Vaccaria hypaphorine alleviates lipopolysaccharide-induced inflammation via inactivation of NFκB and ERK pathways in Raw 264.7 cells

Abstract

Background: Activation of macrophage is involved in many inflammation diseases. Lipopolysaccharide (LPS) is a powerful inflammatory signal contributing to monocytes/macrophages activation associated with increased proinflammatory cytokines expressions. We recently identified that vaccarin was expected to protect endothelial cells from injury. Hypaphorine was abundantly found in vaccaria semen. However, the potential roles and underlying mechanisms of vaccaria hypaphorine on macrophage inflammation have been poorly defined.

Methods: This study was designed to determine the effects of vaccaria hypaphorine on LPS-mediated inflammation in RAW 264.7 cells.

Results: In this study, we demonstrated that vaccaria hypaphorine dramatically ameliorated LPS-induced nitric oxide (NO) release and productions of proinflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, IL-10, monocyte chemoattractant protein 1 (MCP-1) and prostaglandin E2 (PGE₂) in RAW 264.7 cells. LPS-stimulated expressions of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were down-regulated by vaccaria hypaphorine. Furthermore, vaccaria hypaphorine retarded LPS-induced phosphorylation of ERK, nuclear factor kappa beta (NFκB), NFκB inhibitor IκBα, and IKKβ. Immunofluorescence staining revealed that vaccaria hypaphorine eliminated the nuclear translocation of NFκB in LPS-treated RAW 264.7 cells.

Conclusion: It was seen that vaccaria hypaphorine counteracted inflammation via inhibition of ERK or/and NFκB signaling pathways. Collectively, we concluded that vaccaria hypaphorine can be served as an anti-inflammatory candidate.

Keywords: COX-2; ERK; Hypaphorine; Inflammation; NFκB; iNOS.

Fig. 1

Effects of different doses of Vaccaria hypaphorine (12.5, 25 and 50 μM), Dex (100 μM) and Asp (1 mM) on the mRNA expressions of TNF-α (a), IL-1β (b), IL-6 (c), IL-10 (d) and MCP-1(e) response to LPS (1 μg/ml)-treated RAW264.7 cells for 24 h in vitro. Values are mean ± S.D. *P < 0.05 vs. Control, †P < 0.05 vs. LPS, †† P < 0.05 vs. Vaccaria hypaphorine (12.5 μM) + LPS. n = 6 for each group. LPS, lipopolysaccharide; Dex, dexamethasone; Asp, aspirin; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; IL-6, interleukin-6; IL-10, interleukin-10; MCP-1, monocyte chemoattractant protein 1

Fig. 2

Effects of different doses of Vaccaria hypaphorine (12.5, 25 and 50 μM), Dex (100 μM) and Asp (1 mM) on the TNF-α (a), IL-1β (b), IL-6 (c), PGE₂ (d) expressions and NO production (e) response to LPS (1 μg/ml)-treated RAW264.7 cells for 24 h in vitro. Values are mean ± S.D. *P < 0.05 vs. Control, †P < 0.05 vs. LPS, †† P < 0.05 vs. Vaccaria hypaphorine (12.5 μM) + LPS. n = 6 for each group. LPS, lipopolysaccharide; Dex, dexamethasone; Asp, aspirin; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; IL-6, interleukin-6; PGE₂, prostaglandin E2; NO, nitric oxide

Fig. 3

Effects of different doses of Vaccaria hypaphorine (12.5, 25 and 50 μM for 24 h) on the protein expressions of COX-2 or iNOS in response to LPS-stimulated RAW264.7 cells in vitro. a, representative images showing effects of pretreatment of different concentrations of Vaccaria hypaphorine (12.5, 25 and 50 μM), Dex (100 μM) and Asp (1 mM) on COX-2 or iNOS levels of RAW264.7 cells response to LPS (1 μg/ml). b, quantitative analysis of COX-2 or iNOS protein expressions in different groups. Values are mean ± S.D. *P < 0.05 vs. Control, †P < 0.05 vs. LPS, †† P < 0.05 vs. Vaccaria hypaphorine (12.5 μM) + LPS. n = 6 for each group. NO, nitric oxide; LPS, lipopolysaccharide; Dex, dexamethasone; Asp, aspirin. iNOS, induced nitric oxide (NO); COX-2, cyclooxygenase-2

Fig. 4

Effects of different doses of Vaccaria hypaphorine (12.5, 25 and 50 μM for 24 h) on the total or phosphorylated protein expressions of ERK in response to LPS-stimulated RAW264.7 cells in vitro. a, representative images of Western blot showing effects of pretreatment of different concentrations of Vaccaria hypaphorine (12.5, 25 and 50 μM), Dex (100 μM) and Asp (1 mM) on phosphorylated protein expressions of ERK of RAW264.7 cells response to LPS (1 μg/ml). b, quantitative analysis of phosphorylation of ERK. Values are mean ± S.D. *P < 0.05 vs. Control, †P < 0.05 vs. LPS, †† P < 0.05 vs. Vaccaria hypaphorine (12.5 μM) + LPS. n = 6 for each group. LPS, lipopolysaccharide; Dex, dexamethasone; Asp, aspirin

Fig. 5

Effects of different doses of Vaccaria hypaphorine (12.5, 25 and 50 μM for 24 h) on the protein expressions of NFκB、IκBα、IKKβ and their phosphorylated protein in response to LPS-stimulated RAW264.7 cells in vitro. a, representative images of Western blot showing effects of pretreatment of different concentrations of Vaccaria hypaphorine (12.5, 25 and 50 μM), Dex (100 μM) and Asp (1 mM) on NFκB, IκBα, IKKβ and their phosphorylated protein levels of RAW264.7 cells response to LPS (1 μg/ml). b, quantitative analysis of phosphorylation of IκBα. c, quantitative analysis of phosphorylation of IKKβ. d, quantitative analysis of phosphorylation of NF-κB. Values are mean ± S.D. *P < 0.05 vs. Control, †P < 0.05 vs. LPS, †† P < 0.05 vs. Vaccaria hypaphorine (12.5 μM) + LPS. n = 6 for each group. LPS, lipopolysaccharide; Dex, dexamethasone; Asp, aspirin

Fig. 6

Immunofluorescence staining showing the p65-NFκB distribution in RAW264.7 cells. Nuclei were stained by DAPI (blue). These representative photomicrographs indicated that Vaccaria hypaphorine (50 μM for 24) diminished nuclear translocation of p65-NFκB in LPS(1 μg/ml)-incubated RAW264.7 cells