Euphorbia helioscopia Inhibits the LPS-Induced Pro-Inflammatory Response in RAW 264 . 7 Cells via the NF-κ B and MAPK Pathway

Previous studies have characterized the total polyphenolic contents, antioxidant activity, tyrosinase, elastase, and NO production of halophytes. Halophytes are distributed among many countries; however, it has not been properly utilized. In this study, we investigated the inhibitory effect of halophyte Euphorbia helioscopia (E.H.) on the LPS-induced inflammatory response in RAW 264.7 macrophages by MTT assay, NO assay, ELISA, and western blot analysis. Our results demonstrate that E.H. reduced LPS-induced NO and PGE2 in addition to pro-inflammatory mediators, IL-6, IL-1β and TNF-α. Furthermore, E.H. inhibited LPS-induced activation of NF-κB, via IκBα degradation and phosphorylation of JNK and ERK. Our data suggest that the E.H. anti-inflammatory effect is a result of inhibition of LPS-induced NO, PGE2, IL-6, IL-1β, and TNF-α production via downregulation of the NF-κB and MAPK pathway.

About 1500 species of salt-tolerant halophytes are distributed in the costal wetland of river estuaries, saltpans, and reclaimed land.Moreover, about 40-100 species are distributed in Korea.Halophytes are rich in natural minerals owing to their ability to grow in the soil of seawater 6,7 .
We recently characterized the total polyphenolic content, antioxidant activity, tyrosinase, elastase, and nitric oxide (NO) production of 21 halophyte species in vitro 8 .Among them, Euphorbia helioscopia (E.H)., used as traditional Chinese medicine, and its extracts were found to inhibit various carcinomas, in addition to having antifungal and antibacterial activity 9,10 .However, there have been no reports on its anti-inflammatory activity.Therefore, we evaluated the effect of E.H. on NO, iNOS, and pro-inflammatory cytokine production.Further, to establish the anti-inflammatory mechanism for E.H., we examined the NF-κB and MAPK signaling pathways in the murine RAW 264.7 cells.

Nitric oxide assay
Nitrite levels was used as an indicator of NO production in the cell culture medium by the Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, and 2.5% phosphoric acid) 13,14 .Specifically, culture supernatant from each well was mixed with equal volume Griess reagent for 15 min, and optical density was determined at 540 nm.Percent NO production was determined relative to the control LPS only treated group.
Cells were treated with 1 µg/mL of LPS or with LPS plus various concentrations (50 to 200 µg/mL) of E. helioscopia for 24 h.Nitric oxide (NO) production was determined by the Griess reagent method.Cell viability was determined from the 24 h culture of cells treated with LPS (1 µg/mL) in the presence of E. helioscopia.The data represent the mean ± SD of triplicate experiments.*p <0.005, **p<0.001versus LPS alone.

ELISA determination of cytokines
Cells (1 × 10 6 ) were cultured for 24 h in the presence or absence of 1 µg/mL LPS and various concentrations of E. helioscopia (50, 100, 200 µg/mL).Cell supernatants were harvested and centrifuged at 4,500 rpm for 4 min to remove dead cells.Cytokine production percent was determined relative to the control LPS only treated group.

Western blot analysis
Cells were washed twice with cold PBS, followed by lysis in RIPA lysis buffer on ice for 20 min.Cell lysates were centrifuged at 12,000 rpm for 20 min at 4°C and protein concentration was determined using the BCA method.Cell lysates were separated by 8% Bis-Tris gel and transferred with the iBlot 7-minute blotting system to iBlot Gel transfer stacks (ThermoFisher Scientific, USA).The membrane was blocked using 5% skim milk for 1 h at room temperature, followed by an overnight incubation at 4°C 2 h with 1:1000 dilution primary antibody.Following four washes, the membranes were incubated with 1:5,000 dilution of horseradish peroxidase-conjugated secondary antibody for 30 min at room temperature.Membrane proteins were detected using the ECL western blotting detection substrate.
Cells were stimulated with 1 µg/ml of LPS or with LPS plus various concentrations (50, 100, 200 µg/mL) of E. helioscopia for 24 h.IL-6 (A), IL-1b (B), and TNF-a (C) productions were determined by ELISA.The data represent the mean ± SD of triplicate experiments.*p <0.005, **p<0.001versus LPS alone.This study is the first to provide evidence that E. helioscopia inhibits NO, PGE 2 and proinflammatory mediators in LPS-treated RAW 264.7 cells via the blockade of the NF-κB and MAPK pathways.

Fig. 2 :Fig. 3 :
Fig. 2: Effect of Euphorbia helioscopia on the protein level of iNOS in LPS-treated RAW 264.7 cells

Fig. 6 :
Fig. 6: Effect of Euphorbia helioscopia on the phosphorylation of JNK (A) and ERK (B) in LPS-treated RAW 264.7 cells 7 cells NF-κB, we examined phosphorylation in the NF-κB and MAPK pathways.We found that the activity of p65, JNK and ERK was inhibited by E.H. in LPStreated RAW 264.7 cells.These data suggest that the E.H.-induced phosphorylation in the MAPK pathway may contribute to its inhibitory effect on NF-κB in E.H.-treated RAW 264.7 cells.Further, E.H. may block LPS-induced IκBα degradation, inhibiting NF-κB activation.In summary, our findings suggest that E.H. is a potent inhibitor of LPS-induced NO, PGE 2 , IL-6, IL-1β, and TNF-α production in RAW 264.7 cells.Moreover, the inhibitory effect of E.H. was found to be associated with NF-κB inactivation via blockade of IêBá phosphorylation and degradation.