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Pharmaceutical Biology Volume 55 ,Issue 1 ,2017-01-01
Anti-inflammatory effect of pomegranate flower in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages
Research Article
Jianjun Xu 1 , 2 Yongxin Zhao 1 , 3 Haji Akber Aisa 1 , 3
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DOI:10.1080/13880209.2017.1357737
Received 2016-7-22, accepted for publication 2017-7-17, Published 2017-7-17
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摘要

Context: Punica granatum L (Punicaceae) flower is an important diabetes treatment in oriental herbal medicine.Objective: This study investigates the inflammation effects of pomegranate flower (PFE) ethanol extract in LPS-induced RAW264.7 cells.Materials and methods: PFE (10, 25, 50, 100 μg/mL) was applied to 1 μg/mL LPS-induced RAW 264.7 macrophages in vitro. Levels of nitric oxide (NO), prostaglandin E2 (PGE2) and pro-inflammatory cytokines interleukin (IL)-1β (IL-1β), interleukin (IL)-6 (IL-6) and tumor necrosis factor (TNF-α) in the supernatant fraction were determined using enzyme-linked immunosorbent assay (ELISA). Expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), phosphorylation of mitogen-activated protein kinase (MAPK) subgroups extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and P38, as well as nuclear factor-κB (NF-κB) activation in extracts were detected via Western blot.Results: 10–100 μg/mL PFE decreased the production of NO (IC50 value = 31.8 μg/mL), PGE2 (IC50 value = 54.5 μg/mL), IL-6 (IC50 value = 48.7 μg/mL), IL-1β (IC50 value = 71.3 μg/mL) and TNF-α (IC50 value = 62.5 μg/mL) in LPS-stimulated RAW 264.7 cells significantly. A mechanism-based study showed that phosphorylation of ERK1/2, p38, JNK and translocation of the NF-B p65 subunit into nuclei were inhibited by the PFE treatment.Discussion and conclusion: These results show that PFE produced potential anti-inflammatory effect through modulating the synthesis of several mediators and cytokines involved in the inflammatory process.

关键词

MAPK;NF-κB;anti-inflammation;Punica granatum

授权许可

© 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

图表

Total ion chromatograms of PFE in negative ESI mode.

Cytotoxicity of PFE in RAW 264.7 cells. Cells were treated with different concentrations of PFE for 24 h, and viability was assayed by the MTT assay. Data represent mean values of triple determinations ± SEM. PFE at 100 μg/mL was not cytotoxic.

Effects of PFE on LPS-induced NO, PGE2 production and iNOS, COX-2 protein expression levels in LPS-induced RAW264.7 cells. Cells were incubated in the presence of PFE or in combination with 1 μg/mL LPS for 18 h. The culture supernatant was analyzed for NO (A), PGE2 (B) production. The iNOS and COX-2 (C) expression levels were determined by Western blotting. Data show mean ± SEM values of three independent experiments. *p < 0.05 and **p < 0.01 indicate significant differences from LPS-stimulation value.

Effects of PFE on TNF-α (A), IL-6 (B) and IL-1β (C) in LPS-induced RAW264.7 cells. The cells were pretreated with the different concentrations of PFE for 1 h and then exposed to 1 μg/mL LPS for 18 h. The levels of TNF-α, IL-1β and IL-6 in the supernatant were determined by ELISA. Data show mean ± SEM values of three independent experiments. *p < 0.05 and **p < 0.01 indicate significant differences from LPS stimulation value.

Effects of PFE on NF-κB p65 and IκBα activity in LPS-stimulated RAW 264.7 cells. The cells were pretreated with the different concentrations of PFE for 1 h and then exposed to 1 μg/mL LPS for additional 30 min. Cytoplasm and nuclear extracts proteins of cells were harvested for measurements of NF-κB p65 and IκB-α protein by Western blotting. β-Actin and Lamin B were used as the internal control. Data show mean ± SEM values of three independent experiments. *p < 0.05 and **p < 0.01 indicate significant differences from LPS-stimulation value.

Effects of PFE on phosphorylation of MAPKs activity in LPS-stimulated RAW 264.7 cells. The cells were pretreated with the different concentrations of PFE for 1 h and then exposed to LPS for 30 min. Total cellular proteins of cells were harvested for measurements of total or phosphorylated ERK1/2, JNK, and p38 by Western blotting. Data show mean ± SEM values of three independent experiments. *p < 0.05 and **p < 0.01 indicate significant differences from LPS-stimulation value.

通讯作者

Haji Akber Aisa.Key Laboratory of Chemistry of Plant Resources in Arid Regions, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi, China;State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi, Chin.haji@ms.xjb.ac.cn

推荐引用方式

Jianjun Xu,Yongxin Zhao,Haji Akber Aisa. Anti-inflammatory effect of pomegranate flower in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Pharmaceutical Biology ,Vol.55, Issue 1(2017)

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