人参叶调控菌—炎功能轴稳态的抗2型糖尿病物质基础及作用机制研究

Leaves of Panax ginseng (LPG), with abundant natural material resource, is usually neglected for its role of non-traditional medical part. Description of LPG’s medicinal effects found in the ancient Chinese materia medica contained several references to its ability to “quench thirst”. LPG has been documented in Chinese pharmacopoeia (2010 edition) for its “stimulate saliva” and “quench thirst” effects. In our previous study, it has been found that the chemical profile of LPG is totally different from that of roots, which implies the pharmacological activity may be different. These years, scientists found extract of LPG could decrease baseline blood glucose and reduce hyperglycemia in ob/ob mice or diabetic rat caused by STZ. And the leaves extract exhibited significantly more potent anti-diabetic effects than that from the root. However, unclear material base and anti-diabetes mechanism limited exploration of the potential hypoglycemic value of LPG. Recent years, a number of reports indicated that activation of NLRP3 inflammasome involved by gut microbiota plays a central role on the development of type 2 diabetes. Increasing level of gut microbiota-derived lipopolysaccharide (LPS) and ROS induced by hyperglycemia are major factors involved in the onset and progression of inflammation and diabetes. Previously, we found that extract of LPG reduced hyperglycemia and body weight in diabetic rat caused by high-fat diet and STZ. Based on our previous results and the characteristics of gut microbiota and NLRP3 inflammasome, we raise the hypothesis that LPG could regulated the homeostasis of gut microbiota-NLRP3 inflammasome axis, including reducing the level of LPS and ROS, so as to inhibit the activation of NLRP3 and consequently control the development of type 2 diabetes. In this proposal, the material base will be investigated by UPLC-QTOFMS/MS and LC-TQ-MS/MS. The anti-diabetic mechanism will be explored by 16S rRNA PCR、ELISA and other pharmacological technique. As a consequence, the bioactive components of LPG and anti-diabetic mechanism will be revealed, which may promote exploration of anti-diabetic value and contribute to the realization of a resource-saving and environment-friendly recycling resource development.

人参叶资源丰富，我国药典和古代医学论著记载可用于“生津，止消渴”。现代研究表明人参叶有确切的降糖活性。但药效物质基础和作用机制不明已然成为制约人参叶药用价值深度开发的瓶颈问题。肠道菌群介导的NLRP3炎症小体激活是2型糖尿病重要致病原因之一。前期研究发现：人参叶含有侧链异构型达玛烷皂苷等特征性成分；并能有效降低血糖、调节肠道菌稳态和炎症因子。为此，我们提出如下科学假说：人参叶通过调控“菌”(肠道菌)—“炎”(NLRP3炎症小体)轴稳态：即调节肠道菌稳态，减少致病菌数量、脂多糖和ROS水平，从而抑制NLPR3炎症小体激活，发挥降糖作用。拟运用C57BL/KsJ db/db自发性糖尿病小鼠为模型； UPLC-QTOF-MS/MS，16S rRNA RT PCR、ELISA等多学科交叉技术，揭示人参叶调控2型糖尿病的药效物质基础和作用机制，为其降糖价值深度开发和资源充分利用提供科学依据。

人参叶资源丰富，我国药典和古代医学论著记载可用于“生津，止消渴”。现代研究表明人参叶有确切的降糖活性。但药效物质基础和作用机制不明已然成为制约人参叶药用价值深度开发的瓶颈问题。肠道菌群介导的NLRP3炎症小体激活是2型糖尿病重要致病原因之一。本项目基于UPLC-QTOF-MS/MS技术，研究了人参叶体外化学物质基础以及吸收入血成分；运用C57BL/KsJ db/db自发性糖尿病小鼠为模型；16S rRNA RT PCR、ELISA、western blot，代谢组学结合多变量数据统计等多学科交叉技术，揭示人参叶调控2型糖尿病的药效物质基础和作用机制。.本项目主要研究结果如下：.（1）采用UPLC-QTOF-MS/MS的全成分表征手段对人参叶水煎液和醇提液进行整体化学轮廓表征，在人参叶水提物中共鉴定得到45个化合物；人参叶醇提物中鉴定到14个化合物。在煎煮过程中，人参叶皂苷经水解、脱羧等反应，生成了较多短糖链次生苷；.（2）基于优化的UPLC-QTOF-MS/MS技术对人参叶提取物在大鼠体内入血成分进行定性分析，共鉴定得到6个原型化合物；.（3）采用C57BL/KsJ db/db自发糖尿病小鼠为模型，16S rRNA实时荧光定量PCR技术（16S rRNA RT-PCR）对人参叶干预前后小鼠粪便的肠道菌群进行表征，生物信息学分析、鉴定了人参叶干扰后丰度回调的菌群：放线菌门、另枝菌属和毛螺菌属，且COG功能分析及KEGG通路分析表明差异菌群与2型糖尿病的发生发展有密切关联；.（4）结合多学科交叉技术，运用分子生物学（western blot，ELISA）、病理组织学等多种手段进行研究，发现人参叶给药后降低外周循环中促炎因子LBP；减轻附睾脂肪组织中炎细胞浸润；并且抑制附睾脂肪组织中NLRP3，caspase 1的表达。. 综上，我们发现人参叶经水加热提取后其中人参皂苷发生化学转变，较甲醇提取新生成了短糖链次生苷；提取物灌胃给药后，人参皂苷入血成分吸收少；对db/db鼠干预后，发现人参叶可调节肠道菌群稳态，减少致病菌数量；降低外周LPS水平，减轻附睾脂肪组织中炎细胞浸润，抑制NLRP3和caspase 1。本项目将为人参叶降糖药用价值的深入开发及人参叶资源的充分利用提供有力科学依据。