Department of Anatomy, Kunming Medical University, Kunming, Yunnan, People's Republic of China.
PLoS One. 2012;7(8):e44107. doi: 10.1371/journal.pone.0044107. Epub 2012 Aug 31.
Resveratrol is a natural polyphenolic compound that has cardioprotective, anticancer and anti-inflammatory properties. We investigated the capacity of resveratrol to protect RAW 264.7 cells from inflammatory insults and explored mechanisms underlying inhibitory effects of resveratrol on RAW 264.7 cells.
METHODOLOGY/PRINCIPAL FINDINGS: Murine RAW 264.7 cells were treated with resveratrol (1, 5, and 10 µM) and/or LPS (5 µg/ml). Nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by Griess reagent and ELISA. The mRNA and protein levels of proinflammatory proteins and cytokines were analysed by ELISA, RT-PCR and double immunofluorescence labeling, respectively. Phosphorylation levels of Akt, cyclic AMP-responsive element-binding protein (CREB), mitogen-activated protein kinases (MAPKs) cascades, AMP-activated protein kinase (AMPK) and expression of SIRT1(Silent information regulator T1) were measured by western blot. Wortmannin (1 µM), a specific phosphatidylinositol 3-kinase (PI3-K) inhibitor, was used to determine if PI3-K/Akt signaling pathway might be involved in resveratrol's action on RAW 264.7 cells. Resveratrol significantly attenuated the LPS-induced expression of nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in RAW 264.7 cells. Resveratrol increased Akt phosphorylation in a time-dependent manner. Wortmannin, a specific phosphatidylinositol 3-kinase (PI3-K) inhibitor, blocked the effects of resveratrol on LPS-induced RAW 264.7 cells activation. In addition, PI3-K inhibition partially abolished the inhibitory effect of resveratrol on the phosphorylation of cyclic AMP-responsive element-binding protein (CREB) and mitogen-activated protein kinases (MAPKs) cascades. Meanwhile, PI3-K is essential for resveratrol-mediated phosphorylation of AMPK and expression of SIRT1.
This investigation demonstrates that PI3-K/Akt activation is an important signaling in resveratrol-mediated activation of AMPK phosphorylation and SIRT1 expression, and inhibition of phosphorylation of CREB and MAPKs activation, proinflammatory mediators and cytokines production in response to LPS in RAW 264.7 cells.
白藜芦醇是一种天然多酚化合物，具有心脏保护、抗癌和抗炎特性。我们研究了白藜芦醇保护 RAW 264.7 细胞免受炎症损伤的能力，并探讨了白藜芦醇抑制 RAW 264.7 细胞的作用机制。
方法/主要发现：用白藜芦醇（1、5 和 10 µM）和/或 LPS（5 µg/ml）处理鼠源 RAW 264.7 细胞。通过格里希试剂和 ELISA 测定一氧化氮（NO）和前列腺素 E2（PGE2）。通过 ELISA、RT-PCR 和双免疫荧光标记分别分析促炎蛋白和细胞因子的 mRNA 和蛋白水平。通过 Western blot 测定 Akt、环磷酸腺苷反应元件结合蛋白（CREB）、丝裂原活化蛋白激酶（MAPKs）级联、AMP 激活的蛋白激酶（AMPK）和 SIRT1（沉默信息调节因子 T1）的磷酸化水平。用特异性磷脂酰肌醇 3-激酶（PI3-K）抑制剂 wortmannin（1 µM）确定 PI3-K/Akt 信号通路是否参与白藜芦醇对 RAW 264.7 细胞的作用。白藜芦醇显著减弱 LPS 诱导的 RAW 264.7 细胞中一氧化氮（NO）、前列腺素 E2（PGE2）、诱导型一氧化氮合酶（iNOS）、环氧化酶-2（COX-2）、肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）的表达。白藜芦醇呈时间依赖性增加 Akt 的磷酸化。特异性磷脂酰肌醇 3-激酶（PI3-K）抑制剂 wortmannin 阻断白藜芦醇对 LPS 诱导的 RAW 264.7 细胞激活的作用。此外，PI3-K 抑制部分消除了白藜芦醇对环磷酸腺苷反应元件结合蛋白（CREB）和丝裂原活化蛋白激酶（MAPKs）级联磷酸化的抑制作用。同时，PI3-K 对于白藜芦醇介导的 AMPK 磷酸化和 SIRT1 表达至关重要。
本研究表明，PI3-K/Akt 激活是白藜芦醇介导的 AMPK 磷酸化和 SIRT1 表达以及 LPS 诱导的 RAW 264.7 细胞中 CREB 磷酸化和 MAPKs 激活、促炎介质和细胞因子产生抑制的重要信号通路。
