fractions, respectively. b Cells were pretreated with 300 μg/ml of HHS for 1 h prior to stimulation with LPS (100 ng/ml) for 30 min. Localization of NF-κB p65 was visualized with a fluorescence microscope after immunofluorescence staining with anti-NF-κB p65 antibody and an FITC-labeled anti-rabbit IgG antibody (green). Nuclei of the corresponding cells were visualized with DAPI (blue). c Following treatment with 300 μg/ml of HHS for 1 h, the cells were treated with 100 ng/ml of LPS for 30 min. Nuclear extracts were prepared, and the DNA binding activity of NF-κB was analyzed by an EMSA

Fig. 5

Suppression of LPS-induced AP-1 activation by HHS in RAW 264.7 macrophages. a The cells were treated with 300 μg/ml of HHS for 1 h before LPS treatment (100 ng/ml) for 30 min. Nuclear proteins were resolved on 10 % SDS-polyacrylamide gels, followed by Western blotting using the indicated antibodies. Lamin B was used as an internal control for the nuclear fractions. b In a parallel experiment, nuclear extracts were prepared, and the DNA binding activity of AP-1 was analyzed by an EMSA

Fig. 6

Effects of HHS and LPS on the cell viability of RAW 264.7 macrophages. The cells were treated with the indicated concentrations of HHS alone or pretreated with HHS for 1 h before 100 ng/ml of LPS treatment. After 24 h, the cell viability was assessed using an MTT reduction assay. Data are expressed as the mean ± SD of three independent experiments

Fig. 7

Effect of HHS on LPS-induced phosphorylation of MAPKs in RAW 264.7 macrophages. The cells were pretreated with various concentrations of HHS for 1 h prior to exposure to LPS (100 ng/ml) for 30 min, and total proteins were isolated. The proteins were subjected to SDS-polyacrylamide gels, followed by Western blot analysis using the indicated antibodies and an ECL detection system