桑葚提取物体外抗炎作用及机制的研究

In vitro Characterization of the Anti-inflammatory Effects of Mulberry Extract and the Molecular Mechanisms

摘要:

本文采用脂多糖（LPS）刺激小鼠腹腔巨噬细胞RAW264.7，建立细胞体外的炎症模型，研究桑葚提取物对LPS诱导巨噬细胞RAW264.7分泌功能的影响及其作用机制。实验用1 μg/mL LPS刺激RAW264.7细胞，在不同浓度样品的干预下，用MTT法检测不同浓度的样品对RAW264.7细胞的作用；用Griess法检测细胞液中NO的含量；用酶联免疫吸附法（ELISA）检测细胞液中PGE2含量；用免疫印迹法（Western Blot）和RT-PCR法检测桑葚提取物对细胞iNOS和COX-2表达的影响；用HPLC法检测桑葚提取物中白藜芦醇的含量。结果表明桑葚提取物浓度在0.5~2 mg/mL范围内对细胞生长无明显影响；在1~2 mg/mL范围内能有效抑制NO和PGE2的分泌并能有效抑制iNOS和COX-2的表达；桑葚提取物中白藜芦醇的含量为107.44±0.48 μg/g。这表明桑葚提取物抑制炎症相关因子表达量，从而减弱促炎症反应，发挥抗炎功效，其抗炎活性可能与桑葚中含有较高的白藜芦醇相关。

Abstract:

An in vitro model of inflammation was established by stimulating the mouse peritoneal macrophage cell line RAW264.7 with lipopolysaccharide (LPS). This model was employed to investigate the anti-inflammatory effects of mulberry extract and the underlying molecular mechanisms. RAW264.7 cells were stimulated with 1 ?g/mL LPS, followed by treatment with different concentrations of the extract. The effect of the extract on RAW264.7 cell viability was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The nitric oxide (NO) and prostaglandin E2 (PGE2) content were determined by the Griess reagent assay and enzyme-linked immunosorbent assay (ELISA), respectively. The expression of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was determined by reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. At the tested concentrations (0.5~2 mg/mL), the mulberry extract did not affect RAW264.7 cell growth. At concentrations ranging from 1 to 2 mg/mL, the extract significantly inhibited NO and PGE2 secretion, as well as iNOS and COX-2 expression. The resveratrol content in the mulberry extract was 107.44±0.48 ?g/g. In conclusion, the results suggest that mulberry extract attenuates LPS-stimulated inflammatory responses and exerts anti-inflammatory effects by suppressing the expression of inflammation-related cytokines; the anti-inflammatory effect of mulberry extract is probably attributable to its high resveratrol content.