Division of Pharmacy and Pharmaceutical Science, School of Applied Sciences, University of Huddersfield, Queensgate, Huddersfield HD1 3DH, United Kingdom.
J Ethnopharmacol. 2013 Jan 9;145(1):42-9. doi: 10.1016/j.jep.2012.10.031. Epub 2012 Nov 6.
Anacardium occidentale is used in traditional African medicine for the treatment of arthritis, fever, aches, pains, and inflammation of the extremities.
In this study, we investigated the molecular mechanisms responsible for anti-inflammatory effects of a stem bark extract of A. occidentale (ANE) in LPS-stimulated microglia.
Nitric oxide (NO), prostaglandin E(2) and cytokine (TNFα and IL-6) production were evaluated in supernatants from LPS-stimulated BV-2 cells. Cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and microsomal prostaglandin E2 synthase (mPGES-1) protein expressions in rat primary microglia were measured using western blot. The effects of ANE on NF-κB activation and nuclear translocation were evaluated in the luciferase reporter gene assay and ELISA, while ability of ANE to influence IκB phosphorylation was determined using ELISA specific for phospho-IκB. The involvement of MAPK phosphorylation in the anti-inflammatory actions of ANE was evaluated using specific ELISA for phospho-p38, phospho-p42/44 and phospho-JNK. The MTT assay was used to determine the effect of ANE on BV-2 microglia viability.
ANE (25-100 μg/ml) produced significant (p<0.05) reduction in the production of NO, PGE(2), TNFα and IL-6 in BV-2 microglia stimulated with LPS for 24h. Pre-treatment with ANE caused a significant (p<0.05) inhibition of COX-2, iNOS and mPGES-1 protein expressions in the rat primary microglia. Further experiments showed that ANE inhibited COX-2 and iNOS protein expression via IκB-mediated nuclear translocation and transactivation of NF-κB. Our studies also revealed that ANE produced significant (p<0.05) and dose-dependent inhibition of p38, p42/44 and JNK MAPK phosphorylation in LPS-activated BV-2 microglia.
We conclude that ANE has an anti-inflammatory property related to inhibition of inflammation-associated cytokine production as well as iNOS and COX-2 gene expression by blocking NF-κB and MAPK pathways in the microglia. It is also suggested that mPGES-1 inhibition contributes to the effect of ANE on PGE(2) production in the microglia.
在传统的非洲医学中，鳄梨被用于治疗关节炎、发热、疼痛和四肢炎症。
本研究旨在探讨鳄梨茎皮提取物（ANE）对脂多糖刺激的小胶质细胞抗炎作用的分子机制。
通过 LPS 刺激的 BV-2 细胞上清液评估一氧化氮（NO）、前列腺素 E（2）和细胞因子（TNFα 和 IL-6）的产生。使用 Western blot 测量大鼠原代小胶质细胞中环氧化酶-2（COX-2）、诱导型一氧化氮合酶（iNOS）和微粒体前列腺素 E2 合酶（mPGES-1）的蛋白表达。通过荧光素酶报告基因测定和 ELISA 评估 ANE 对 NF-κB 激活和核易位的影响，通过 ELISA 测定 ANE 对 IκB 磷酸化的影响。通过特定的 ELISA 测定 MAPK 磷酸化在 ANE 抗炎作用中的参与，用于磷酸化 p38、磷酸化 p42/44 和磷酸化 JNK。MTT 测定用于确定 ANE 对 LPS 刺激的 BV-2 小胶质细胞活力的影响。
ANE（25-100μg/ml）可显著（p<0.05）减少 LPS 刺激 24 小时的 BV-2 小胶质细胞中 NO、PGE（2）、TNFα 和 IL-6 的产生。ANE 预处理可显著（p<0.05）抑制大鼠原代小胶质细胞中 COX-2、iNOS 和 mPGES-1 蛋白的表达。进一步的实验表明，ANE 通过 IκB 介导的核易位和 NF-κB 的转录激活抑制 COX-2 和 iNOS 蛋白的表达。我们的研究还表明，ANE 可显著（p<0.05）并呈剂量依赖性抑制 LPS 激活的 BV-2 小胶质细胞中 p38、p42/44 和 JNK MAPK 的磷酸化。
我们得出结论，ANE 具有抗炎特性，与通过阻断小胶质细胞中的 NF-κB 和 MAPK 途径抑制炎症相关细胞因子的产生以及 iNOS 和 COX-2 基因表达有关。此外，mPGES-1 抑制有助于 ANE 对小胶质细胞中 PGE（2）产生的影响。
