Syk/NF-κB-targeted anti-inflammatory activity of Melicope accedens (Blume) T.G. Hartley methanol extract,Journal of Ethnopharmacology

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Syk/NF-κB-targeted anti-inflammatory activity of Melicope accedens (Blume) T.G. Hartley methanol extract
Journal of Ethnopharmacology ( IF 4.8 ) Pub Date : 2021-02-01 , DOI: 10.1016/j.jep.2021.113887
Jin Kyeong Kim ₁ , Eunju Choi ₁ , Yo Han Hong ₁ , Haeyeop Kim ₁ , Young-Jin Jang ₂ , Jong Sub Lee ₃ , Eui Su Choung ₃ , Byoung Young Woo ₄ , Yong Deog Hong ₄ , Sarah Lee ₅ , Byoung-Hee Lee ₅ , Tran The Bach ₆ , Ji Hye Kim ₁ , Jong-Hoon Kim ₂ , Jae Youl Cho ₁

Affiliation

Ethnopharmacological relevance

Melicope accedens (Blume) Thomas G. Hartley is a plant included in the family Rutaceae and genus Melicope. It is a native plant from Vietnam that has been used for ethnopharmacology. In Indonesia and Malaysia, the leaves of M. accedens are applied externally to decrease fever.

Aim of the study

The molecular mechanisms of the anti-inflammatory properties of M. accedens are not yet understood. Therefore, we examined those mechanisms using a methanol extract of M. accedens (Ma-ME) and determined the target molecule in macrophages.

Materials and methods

We evaluated the anti-inflammatory effects of Ma-ME in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and in an HCl/EtOH-triggered gastritis model in mice. To investigate the anti-inflammatory activity, we performed a nitric oxide (NO) production assay and ELISA assay for prostaglandin E2 (PGE₂). RT-PCR, luciferase gene reporter assays, western blotting analyses, and a cellular thermal shift assay (CETSA) were conducted to identify the mechanism and target molecule of Ma-ME. The phytochemical composition of Ma-ME was analyzed by HPLC and LC-MS/MS.

Results

Ma-ME suppressed the production of NO and PGE₂ and the mRNA expression of proinflammatory genes (iNOS, IL-1β, and COX-2) in LPS-stimulated RAW264.7 cells without cytotoxicity. Ma-ME inhibited NF-κB activation by suppressing signaling molecules such as IκBα, Akt, Src, and Syk. Moreover, the CETSA assay revealed that Ma-ME binds to Syk, the most upstream molecule in the NF-κB signal pathway. Oral administration of Ma-ME not only alleviated inflammatory lesions, but also reduced the gene expression of IL-1β and p-Syk in mice with HCl/EtOH-induced gastritis. HPLC and LC-MS/MS analyses confirmed that Ma-ME contains various anti-inflammatory flavonoids, including quercetin, daidzein, and nevadensin.

Conclusions

Ma-ME exhibited anti-inflammatory activities in vitro and in vivo by targeting Syk in the NF-κB signaling pathway. Therefore, we propose that Ma-ME could be used to treat inflammatory diseases such as gastritis.

中文翻译：

Melicope accedens (Blume) TG Hartley 甲醇提取物的 Syk/NF-κB 靶向抗炎活性

民族药理学相关性

Melicope accedens (Blume) Thomas G. Hartley 是包含在芸香科和Melicope属的植物。它是一种来自越南的本地植物，已用于民族药理学。在印度尼西亚和马来西亚，M. accedens的叶子外用以退烧。

研究目的

M. accedens抗炎特性的分子机制尚不清楚。因此，我们使用M. accedens (Ma-ME)的甲醇提取物检查了这些机制，并确定了巨噬细胞中的目标分子。

材料和方法

我们评估了 Ma-ME 在脂多糖 (LPS) 刺激的 RAW264.7 细胞和小鼠 HCl/EtOH 引发的胃炎模型中的抗炎作用。为了研究抗炎活性，我们对前列腺素 E2 (PGE ₂ )进行了一氧化氮 (NO) 产生测定和 ELISA 测定。进行RT-PCR、荧光素酶基因报告分析、蛋白质印迹分析和细胞热位移分析(CETSA)来鉴定Ma-ME的机制和靶分子。Ma-ME 的植物化学成分通过 HPLC 和 LC-MS/MS 进行分析。

结果

Ma-ME在 LPS 刺激的 RAW264.7 细胞中抑制 NO 和 PGE _{2 的产生}以及促炎基因（iNOS、IL-1β 和 COX-2）的 mRNA 表达，而没有细胞毒性。Ma-ME 通过抑制 IκBα、Akt、Src 和 Syk 等信号分子来抑制 NF-κB 活化。此外，CETSA 分析显示 Ma-ME 与 Syk 结合，Syk 是 NF-κB 信号通路中最上游的分子。Ma-ME 的口服给药不仅减轻了炎症病变，而且还降低了 HCl/EtOH 诱导的胃炎小鼠中 IL-1β 和 p-Syk 的基因表达。HPLC 和 LC-MS/MS 分析证实，Ma-ME 含有多种抗炎类黄酮，包括槲皮素、黄豆苷元和内华登素。