Purpose: Euphorbia neriifolia  Linn. has important medicinal value in the treatment of ulcers, tumors, inflammation, chronic respiratory troubles, and so on. Although many ingredients with anti-inflammatory activity have been discovered and isolated from the Euphorbia neriifolia , the current research still cannot explain its multivariate effects on the immune response. This article aims to introduce two Ingenane-type diterpenoids from Euphorbia neriifolia  with macrophage regulatory effects and to investigate the mechanism of their action.
Methods: The stem bark of E. neriifolia  was extracted with various separation methods to obtain ingenane-type diterpenoids. The RAW264.7 cells were treated with lipopolysaccharide (LPS, 1 μg/mL) to establish an inflammatory cell model. The cell viability was detected by MTT assay. The secretion of PGE2, TNF-α, IL-1β, and IL-6 was tested with ELISA. The levels of iNOS, COX-2, IκBα, JNK, ERK, p38, p-IκBα, p-JNK, p-ERK, and p-p38 in cells were detected by Western blotting. The translocation of nuclear factor-kappa B (NF-κB)/p65 subunit were evaluated by Immunofluorescence staining.
Results: Ingenane-type diterpenoids, eurifoloid A (Euri A) and a new compound euphorneroid E (Euph E), were isolated from the EtOAc fraction of E. neriifolia  stem bark extracts. Euph E and Euri A exhibited significant inhibition on the levels of pro-inflammatory mediators NO, IL-1β, IL-6, and iNOS on LPS-induced macrophage RAW264.7. Cellular signaling pathway studies showed that they prevented the degradation of IκBα and the translocation of NF-κB/p65 subunit. Furthermore, the production of PGE2, TNFα, and COX-2 was dramatically increased under the influence of the compounds, which were closely related to the phosphorylation of protein kinase C δ (PKCδ) and activation of mitogen-activated protein kinase (MAPKs) signaling pathway.
Conclusion: These results demonstrated that Euph E and Euri A exhibited multidirectional regulation on cytokines and immune function of macrophages, in addition to a good anti-inflammatory activity, and which was closely related to the regulation of PKCδ/MAPKs and NF-κB signal pathways.
Keywords: euphorneroid E, eurifoloid A, ingenane-type diterpenoids, Euphorbia neriifolia , M-macrophage, immunoregulation