Inhibitory effects of Euphorbia supina on Propionibacterium acnes-induced skin inflammation in vitro and in vivo

Abstract

Background: Euphorbia supina (ES) plant has been used as treatment for inflammatory conditions. The antibacterial effect and the anti-inflammatory mechanism of ES for Propionibacterium (P.) acnes-induced inflammation in THP-1 cells and acne animal model remain unclear. Therefore, the objective of the present study was to determine the antibacterial and anti-inflammatory activities of ES against P. acnes, the etiologic agent of skin inflammation.

Method: The antibacterial activities of ES were tested with disc diffusion and broth dilution methods. Cytotoxicity of ES at different doses was evaluated by the MTT assay. THP-1 cells were stimulated by heat-killed P. acnes in the presence of ES. The pro-inflammatory cytokines and mRNA levels were measured by ELISA and real-time-PCR. MAPK expression was analyzed by Western blot. The living P. acnes was intradermally injected into the ear of BLBC/c mice. Subsequently, chemical composition of ES was analyzed by liquids chromatography-mass spectrometry (LC-MS).

Result: ES had stronger antibacterial activity against P. acnes and inhibitory activity on lipase. ES had no significant cytotoxicity on THP-1 cells. ES suppressed the mRNA levels and production of IL-8, TNF-a, IL-1β in vitro. ES inhibited the expression levels of pro-inflammatory cytokines and the MAPK signaling pathway. Ear thickness and inflammatory cells were markedly reduced by ES treatment. Protocatechuic acid, gallic acid, quercetin, and kaempferol were detected by LC-MS analysis in ES.

Conclusions: Our results demonstrate antibacterial and anti-inflammatory activities of ES extract against P. acnes. It is suggested that ES extract might be used to treatment anti-inflammatory skin disease.

Fig. 1

Antibacterial activity of ES against P. acnes (a; tetracycline 50 μg/ml, b; DMSO, c; ES 200 mg/ml, d; ES 100 mg/ml)

Fig. 2

Effect of ES on cell viability and P. acnes-induced pro-inflammatory cytokines in THP-1 cells. (a) Cell viability of ES was determined by MTT assay in THP-1 cells. THP-1 cells were treated with 0.1, 1 and 10 μg/ml of ES for 24 hrs. ELISA results demonstrate that ES suppressed the secretion of (b) IL-1β, (c) IL-8 and (d) TNF-α in P. acnes-stimulated THP-1 cells. Values represent mean ± S.E.M. Data were analyzed by Tukey post hoc test ( * p < 0.05 versus control and ** p < 0.05 versus P.acnes alone)

Fig. 3

Effect of ES on the gene expression of (a) IL-1β, (b) IL-8 and (c) TNF-α in P. acnes-induced THP-1 cells. The expression level of mRNA was determined using a Real-time PCR. THP-1 cells were pre-treated with 0.1, 1 and 10 μg/ml of ES for 4 hrs incubation and then stimulated with P. acnes for 18 hrs incubation. Values represent mean ± S.E.M. Data were analyzed by Tukey post hoc test ( * p < 0.05 versus control and ** p < 0.05 versus P.acnes alone)

Fig. 4

Effect of ES on the MAPK signaling pathway in P. acnes-indcued THP-1 cells. THP-1 cells were pre-treated with 0.1, 1 and 10 μg/ml of ES for 1 hrs incubation and then stimulated with P. acnes for 1 hrs incubation. (a) Western blot analysis shows that phosphorylation of p38, ERK and JNK is suppressed by ES treatment. (b) MAPKs/β-actin ratio were determined by densitometry. Values represent mean ± S.E.M. Data were analyzed by Tukey post hoc test ( *P < 0.05 versus non-treatment and **P < 0.05 versus P.acnes alone)

Fig. 5

Effect of ES on ear thickness in living P. acnes-injected mice ears. (a) The suppress effects with 1, 10 mg/ml of ES on P. acnes-induced ear edema in mice were evaluated by measuring the ear thickness. (b) Paraffin sections of Ear tissue were stained with H&E observed by microscope. Values represent mean ± S.E.M. (n = 5). Data were analyzed by Tukey post hoc test ( * p < 0.05 versus control and ** p < 0.05 versus P.acnes alone)

Fig. 6

HPLC chromatogram of standards and Euphorbia supina (a), mass scan of quercetin, kaempferol, gallic acid and protocatechuic acid (b)