山药多酚6,7-Dihydroxy-2,4-dimethoxyphenan基于COX-2信号通路保护肠粘膜损伤的机制

Chinese yam is a traditional medicinal and edible plant in China, which is commonly used in gastrointestinal health care, while its material basis and mechanism lack of scientific interpretation. During the early stage of the project, the applicant found that the ethyl acetate extract of Chinese yam effectively protected intestinal mucosal cell injury induced by Dextran Sodium Sulfate (DSS) in ulcerative colitis (UC). Then, cyclooxygenase-2 (COX-2) inhibitory activity was used as a screening index, the characteristic polyphenol 6,7-Dihydroxy-2,4-dimethoxyphenan (PC4) with better activity than ibuprofen and the highest content in active component, was isolated and identified from Chinese yam for the first time. As a result, it is speculated that PC4 is most likely related to the health benefit on gastrointestinal. On this basis, the present project intends to confirm the core role of PC4 in protecting intestinal mucosal cell injury in UC model mice experiments. RT-qPCR and Western blot will be employed to analysis the gene transcription and protein factor expression related to intestinal mucosa protection based on COX-2 signal pathway. Further, COX-2 silencing and normal Caco-2 intestinal epithelial cells will be treated with PC4, molecular docking software will be applied to analyze the mechanism of COX-2 inhibition by PC4 absorption and metabolism. As a result, the effect of PC4 on intestinal mucosal injury based on COX-2 signaling pathway will be revealed, the material basis and molecular mechanism of Chinese yam on gastrointestinal health care will be elucidated. The results of the study will provide a theoretical basis for the scientific interpretation of the gastrointestinal health effects of Chinese yam.

山药为我国传统药食两用植物，民间常用于健肠胃的养生保健，但其物质基础和作用机制缺乏科学阐释。申请人前期发现山药乙酸乙酯提取物可有效保护右旋葡聚糖硫酸钠诱导的溃疡性结肠炎(UC)小鼠肠粘膜细胞损伤，继而以环氧化酶-2（COX-2）抑制活性为筛选指标，首次从其中分离鉴定出活性优于布洛芬的多酚PC4，活性组分中含量亦最高，由此推测PC4极可能与山药健肠胃相关。据此，本项目拟进一步设计动物实验确证PC4在保护UC小鼠肠粘膜细胞损伤的核心作用，RT-qPCR和蛋白质印迹法解析PC4基于COX-2相关信号通路在基因转录水平和蛋白因子表达的变化，筛选其保护肠粘膜损伤的通路途径；再将PC4作用于COX-2沉默和正常的Caco-2肠上皮细胞，通过分子对接分析PC4经肠细胞吸收代谢发挥COX-2抑制活性机理，揭示基于COX-2信号通路保护肠粘膜损伤的分子机制。研究结果将为科学解释山药肠胃健康效应提供理论依据。

山药在民间养生中被广泛用于“健肠胃”，改善“久泻不止、泄泻便溏”，其中的物质基础和作用机制一直未有明确定论。申请前期研究发现山药多酚提取物可有效保护右旋葡萄糖磷酸钠诱导的溃疡性结肠炎(UC)小鼠肠粘膜细胞损伤，继而以环氧化酶-2（COX-2）抑制活性为筛选指标，首次从其中分离鉴定出活性优于布洛芬的多酚6,7-二羟基-2,4-二甲基菲(6,7-Dihydroxy-2,4-dimethoxyphenan) PC4，活性组分中含量亦最高。本项目进一步通过动物实验确证了PC4在保护UC小鼠肠粘膜细胞损伤的核心作用。结果发现PC4处理组结肠损伤程度、NO含量、MPO活性、凋亡因子表达量、炎症因子表达量、上皮细胞凋亡率均低于MC组；PC4处理组NF-кB/COX-2信号通路中ERK1/2、NF-кB、pNF-кB、COX-2等蛋白表达量与MC组均具有显著性差异。说明PC4可有效抑制NF-κB/COX-2信号通路的激活，从而预防DSS诱导的结肠炎，保护肠黏膜损伤。进一步通过分子对接、动力学模拟和基因沉默研究PC4作用于NF-κB/COX-2信号通路的机制和靶点，结果表明PC4处理的Caco-2细胞中IL-8和TNF-α的低表达表明PC4比PC2具有更强的抗炎活性。由于羟基和Tyr385之间的氢键，PC4和COX-2的结合更强。COX-2基因沉默后，对照组和PC4组之间的磷酸化核因子κB抑制剂α（pIkBα）、磷酸化NF-κB（pNF-κB）和磷酸化细胞外信号调节激酶1/2（pERK1/2）表达没有显著差异，而这些蛋白表达在未沉默的PC4组中显著降低，这证实COX-2是PC4缓解溃疡性结肠炎的重要靶点，PC4可能通过抑制COX-2的正反馈来抑制NF-κB通路介导的炎症。本研究在提高山药资源利用多元化的同时，也为山药健肠胃的说法提供了科学依据，为可在预防结肠炎方向开发预防结肠炎保健功能食品或辅助药物奠定理论基础。