Ganoderic acid C1 isolated from the anti-asthma formula, ASHMI™ suppresses TNF-α production by mouse macrophages and peripheral blood mononuclear cells from asthma patients

Abstract

Asthma is a heterogeneous airway inflammatory disease, which is associated with Th2 cytokine-driven inflammation and non-Th2, TNF-α mediated inflammation. Unlike Th2 mediated inflammation, TNF-α mediated asthma inflammation is generally insensitive to inhaled corticosteroids (ICS). ASHMITM, aqueous extract of three medicinal herbs-Ganoderma lucidum (G. lucidum), Sophora flavescens Ait (S. flavescens) and Glycyrrhiza uralensis Fischer (G. uralensis), showed a high safety profile and was clinically beneficial in asthma patients. It also suppresses both Th2 and TNF-α associated inflammation in murine asthma models. We previously determined that G. uralensis flavonoids are the key active compounds responsible for ASHMITM suppression of Th2 mediated inflammation. Until now, there are limited studies on anti-TNF-α compounds presented in ASHMITM. The objective of this study was to isolate and identify TNF-α inhibitory compounds in ASHMITM. Here we report that G. lucidum, but not the other two herbal extracts, S. flavescens or G. uralensis inhibited TNF-α production by murine macrophages; and that the methylene chloride (MC)-triterpenoid-enriched fraction, but not the polysaccharide-enriched fraction, contained the inhibitory compounds. Of the 15 triterpenoids isolated from the MC fraction, only ganoderic acid C1 (GAC1) significantly reduced TNF-α production by murine macrophages (RAW 264.7 cells) and peripheral blood mononuclear cells (PBMCs) from asthma patients. Inhibition was associated with down-regulation of NF-κB expression, and partial suppression of MAPK and AP-1 signaling pathways. Ganoderic acid C1 may have potential for treating TNF-α mediated inflammation in asthma and other inflammatory diseases.

Keywords: ASHMITM; Asthma; Ganoderic acid C1; Ganoderma lucidum; Inflammation; Macrophages; PBMCs; Peripheral blood mononuclear cells; RAW 264.7 cells; TNF-α; Traditional Chinese medicines.

Fig. 1. Effect of ASHMI^TM and extracts of its constituent herbs on TNF-α production

(A) RAW 264.7 cells were cultured with or without extracts for 24 hours, and then cultured in the presence of 1 μg/mL LPS for 24 hours. TNF-α levels in cell culture supernatants were determined by ELISA. Data are expressed as mean ± S.D. of three independent experiments. #, p<0.05 vs medium alone culture; **p<0.01 vs. LPS stimulated, but untreated (LPS alone) culture. (B) Percent inhibition was calculated in relation to LPS stimulated untreated cells.

Fig. 2. Effect of 15 compounds isolated from G. lucidum on TNF-α production

(A). RAW 264.7 cells were pre-incubated with or without isolated G.lucidum triterpenoids (10 and 20μg/mL) for 24 hours, followed by addition of LPS (1 μg/mL) and culture for additional 24 hours. TNF-α levels in cell culture supernatants were measured by ELISA. Data are expressed as percent inhibition compared with LPS only group. (B). Concentration dependent GAC₁ inhibition of TNF-α production by LPS-stimulated RAW 264.7 cells. (C). Cell viability of GAC₁-treated and LPS-stimulated macrophages. Data are expressed as mean ± S.D. of 3–6 independent experiments. **p<0.01,*p<0.05 Vs LPS alone. (D). Cells were exposed to 0, 10, 20, 40 μg/mL GAC₁ for 24 hours, followed by addition of LPS (1.0 μg/mL) and cultured for an additional 24 hours. Cells were then collected and subjected to Annexin V-FITC/PI staining and analysis by flow cytometry.

Fig. 3. GAC₁ inhibited TNF-α production when simultaneously co-incubated with LPS

(A). RAW 264.7 cells were treated by simultaneous co-incubation with various concentrations of GAC₁ and LPS (1.0 μg/mL) for 24 hours. TNF-α levels in cell culture supernatants were determined by ELISA. Data are expressed as mean ± S.D. of 3–6 independent experiments. **p<0.01,*p<0.05 vs medium and LPS control groups. (B). RAW264.7 cells were treated with 0, 2.5, 5, 10, 20 and 40 μg/mL GAC₁for 24 hours then subjected to MTT cell proliferation assay. Cell proliferation percentage was calculated by comparison with the medium alone group, which was set at 100%.

Fig. 4. GAC₁ inhibited TNF-α production by PBMCs from asthma patients

Purified PBMCs were treated with or without GAC₁ (20 μg/mL) for 24 hours. LPS (2 μg/mL) was added and culture conditions maintained for additional 24 hours. Supernatants were harvested and TNF-α levels were determined by ELISA. (A). GAC₁ (20 μg/mL) inhibition of TNF-α production by LPS-stimulated PBMCs from asthma patients. (B). PBMC viability after treatment with GAC₁ (20 μg/mL). * p<0.05, paired t-test, (n=11).

Fig. 5. GAC₁ regulation of NF-κB, AP-1 and MAPK

RAW 264.7 macrophages were pretreated with GAC₁ (0, 10, or 20 μg/mL) for 24 hours, followed by stimulation with LPS (1μg/mL) for 30 minutes. (A). p65, and p-IκBα expression were evaluated by Western-blot analysis of whole cell extracts. (B). Nuclear translocation of p-p65 was evaluated by Western-blot analysis of nuclear extracts. Equal protein loading was verified with Histone-H3 antibody. (C). c-Fos, c-Jun, ERK1/2, p-ERK1/2, JNK, p-JNK, p38, and p-p38 were evaluated by Western-blot analysis of whole cell extracts. Results are representative of three independent experiments. Purified PBMCs were pretreated with GAC₁ (20 μg/mL) for 24 hours, followed by stimulation with LPS (2μg/mL) for 30 minutes. (D). Representative Western blotting analysis of NF-κB (p-p65) expression in PBMCs cultured in medium alone, stimulated with LPS, with and without GAC₁ treatment. GAPDH expression was used as control. (E). Quantitation of Western blotting analysis of p-NF-κB (p-p65) expression by PBMCs cultured in medium alone, stimulated with LPS, and stimulated and treated with GAC₁ (p<0.05 Vs LPS alone, Mean ± S.D., n=3).