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、25、50和100 μg/mL四组质量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 this study, we aimed to investigate the anti-inflammatory activity of polypeptides Asp-Asp-Asp-Tyr (DDDY) and Asp-Tyr-Asp-Asp (DYDD) discovered in fermentation media from Dendrobium aphyllum towards LPS-induced RAW264.7 macrophages. Synthetic D. aphyllum fermentation polypeptides were prepared and subjected to experimentation. Concentrations of fermentation polypeptides that resulted in the highest cell proliferative activity were screened for using the methyl thiazolyl tetrazolium (MTT) assay. The effects of fermentation polypeptides on the phagocytic activity of RAW264.7 cells were determined using neutral red phagocytosis assay. Cell differentiation and changes in morphology were observed under an inverted microscope. Secretion levels of NO, cytokines IL-1β, IL-6, and IL-10, and tumor necrosis factor-alpha (TNF-α) were determined by enzyme-linked immunosorbent assay (ELISA). Fermentation polypeptide concentrations of 12.5, 25, 50, and 100 μg/mL exerted no cytotoxic effects and promoted cell proliferation. Approximately 100 μg/mL DDDY and DYDD and 1 μg/mL LPS enabled cell activation and enhanced RAW264.7 cell phagocytic ability, with relative phagocytic rates of 2.05%, 1.97%, and 2.19%, respectively. An LPS-induced RAW264.7 cell inflammation model was constructed, in which both types of fermentation polypeptides effectively inhibited cell differentiation and restored normal cell morphology. The respective NO secretion capacities of cells in the 100 μg/mL DDDY and DYDD treatment groups decreased to 0.41- and 0.49-times, respectively, compared with that of the LPS group. DDDY and DYDD also significantly and dose-dependently increased anti-inflammatory cytokine secretion and decreased the secretion of pro-inflammatory cytokines. Therefore, DDDY and DYDD exert anti-inflammatory effects on the LPS-induced RAW264.7 macrophages. The results of this study may provide a scientific basis for future investigations of the anti-inflammatory mechanisms of fermentation polypeptides.

广东省自然科学基金项目（2019A1515011283）；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）；国家自然科学基金项目（32001622）