基于RAS信号通路薏苡仁生物肽干预痰湿型高血压作用机制研究

With the improvement of people’s quality of life, the proportion of patients with damp phlegam type hypertension is gradualy increasing. The seed of adlay (Coix lachryma-jobi L.var.ma-yuen Stapf) , one of effective herbs inducing diuresis to remove dampness shows distinct advantage in treating this disease. As for the effective components, only Coix seed oil and coixol were fewly reported, while bioactive peptide which is rich in Coix seed and its funtion haven’t been aroused attention at present. Renin-angiotensin system (RAS) plays a vital role in blood pressure regulation. In our previous research, several peptide fractions and monomers with satisfied angiotensin converting enzyme inhibitory activity in vitro and antihypertension effect in vivo had been obtained from Coix glutelin. Therefore, we draw a hypothesis that hypotensive effect of Coix peptides might be mainly due to its intervening renin-angiotensin system (RAS). In this research, glutelin will be firstly hydrolyzed under the simulated digestion condition, and then the detailed quantity and sequence of antihypertensive peptides will be analyzed by combining the methods of the chromatography and mass spectrometry. Rats with phlegm type hypertension and human vascular endothelial cell will then be appied in this project. The effects of Coix biaoctve peptides on the expression pattern of RAS pathway will be inspected in body, organ and cell level, respectively based on the techniques involved immunohistochemistry, RNA-seq, shRNA and gene overexpression. Meanwhile, target genes will be detected. From definitely new perspective, this project will fully unveil the profound mechanism of Coix seed treating damp phlegam type hypertension and perfect its effective substance theory. New drugs for the prevention and treatment of hypertension will finally be obtained from bioactive peptides of Chinese herbs.

随着生活水平的提高，痰湿型高血压病人比例逐渐提高。薏苡仁为利水渗湿之良药，治疗该病具有明显优势，其药效成分仅见薏苡仁素和薏苡仁油等少量研究，然而该药含量丰富的多肽组分及功能尚未引起关注。肾素-血管紧张素系统（RAS）为血压调控重要信号通路。本课题组从薏苡仁谷蛋白中获得了高血管紧张素转化酶抑制活性的多肽组分和单体，且体内降压作用明显。由此我们推测：薏苡仁生物肽组分通过调控RAS系统而发挥降压作用。为此，我们首先模拟胃肠道消化薏苡仁谷蛋白，采用色谱、质谱等方法，深刻解析薏苡仁降压肽组成与结构。然后以痰湿型高血压大鼠和人血管内皮细胞为研究对象，采用免疫组化、转录组测序、shRNA和基因过表达等多种技术，从整体-器官-细胞三个层次，探讨薏苡仁生物肽对于RAS通路的影响，发现其靶基因。本课题将从新视角揭示该药治疗痰湿型高血压作用机制，丰富其药效物质研究, 为中医药生物肽预防和治疗高血压提供新型药物。

高血压是临床常见慢性病之一，严重威胁着人类健康，随着生活水平的提高，痰湿型高血压病人比例逐渐提高。薏苡仁为中医临床利水渗湿之良药，治疗该病具有明显优势。课题组前期研究表明薏苡仁生物肽降压作用明确，但对于痰湿型高血压疗效和作用机制尚不明确。. 本课题研究表明，不同产地薏苡仁主成分含量存在一定差异，其中贵州和河北产地样本蛋白含量较高，辽宁和云南产地样本降压药用潜能较大。构建了薏苡仁转录组数据库，共获得76164个Unigene，并注释到七大数据库(NR、NT、Swissprot、COG、KEGG、GO及Interpro)，共计得到51160个CDS，从而成功构建薏苡仁个性化蛋白数据库。生物肽组分经分离纯化和质谱鉴定后，获得高活性生物肽单体。采用4种不同方法构建痰湿壅盛型高血压大鼠模型，其中模型3成功率高，且更符合临床表征。薏苡仁生物肽组分和单体显著降低痰湿型高血压大鼠血压，其中生物肽单体BUCM_L01和BUCM_L02降压作用与临床常用降压药物卡托普利相当，且心脏结构明显改善。相比对照组，该疾病动物主要脏器和血管内皮细胞RAS信号通路关键成员基因和蛋白表达发生改变，薏苡仁生物肽可部分逆转其表达改变，且显著抑制IL-1β、VCAM等炎症基因的表达。其血流变、血脂指标和主动脉结构显著改善，同时肝脏细胞中脂肪堆积明显降低。薏苡仁生物肽对于LPS、AngII、高糖、辐射等损伤的血管内皮细胞具有一定保护作用。转录组学研究找到了差异表达基因和所涉及的主要信号通路。本研究为后续薏苡仁的临床用药和中药源生物肽的开发提供了进一步的理论依据。