Department of Basic Medical Sciences, School of Medicine, University of Missouri, 2411 Holmes Street, Kansas City, MO 64108, USA.
Lipids Health Dis. 2012 Jul 10;11:76. doi: 10.1186/1476-511X-11-76.
Altered immune function during ageing results in increased production of nitric oxide (NO) and other inflammatory mediators. Recently, we have reported that NO production was inhibited by naturally-occurring proteasome inhibitors (quercetin, δ-tocotrienol, and riboflavin) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, and thioglycolate-elicited peritoneal macrophages from C57BL/6 mice. In a continuous effort to find more potent, non-toxic, commercially available, naturally-occurring proteasome inhibitors that suppress inflammation, the present study was carried out to describe the inhibition of NF-κB activation and NO, TNF-α, IL-6, IL-1β, and iNOS expression by trans-resveratrol, trans-pterostilbene, morin hydrate, and nicotinic acid in LPS-induced RAW 264.7 cells and thioglycolate-elicited peritoneal macrophages from C57BL/6 and BALB/c mice.
The present results indicate that resveratrol, pterostilbene, and morin hydrate caused significant inhibition (>70% to 90%; P < 0.02) in the activities of chymotrypsin-like, trypsin-like, and post-acidic (post-glutamase) proteasome sites in RAW 264.7 cells at a dose of only 20 μM. These compounds also inhibited the production of NO by RAW-264.7 cells stimulated with LPS alone (>40%; P < 0.05), or LPS + interferon-γ (IFN-γ; >60%; P < 0.02). Furthermore, resveratrol, pterostilbene, morin hydrate, and quercetin suppressed secretion of TNF-α (>40%; P < 0.05) in LPS-stimulated RAW 264.7 cells, and suppressed NF-κB activation (22% to 45%; P < 0.05) in LPS-stimulated HEK293T cells. These compounds also significantly suppressed LPS-induced expression of TNF-α, IL-1β, IL-6, and iNOS genes in RAW 264.7 cells, and also in thioglycolate-elicited peritoneal macrophages from C57BL/6 and BALB/c mice.
The present results clearly demonstrate that resveratrol and pterostilbene are particularly potent proteasome inhibitors that suppress expression of genes, and production of inflammatory products in LPS-stimulated RAW 264.7 cells, and macrophages from C57BL/6 and BALB/c mice. Resveratrol and pterostilbene which are present in grapes, blueberries, and red wine, have been implicated as contributing factors to the lower incidence of cardiovascular disease in the French population, despite their relatively high dietary fat intake. Consequently, it appears likely that the beneficial nutritional effects of resveratrol and pterostilbene are due at least in part, to their ability to inhibit NF-κB activation by the proteasome, thereby suppressing activation of pro-inflammatory cytokines and iNOS genes, resulting in decreased secretion of TNF-α, IL-1β, IL-6, and NO levels, in response to inflammatory stimuli. This is the first report demonstrating that resveratrol and pterostilbene act as proteasome inhibitors, thus providing a mechanism for their anti-inflammatory effects.
衰老过程中免疫功能的改变导致一氧化氮（NO）和其他炎症介质的产生增加。最近，我们报道脂多糖（LPS）刺激的 RAW264.7 细胞和 C57BL/6 小鼠的硫代乙醇酸盐诱导的腹腔巨噬细胞中，天然存在的蛋白酶体抑制剂（槲皮素、δ-生育三烯酚和核黄素）抑制了 NO 的产生。为了寻找更有效、无毒、商业可用、天然存在的蛋白酶体抑制剂来抑制炎症，本研究旨在描述反式白藜芦醇、反式紫檀芪、密勒醇和烟酸对 LPS 诱导的 RAW264.7 细胞和 C57BL/6 和 BALB/c 小鼠的硫代乙醇酸盐诱导的腹腔巨噬细胞中 NF-κB 激活和 NO、TNF-α、IL-6、IL-1β 和 iNOS 表达的抑制作用。
本研究结果表明，白藜芦醇、紫檀芪和密勒醇在 20 μM 时对 RAW 264.7 细胞中的糜蛋白酶样、胰蛋白酶样和酸性后（谷氨酰胺酶后）蛋白酶体位点的活性有显著抑制作用（>70%至 90%；P<0.02）。这些化合物还抑制了 LPS 单独刺激的 RAW-264.7 细胞中 NO 的产生（>40%；P<0.05），或 LPS+干扰素-γ（IFN-γ；>60%；P<0.02）。此外，白藜芦醇、紫檀芪、密勒醇和槲皮素抑制了 LPS 刺激的 RAW 264.7 细胞中 TNF-α的分泌（>40%；P<0.05），并抑制了 LPS 刺激的 HEK293T 细胞中的 NF-κB 激活（22%至 45%；P<0.05）。这些化合物还显著抑制了 LPS 诱导的 RAW 264.7 细胞中 TNF-α、IL-1β、IL-6 和 iNOS 基因的表达，以及 C57BL/6 和 BALB/c 小鼠的硫代乙醇酸盐诱导的腹腔巨噬细胞中这些基因的表达。
本研究结果清楚地表明，白藜芦醇和紫檀芪是特别有效的蛋白酶体抑制剂，可抑制 LPS 刺激的 RAW 264.7 细胞和 C57BL/6 和 BALB/c 小鼠的巨噬细胞中基因的表达和炎症产物的产生。白藜芦醇和紫檀芪存在于葡萄、蓝莓和红酒中，与法国人患心血管疾病的发病率较低有关，尽管他们的饮食中脂肪含量相对较高。因此，白藜芦醇和紫檀芪的有益营养作用似乎至少部分归因于它们抑制蛋白酶体 NF-κB 激活的能力，从而抑制促炎细胞因子和 iNOS 基因的激活，导致 TNF-α、IL-1β、IL-6 和 NO 水平的分泌减少，从而对炎症刺激产生反应。这是第一个证明白藜芦醇和紫檀芪作为蛋白酶体抑制剂发挥作用的报告，从而为它们的抗炎作用提供了一种机制。
