Prunus yedoensis bark inhibits lipopolysaccharide-induced inflammatory cytokine synthesis by IκBα degradation and MAPK activation in macrophages

Abstract

The bark of Prunus yedoensis is used in antitussive medicines and in oral herbal formulations for inflammatory skin disorders. In the present study, we explored whether P. yedoensis bark extract (PYE) and its solvent partitioned fractions could modulate lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α and interleukin (IL)-6 in vivo and in vitro. In addition, we examined the effect of PYE extract and its fractions on LPS-induced NF-κB and mitogen-activated protein kinase (MAPK) signaling in mouse peritoneal macrophages. Oral treatment of PYE decreased serum levels of TNF-α and IL-6 in LPS injected mice. PYE inhibited LPS-induced TNF-α and IL-6 in macrophages at the transcriptional level and also suppressed LPS-induced IκBα degradation and MAPK activation in vitro. Among the fractions, the chloroform fraction, which contains genistein, naringenin, sakuranetin, prunetin, and amygdalin, showed inhibitory effects at much lower concentrations than the water and ethyl acetate fractions. Taken together, our results indicate that PYE was able to inhibit LPS-induced expression of TNF-α and IL-6, the latter of which was more prominent. The effects of PYE on inflammatory cytokine synthesis may involve modulation of NF-κB and MAPK activation.

FIG. 1.

High performance liquid chromatography (HPLC) chromatograms of Prunus yedoensis bark extract (PYE) and its water, ethyl acetate, and chloroform fractions. 1, genistein; 2, naringenin; 3, sakuranetin; 4, prunetin; 5, amygdalin. (A) PYE, (B) water fraction, (C) chloroform fraction, and (D) ethyl acetate fraction. Color images available online at www.liebertpub.com/jmf

FIG. 2.

Effect of PYE on systemic inflammatory responses. PYE (10, 50, and 250 mg/kg) was administered orally to mice for seven days before intraperitoneal injection of lipopolysaccharide (LPS; 1.3 mg/kg). Dexamethasone (Dex) was intraperitoneally injected 16 h before LPS stimulation. Serum was obtained 1 h after LPS stimulation, and the levels of TNF-α and IL-6 were measured by enzyme-linked immunosorbant assay (ELISA). Data are expressed as the means±standard deviation (SD; n=9–12). *P<.05, **P<.01, ***P<.005 compared with the control group.

FIG. 3.

Cytotoxicity of PYE and its solvent partitioned fractions in vitro. Peritoneal macrophages were isolated from thioglycollate-injected mice and cultured in the presence of PYE, water, ethyl acetate, or chloroform for 24 h. Extracellular lactate dehydrogenase (LDH) release was presented as optical density. Each bar represents means±SD of four independent experiments. (A) PYE, (B) water fraction, (C) chloroform fraction, and (D) ethyl acetate fraction. *P<.05, **P<.01 compared with untreated cells.

FIG. 4.

Effect of PYE and its fractions on LPS-induced TNF-α and IL-6 gene expression. Peritoneal macrophages were pretreated 1 h with PYE (A) or its fractions (B) and (C), and then stimulated for 4 h with LPS (100 ng/mL); 1 μM dexamethasone was used as a reference drug. Total RNA was extracted and real-time polymerase chain reaction (PCR) was performed. GAPDH was used as an internal control. Data are expressed as means±SD of three independent experiments. *P<.05, **P<.005, ***P<.001 compared with cells treated with LPS only (control).

FIG. 5.

Effect of PYE on LPS-induced IκBα degradation and MAPK activation. Peritoneal macrophages were pretreated 1 h with PYE and then stimulated for 30 min with LPS. Whole protein was extracted and examined by Western blot analysis. Tubulin was used as an internal control. One of the three experiments is shown.

FIG. 6.

Effect of water, chloroform, and ethyl acetate fractions from PYE on LPS-induced IκBα degradation and MAPK activation. Peritoneal macrophages were pretreated 1 h with 1 μM dexamethasone (Dex), 200 μg/mL water fraction (W), 10 μg/mL chloroform fraction (Ch), and 200 μg/mL ethyl acetate fraction (EA), and then stimulated for 4 h with LPS. Whole protein was extracted and examined by Western blot analysis. Tubulin was used as an internal control. One of the three experiments is shown.