Anti-inflammatory effects of Cordyceps mycelium (Paecilomyces hepiali, CBG-CS-2) in Raw264.7 murine macrophages

Abstract

Cordyceps (CS) is a traditional Chinese herb with various biological effects that include immune modulation. CBG-CS-2 is a strain, Paecilomyces hepiali, of the Cordyceps spp. The anti-inflammatory effects of CBG-CS-2 were investigated. The water-soluble fraction of CBG-CS-2 has high anti-inflammatory activity in LPS-induced Raw264.7 macrophages. We tested the role of CBG-CS-2 on the anti-inflammation cascade in LPS-stimulated Raw264.7 cells. CBG-CS-2 significantly decreased NO production, iNOS expression, and pro-inflammatory cytokine secretion in a dose-dependent manner. To investigate the mechanism by which CBG-CS-2 inhibits NO, iNOS, and pro-inflammatory cytokines, we examined the activities of NF-κB and AP-1 in LPS-activated macrophages. The results demonstrate that CBG-CS-2 suppresses the production and expression of NO, iNOS, and pro-inflammatory cytokines in LPS-activated macrophages via inhibition of NF-κB and AP-1, which may play an important role in inflammation. These findings suggest that CBG-CS-2 has modulatory effects on the inflammatory system in macrophages, and that it can serve as a useful anti-inflammatory dietary supplement or drug.

Keywords: Anti-inflammation; CBG-CS-2; Cordyceps; Paecilomyces hepiali.

Fig. 1

Effects of CBG-CS-2 on LPS-induced NO production in mouse macrophage Raw264.7 cells. a Cells were pretreated with 100S–2000S μg/ml of CBG-CS-2 before administration of LPS (1 μg/ml). Supernatants were analyzed following 20 h in culture to determine their concentrations of nitric oxide. b Cell cytotoxicity of CBG-CS-2 against mouse macrophage Raw264.7 cells. Cell viability was estimated via crystal violet assay. The results are expressed as mean with SEM from three independent experiments. *P < 0.05 and **P < 0.01, Student’s t-test

Fig. 2

Effects of CBG-CS-2 on the expression of iNOS protein and mRNA in macrophage Raw264.7 cells. Cells were treated with two concentrations (500S and 1000S μg/ml) of CBG-CS-2, with or without LPS (1 μg/ml) for 20 h. a Total cellular proteins (50 μg) were separated and blots were probed with specific antibodies. b iNOS mRNA in Raw264.7 cells was assessed by RT-PCR. The experiments were repeated three times, and similar results were obtained

Fig. 3

Effects of CBG-CS-2 on LPS-induced pro-inflammatory cytokine production in macrophage Raw264.7 cells. Cultures were treated with several concentrations (100S, 500S, and 1000S μg/ml) of CBG-CS-2, with LPS (1 μg/ml). a TNF-α secretion was measured in culture media using an ELISA kit. The results are expressed as mean with SEM from three independent experiments. Double asterisks (**) represent P < 0.01, Student’s t-test. b The mRNA expression of the pro-inflammatory cytokines, TNF-α and IL-6, was analyzed via RT-PCR. The GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene was used as a control

Fig. 4

CBG-CS-2 inhibited LPS-induced NF-kB activation and AP-1 activity in macrophage Raw264.7 cells. Raw264.7 macrophages transfected with pGL3-NF-kB-Luc or pGL3-AP-1-Luc were stimulated with LPS (1 μg/ml) for 1 h with or without a variety of concentrations of CBG-CS-2 (100S, 500S, 1000S, and 2000S μg/ml). Lysates of these cells were subjected to a luciferase assay. The results are expressed as mean with SEM from three independent experiments. Double asterisks (**) represent P < 0.01, Student’s t-test