为了研究兜唇石斛发酵多肽Asp-Asp-Asp-Tyr（DDDY）、Asp-Tyr-Asp-Asp（DYDD）对LPS诱导的RAW264.7巨噬细胞抗炎活性。以合成的兜唇石斛发酵多肽为研究对象，采用噻唑蓝法筛选增殖活性最高的发酵多肽浓度，通过中性红吞噬实验和倒置显微镜观察发酵多肽对细胞的吞噬作用和分化形态变化，使用ELISA试剂盒测定细胞中NO及细胞因子IL-1β、IL-6、IL-10和肿瘤坏死因子TNF-α的分泌量。结果表明：12.5 μg/mL、25 μg/mL、50 μg/mL和100 μg/mL四种浓度的发酵多肽对细胞无毒性并有增殖作用；100 μg/mL的DDDY和DYDD和1 μg/mL的LPS处理可以激活细胞，增强细胞吞噬能力，相对吞噬率为2.05%、1.97%和2.19%；构建LPS诱导的RAW264.7细胞炎症模型，发现两种发酵多肽处理有效抑制细胞分化，使细胞恢复正常形态；抑制细胞NO分泌能力，100 μg/mL DDDY和DYDD处理组的NO分泌能力降低到LPS组的0.41倍和0.49倍；并且对抑炎细胞因子分泌能力的提高和促炎细胞因子的降低有显著效果，均表现出剂量反应关系。由此可知，DDDY和DYDD对LPS诱导的RAW264.7巨噬细胞炎症反应具有抗炎作用，为后面探究发酵多肽的炎症机制提供理论支持。

In order to investigate the anti-inflammatory activity of fermentation polypeptides Asp-Asp-Asp-Tyr (DDDY) and Asp-Tyr-Asp-Asp (DYDD) of Dendrobium aphyllumin on LPS induced RAW264.7 macrophages. In this study, synthetic fermentation polypeptides of Dendrobium aphyllumin were selected as the object, the methyl thiazolyl tetrazolium (MTT) assay was used to screen the concentration of the fermentation polypeptides with the highest proliferation activity, and neutral red phagocytosis assay was used to determine phagocytosis of cells, microscope was used for examining morphological changes in these cells, and the secretion levels of NO and interleukin IL-1β, IL-6, IL-10 and tumor necrosis factor TNF-α were determined by enzyme-linked immunosorbent assay (ELISA). The results showed that the concentrations of 12.5 μg/mL, 25 μg/mL, 50 μg/mL and 100 μg/mL fermentation polypeptides had no toxic and increased proliferation. RAW264.7 cells were activated by 100 μg/mL DDDY and DYDD and 1 μg/mL LPS, and the phagocytosis of RAW264.7 cells was 2.05%, 1.97% and 2.19%. The inflammatory model of RAW264.7 cells induced by LPS was established, and it was found that two kinds of fermentation polypeptides could effectively inhibit cell differentiation, restore normal morphology of cells, and inhibit the NO secretion capacity of cells, which the NO secretion capacity of 100 μg/mL DDDY and DYDD groups decreased to 0.41 times and 0.49 times of LPS group. In addition, it had a significant effect on the secretion of the increase anti-inflammatory cytokines and the decrease of pro-inflammatory cytokines, and all showed a does response. In conclusion, DDDY and DYDD have anti-inflammatory effects on LPS-induced RAW264.7 inflammatory, which provides theoretical support for future research of the inflammatory mechanism of fermentation polypeptides.

2019年广东省自然科学基金项目（2019A1515011283）；2020年广州市基础研究计划基础与应用研究基础项目（202002030383）；江门市基础与应用基础研究重点项目（2021030103330007214）；国家自然科学基金资助项目（32001622）