乌梅散抗仔猪大肠杆菌性腹泻药效物质基础及作用机制的研究

Wumei san was used for treatment of diarrhea of young animals in veterinary medicine, which came from Yuan-Heng's Therapeutic Treatise of Horses. It was of obvious curative effect in treating piglet diarrhea. Up to now, little information has been reported about effective substance and mechanism of wumei san. The spectrum-effect relationships between HPLC fingerprints and anti-diarrheic activities were investigated using canonical correlation analysis (CCA). The active components were screened from wumei san using bioactivity and fingerprint as index. The effect of effective components was verified by diarrhea model of Escherichia coli infecting mice. Epithelial cells were scraped from mice proximal colon and total RNA was extracted. Semi RT-PCR was adopted to detect the expression of ion channels and carriers which affect the electrolyte transport. Furthermore, immunohistochemistry was adopted to confirm the result of RT-PCR. Piglet intestinal epithelium cell line (IPEC-J2) was treated with ETEC or co-treated with ETEC and effective components. The expression of ion channels and carriers were detected by Semi RT-PCR and western blot analysis. The potential protecting mechanism of wumei san will be elucidated. This work provided new viewpoints and inspirations to search for effective substance from traditional veterinary medicine.

乌梅散为《元亨疗马集》收载的固涩名方，对仔猪腹泻具有明显的疗效，但其药效物质基础及作用机制尚未明确。本项目拟在建立乌梅散指纹图谱基础上，结合乌梅散抗腹泻的药效学试验，经谱效关系分析辨识出与活性相关联的成分，对各活性成分进行分离、鉴定，并依据各活性成分在乌梅散中的含量组配药效组分，进一步采用大肠杆菌感染小鼠腹泻的模型对药效组分的抗腹泻作用进行验证；刮取小鼠近端结肠上皮细胞，采用半定量RT-PCR检测影响肠道吸收和分泌的主要离子通道，利用免疫组织化学的方法验证RT-PCR的结果，应用RT-PCR和western blot方法在猪肠上皮细胞（IPEC-J2）中作进一步证实，从药效组分调控IPEC-J2离子通道的角度，揭示乌梅散抗仔猪大肠杆菌性腹泻的作用机制。本研究成果将为乌梅散质量控制的研究提供必要基础，为抗腹泻中兽药药效物质基础研究提供新的方法和模式。

乌梅散为《元亨疗马集》收载的固涩名方，对仔猪腹泻具有明显的疗效，但其药效物质基础及作用机制尚未明确。本项目在建立乌梅散指纹图谱基础上，结合乌梅散抗腹泻的药效学试验，经谱效关系分析辨识出有9个色谱峰与抗腹泻活性密切相关。进一步通过拆方研究，确定乌梅散中诃子、黄连、乌梅为发挥抗腹泻作用的主要药物，从中筛选出小檗碱、黄连碱、巴马亭、没食子酸、鞣花酸、柯里拉京、诃子素等7个成分为此三味药的主要抗腹泻成分，并且此7个成分均来自于谱效分析中9个关键组分。采用半定量RT-PCR的方法，检测正常组小鼠、腹泻模型组小鼠以及药效组分处理组小鼠的近端结肠上皮细胞主要离子通道的表达变化，结果显示NHE1、NHE2、NHE3、AQP4、AQP8、AE1、CFTR和SGLT1在小鼠近端结肠上皮细胞均存在一定程度的表达。与空白对照组小鼠相比，腹泻组小鼠 NHE1、NHE2、NHE3、AQP4、AQP8、CFTR和SGLT1的表达明显下调，而AE1的表达没有显著性变化。与腹泻组小鼠相比，有效组分处理组小鼠 NHE2、NHE3、AQP4和AQP8的表达明显上调（p<0.05），而NHE1、CFTR和SGLT1的表达不显著。免疫组化结果显示NHE3和AQP4蛋白主要表达于近端结肠上皮刷状缘，腹泻组小鼠NHE3和AQP4蛋白呈少量表达乃至阴性表达，而接受有效组分处理的小鼠，其近端结肠上皮细胞NHE3和AQP4蛋白呈中度表达。本研究首次确定了乌梅散抗腹泻的药效物质基础，并首次发现乌梅散有效组分可上调NHE3和AQP4的表达，提示乌梅散可能通过增强水钠的吸收来发挥治疗腹泻的作用，首次从药效组分调控小肠上皮细胞离子通道的角度，揭示乌梅散抗腹泻的机制。本研究成果将为乌梅散质量控制的研究提供必要基础，为抗腹泻中兽药药效物质基础研究提供新的方法和模式。