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目的:以园蓝为研究材料,鉴定园蓝花青素提取物(GBBAEs)中的功能性成分的结构,建立脂多糖(LPS)诱导的体外炎症模型,并评价其抗炎作用和初步机制。创新点:首次探究了蓝莓花青素对建立的LPS诱导体外炎症模型的营养干预作用,并初步探究了发挥抗炎机制的作用通路。方法:将RAW 264.7细胞分为对照组(不作处理)和实验组(1μg/ml LPS刺激建模)。实验组进一步分为3个不同浓度组:400μg/ml GBBAEs组、800μg/ml GBBAEs组和1200μg/ml GBBAEs组。用酶联免疫吸附测定(ELISA)试剂盒检测一氧化氮(NO)、前列腺素E2(PGE2)、白细胞介素1β(IL-1β)、白细胞介素6(IL-6)、干扰素γ(INF-γ)等炎症因子的释放量;用实时定量聚合酶链反应(RT-PCR)分析IL-1β、IL-6、TNF-α、环氧合酶-2(COX-2)及单核细胞趋化蛋白-1(MCP-1)的炎症相关基因m RNA的表达水平;用蛋白质印迹法(Western blot法)测定相关炎症蛋白COX-2和NF-κBp65表达水平。结论:试验结果表明,通过ELISA法测定GBBAEs可以显著性抑制NO、PGE2、IL-1β、IL-6、INF-γ等炎症因子的释放;RT-PCR分析阐明在LPS诱导的单核-巨噬细胞RAW 264.7中,GBBAEs可以显著性抑制IL-1β、IL-6、TNF-α、COX-2及MCP-1的炎症相关基因m RNA的表达水平。此外,Western blot法进一步显示GBBAEs对相关炎症蛋白COX-2和NF-κBp65表达具有明显抑制作用,进一步证实GBBAEs通过NF-κB机制通路来发挥抗炎作用。
OBJECTIVE: To identify the structure of functional components of gardening anthocyanin extracts (GBBAEs) using Parkland as research material, establish an in vitro inflammation model induced by lipopolysaccharide (LPS) and evaluate its anti-inflammatory effects and primary mechanisms. Innovation point: This study explores the nutritional intervention of blueberry anthocyanins on the model of LPS-induced inflammation in vitro for the first time and explores the pathway of anti-inflammatory mechanism. Methods: RAW 264.7 cells were divided into control group (untreated) and experimental group (1μg / ml LPS stimulation model). The experimental group was further divided into three different concentration groups: 400μg / ml GBBAEs group, 800μg / ml GBBAEs group and 1200μg / ml GBBAEs group. The levels of nitric oxide (NO), prostaglandin E2 (PGE2), interleukin 1β (IL-1β), interleukin 6 (IL-6), interferon gamma INF-γ) and other inflammatory cytokines. The levels of IL-1β, IL-6, TNF-α, COX-2 and monocytes were determined by real-time quantitative polymerase chain reaction The expression of m-RNA was detected by Western blotting. The expression of COX-2 and NF-κBp65 were detected by Western blot. CONCLUSION: The results showed that GBBAEs could significantly inhibit the release of NO, PGE2, IL-1β, IL-6 and INF-γ by ELISA. RT-PCR analysis demonstrated that LPS-induced monocyte- In RAW 264.7 cells, GBBAEs significantly inhibited the mRNA expression of IL-1β, IL-6, TNF-α, COX-2 and MCP-1. In addition, Western blot further showed that GBBAEs significantly inhibited the expression of COX-2 and NF-κBp65, and further confirmed that GBBAEs exert anti-inflammatory effects through NF-κB pathway.