Bone-marrow derived dendritic cells (BMDCs) play a key role in the interface between the innate and acquired immune systems [22
]. Activated BMDCs perform crucial functions in immune and inflammatory responses via the pathogen-associated molecular patterns (PAMPs)-stimulated production of pro-inflammatory cytokines such as IL-12 p40, IL-6, and TNF-α.
Among its many biological activities, IL-12 provides an obligatory signal for the differentiation of effector T-helper 1 (Th1) cells and the secretion of Th1 cytokines, gamma interferon (IFN-γ) and IL-2. IL-12 plays an important role in the generation of a Th1 response against human pathogens [23
]. Although the induction of IL-12 by intracellular organisms is necessary for a protective host Th1 response, overexpression of Th1 cytokines and IL-12 may contribute to the development and perpetuation of chronic inflammatory and autoimmune diseases. Thus, understanding the regulated expression of IL-12 in macrophages may provide insight into the pathogenesis of infectious and inflammatory diseases, and could reveal novel approaches to alter immune responses [25
In this study, the MeOH extract significantly inhibited the production of all pro-inflammatory cytokines, while the dichloromethane fraction showed more potent inhibitory effects ( and ). The methanol extract from the starfish A. polyacanthus showed inhibitory activity on IL-12 p40, IL-6, and TNF-α production (IC50 = 11.47 ± 0.16, 20.28 ± 0.22, and 36.99 ± 0.24 μg/mL, respectively). Since the methanol extract had inhibitory activity on IL-12 p40 production, it was partitioned in dichloromethane/water to obtain a dichloromethane soluble portion and an aqueous phase. Based on , the dichloromethane soluble extract showed potent inhibitory activity towards LPS-stimulated IL-12 p40 production (IC50 = 1.27 ± 0.11 μg/mL), which was higher than that by the methanol extract (IC50 = 11.47 ± 0.16 μg/mL).
Anti-inflammatory effects of the extracts on LPS-stimulated BMDCs.
Effect of crude extracts (5, 10, 25, 50 μg/mL) on IL-12 p40, IL-6, and TNF-α production by LPS-stimulated BMDCs. The data were presented as inhibition rate (%) compared to the value of vehicle-treated DCs.
Subsequently, all isolated compounds (1–7) from the CH2Cl2 fraction of A. polyacanthus were tested for inhibitory effects on the production of the pro-inflammatory cytokines IL-12 p40, IL-6, and TNF-α. Of those tested, compounds 1, 3–5, and 7 showed potent inhibition on IL-12 p40 production with IC50 values of 3.96 ± 0.12, 6.55 ± 0.18, 5.06 ± 0.16, 1.82 ± 0.11, and 3.90 ± 0.14 μM, respectively (). This variability in inflammatory response inhibition by 1, 3–5, and 7 may be explained by the different levels of secreted inflammatory factors upon LPS stimulation. Among those tested, compound 5 (a steroid with hydroxyl group at C-6) had the greatest inhibitory activity towards LPS-stimulated IL-12 p40 production (IC50 = 1.82 ± 0.11 μM), which was comparable to that of the positive control, SB203580 (IC50 = 5.00 ± 0.16 μM). Compound 5 is an isomer of 4, but 5 showed potent inhibitory activity at the tested concentrations. Compounds 2 and 6 showed moderate suppressive effects on the production of IL-12 p40 by 34.86 ± 1.31, 79.05 ± 2.05 μM respectively ( and ), relative to the vehicle group. These compounds were subjected to evaluation of their effects at various concentrations on the production of the pro-inflammatory cytokines in LPS-stimulated BMDCs.
Anti-inflammatory effects of compounds 1 to 7 on LPS-stimulated BMDCs.
Effect of steroids 1 to 7 (1, 2, 5, 10 μM) on IL-12 p40 production by LPS-stimulated BMDCs. The data were presented as inhibition rate (%) compared to the value of vehicle-treated DCs.
As a result, the dichloromethane-soluble extract showed inhibitory cilliary neurotropic factor activity of LPS-stimulated IL-6 production (IC50 = 8.82 ± 0.18 μg/mL). Compounds 1, 4, 5, and 7 considerably decreased the production of IL-6 in the LPS-stimulated BMDCs with IC50 values ranging from 2.61 ± 0.10 to 16.73 ± 0.25 μM (). Remarkably, compound 7 significantly inhibited IL-6 production with IC50 values of 2.61 ± 0.10 μM. The inhibitory effects of compounds 1 and 5 were similar to the positive control (IC50 = 3.50 ± 0.12 μM), with IC50 values of 4.07 ± 0.13 and 5.76 ± 0.14 μM, respectively. Compound 4 showed moderate inhibition of IL-6 production, with IC50 values of 16.73 ± 0.25 μM. The remaining compounds did not show significant activity (IC50 > 100 μM) against IL-6 production ().
Effect of steroids 1 to 7 (1, 2, 5, 10 μM) on IL-6 production by LPS-stimulated BMDCs. The data were presented as inhibition rate (%) compared to the value of vehicle-treated DCs.
Overexpression of pro-inflammatory cytokines TNF-α and IL-6 is associated with the development of autoimmune, inflammatory, and immunopathological diseases. Therefore, blocking TNF-α, IL-6, and their respective signaling pathways can be effective for treatment of inflammatory diseases. The TNF-α assay results are shown in . Compounds 5 and 7 showed potent inhibitory effects on the production of TNF-α, with IC50 values of 4.94 ± 0.12 and 7.00 ± 0.16 μM, respectively. The inhibitory effects of compound 3 (IC50 = 22.80 ± 0.21 μM) was moderate compared to the positive control (IC50 = 7.20 ± 0.13 μM), while compounds 1, 2, 4, and 6 did not show significant inhibitory effects on TNF-α production (IC50 > 100 μM, ).
Effect of steroids 1 to 7 (1, 2, 5, 10 μM) on TNF-α production by LPS-stimulated BMDCs. The data were presented as inhibition rate (%) compared to the value of vehicle-treated DCs.