Nutr Res Pract.  2014 Feb;8(1):11-19.

Ethanol extract of Synurus deltoides (Aiton) Nakai suppresses in vitro LPS-induced cytokine production in RAW 264.7 macrophages and in vivo acute inflammatory symptoms

• Jiang Y
• Wang MH

Abstract

Synurus deltoides (Aiton) Nakai, belonging to the Compositae family, is an edible plant widely distributed in Northeast Asia. In this study, we examined the mechanisms underlying the immunomodulative effects of the ethanol extract of S. deltoides (SDE). The SDE extract strongly down-regulated the mRNA expression of the inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and tumour necrosis factor (TNF)-alpha, thereby inhibiting the production of nitric oxide (NO), prostaglandin E2 (PGE2), and TNF-alpha in the lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Furthermore, SDE also suppressed the nuclear translocation of the activation protein (AP)-1 and the nuclear factor-kappaB (NF-kappaB), and simultaneously decreased the phosphorylation of extracellular signal-regulated protein kinases (ERK), p38, and Akt. In agreement with the in vitro observations, the orally administered SDE ameliorated the acute inflammatory symptoms in the arachidonic acid-induced ear edema and the EtOH/HCl-induced gastritis in mice. Therefore, S. deltoides have a potential anti-inflammatory capacity in vitro and in vivo, suggesting the potential therapeutic use in the inflammation-associated disorders.

Keyword

Anti-inflammatory; macrophages; Synurus deltoides; cyclooxygenase-2; nitric oxide

MeSH Terms

Animals
Asia
Asteraceae
Cyclooxygenase 2
Dinoprostone
Ear
Edema
Ethanol*
Gastritis
Humans
Macrophages*
Mice
Necrosis
Nitric Oxide
Nitric Oxide Synthase Type II
Phosphorylation
Plants, Edible
Protein Kinases
RNA, Messenger
Tumor Necrosis Factor-alpha
Cyclooxygenase 2
Dinoprostone
Ethanol
Nitric Oxide
Nitric Oxide Synthase Type II
Protein Kinases
RNA, Messenger
Tumor Necrosis Factor-alpha

Figure

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  Fig. 1 The effects of the ethanol extract of Synurus deltoides (SDE) on in vitro inflammatory symptoms. (A) RAW264.7 cells (1×106 cells/ml) were incubated with SDE for 24 h, and the cell viability was determined using an MTT assay. (B-D) RAW264.7 cells (1×106 cells/ml) were treated with SDE in the presence or absence of lipopolysaccharide (LPS) (1 µg/ml) for 24 h. The supernatants were collected; and the nitric oxide (NO), prostaglandin E2 (PGE2), and tumour necrosis factor-α (TNF-α) concentrations were determined in the supernatants by the Griess assays and the enzyme-linked immunosorbent assays (ELISAs). (E) RAW264.7 cells (1×106 cells/ml) were treated with SDE in the presence or absence of LPS (1 µg/ml) for 24 h, and the cytoprotective effect was determined using an MTT assay.

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  Fig. 2 Suppression of the LPS-induced reactive oxygen species (ROS) in the RAW 264.7 cells, in the presence of different concentrations of SDE. Each value is the mean ± standard deviation (n = 3). Values with the same superscript letters are not significantly different from each other at P < 0.05.

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  Fig. 3 Effect of the ethanol extract of Synurus deltoides (SDE) on inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and tumour necrosis factor-α (TNF-α) expression in the lipopolysaccharide (LPS)-treated RAW264.7 cells. RAW264.7 cells (5×106 cells/ml) were incubated with SDE in the presence or absence of LPS (1 µg/ml) for 6 h. The levels of iNOS, COX-2, TNF-α, and GAPDH mRNA were determined by semi-quantitative polymerase chain reaction (PCR). Relative intensity (RI) was calculated by a ratio of GAPDH band intensity. The results shown are representative of three independent experiments.

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  Fig. 4 Effect of the ethanol extract of Synurus deltoides (SDE) on the translocation of the transcription factors. RAW264.7 cells (5×106 cells/ml) pre-treated with SDE for 0.5 h were stimulated with lipopolysaccharide (LPS) (1 µg/ml) for 15 and 60 min. After preparation of the nuclear fraction, levels of p65, c-Jun, c-fos, and Lammin A/C were determined by immunoblotting analysis. Results shown are representative of three independent experiments.

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  Fig. 5 HEK 293 cells co-transfected with the plasmid constructs activating protein luciferase (NF-κB) (1 µg/ml) (A) or (AP-1-Luc) (1 µg/ml) (B) and β-gal (as a transfection control) were treated with the ethanol extract of Synurus deltoides (SDE) in the presence or absence of phorbol myristic acid (PMA) (100 nM) for 24 h. RAW 264.7 cells co-transfected with the plasmid constructs activating protein luciferase (NF-κB) (1 µg/ml) (C) or (AP-1-Luc) (1 µg/ml) (D) and β-gal (as a transfection control) were treated with the ethanol extract of Synurus deltoides (SDE) in the presence or absence of lipopolysaccharide (LPS) (1 µg/ml) for 24 h. Luciferase activity was determined by luminometry. Values with the same superscript letters are not significantly different from each other at P < 0.05.

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  Fig. 6 Effect of the ethanol extract of Synurus deltoides (SDE) on the upstream signaling pathways for the activating protein (AP-1) and the nuclear factor-κB (NF-κB) activation. RAW264.7 cells (5×106 cells/ml) pre-treated with SDE for 30 min were stimulated with lipopolysaccharide (LPS) (1 µg/ml) for the indicated times. After immunoblotting, the levels of phospho- or total mitogen activated protein kinases (MAPKs) (ERK, p38, and JNK) (A) or IκBα and Akt (B) were identified based on their antibodies. Results are representative of three experiments.

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  Fig. 7 Effect of the ethanol extract of Synurus deltoides (SDE) on the arachidonic acid-induced mouse ear edema. ICR mice were orally administered with SDE (40 mg/kg) or indomethacin (1 mg/kg) for 3 days. Arachidonic acid solution was topically applied (30 µl/ear) to the ear of ICR mice (n = 6). The thickness of the edema was measured with a dial thickness gauge 1 h after the arachidonic acid treatment. The ear thickness of the arachidonic acid-treatment group is represented by 100%. Values with the same superscript letters are not significantly different from each other at P < 0.05.

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  Fig. 8 Effect of the ethanol extract of Synurus deltoides (SDE) and ranitidine on EtOH/HCl-induced gastritis. ICR mice (n = 6), orally administered with SDE or ranitidine for 3 days, were orally-treated with EtOH/HCl. After 1 h, photos of gastric lesion were taken by a camera (A), and the gastric lesions in the stomach were measured with a ruler (B). Values with the same superscript letters are not significantly different from each other at P < 0.05.

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