补肾虫类中药九香虫抗糖尿病肾病活性成分及其作用机理研究

Diabetic nephropathy (DN) has become a public health concern in recent years because it can develop into renal failure, with a high risk of morbidity and mortality. Diabetes mellitus is a primary driver of DN. With changes in lifestyle and dietary patterns, the number of patients with DN will increase in the coming years. Although some preventive measures, such as glycemic and blood pressure control, are currently used to reduce the kidney damage, the cause of DN is largely unknown, the effective drugs for DN are also scarce, which makes the treatment of DN challenging. Before, we have conducted much related research on DN targeting on oxidative stress and chronic inflammation by collaborating with physicians of South Hospital of China. More recently, we hypothesized that insects are potential sources of DN basing on TCM theory or philosophy of "insects being likely used to promoting blood circulation" and "cell mobility being closely related with Yang-Qi". Our preliminary oriented study indicated that both petroleum ether (PE)and acetone (AC) extracts of Aspongopus chinensis could improve the mobility of high-glucose-induced mesangial cells similar to those of normal-glucose-induced mesangial cells. Furthermore, we found that novel dopamine dimers are active compounds,which could also significantly down-regulated mRNA of IL-2; LC/MS analysis indicated that AC extract still contains diverse dopamine oligomers. However, whether these dopamine oligomers are active or not, their potency and structure-activity relationship (SAR) remain largely unknown. In addition, the active lipophilic compounds in the PE extract are also to be explored. This study will first focus on the isolation and structure identification of novel dopamine oligomers from AC extract by using LC/MS strategy, and compounds from PE extract. We will then study the SAR of compounds and explore the mechanisms of action of a potential compound. This study is expected not only to clarify the anti-diabetic nephropathy agents of Aspongopus chinensis, but to track a promising drug lead or candidate of DN from a unique insect source which will greatly benefit DN patients.

糖尿病肾病（DN）是糖尿病的主要并发症，也是导致终末期肾衰竭的主要原因。目前，临床上以血糖和血压控制为主，尚缺乏有效的肾保护药物。前期，我们和南方医院肾内科合作从氧化应激、慢性炎症等方面开展了大量工作。新近，基于"藤类搜风，虫类通络（微血管）"，的中医理论，初步定向筛选发现九香虫石油醚（PE）及丙酮萃取部位(AC)均能显著增强高糖诱导的病态肾系膜细胞生命活力，并首次发现AC中结构新颖的多巴胺二聚体为其活性成分之一，可显著抑制IL-2基因表达；LC/MS分析显示AC部位富含多巴胺寡聚体，但其是否具有活性、活性强弱及构效关系，以及PE部位的活性成分类型等尚不清楚。本项目拟采取LC/MS方法针对性获取AC部位的多巴胺寡聚体，并研究PE部位的活性成分，以氧化应激、胶原、细胞因子等研究其肾保护作用及信号转导途径，以期在阐明九香虫抗DN活性成分基础上，为从自然界昆虫药中寻找有效的抗DN药物做出贡献。

九香虫作为一种药食兼用的昆虫具有理气止痛、温中助阳作用，常被用来治疗疼痛，消化不良，肾脏疾病等。但长期以来针对药用昆虫的基础研究国内外长期薄弱，九香虫也是如此，其制约了以九香虫为原料的新药研发和应用。我们前期发现九香虫提取物具有抗糖尿病肾病作用，故此有必要弄清该昆虫的化学成分及其结构特点、药效物质及其作用特点等。为此，本项目分别对5 kg和20 kg干燥虫体进行了研究。从干燥虫体中先后获得40个和81个非肽类小分子，合计共121个（不重复者103个），其中新成分为47个，结构类型涉及生物碱与氨基酸的杂合体、N-乙酰化的多巴胺及其以不同方式形成的寡聚体和与碱基形成的杂聚体、碱基和脂肪链形成的杂聚体、生物碱类、倍半萜等等。抗糖尿病肾病活性的研究结果显示多巴胺衍生物多具有活性；鉴于炎症在糖尿病肾病中的作用，我们对其中部分化合物也进行了抗炎活性研究，发现COX-2抑制活性9个，TNF-α抑制活性4个，JAK3抑制活性2个；并从TGF-β/Smads等角度初步探讨了其作用机制。除肾病活性方面，还发现核苷碱基衍生物具有促进成体神经干细胞增殖作用，提示这些化合物对神经系统损伤性疾病具有潜在价值。值得一提的是，我们对所有消旋体进行了手性拆分并通过量子化学计算确定了其绝对构型，对多巴胺和腺嘌呤杂合化合物进行了全合成研究，并且富集量进行了振动圆二色谱（VCD）测试及计算，确定了具有挑战性的手性中心的绝对构型。目前部分工作发表SCI 论文3篇（其中1篇Org Lett），获授权专利1件；新颖杂合体的全合成工作等尚待整理发表。综上，本项目从九香虫中获得103个小分子，其有利于摸清该昆虫化学成分的家底，也将对推动动物药学科发展发挥积极作用。发现的药理活性一定程度上求证了传统疗效的科学性，同时也提示昆虫内源性活性物质的重要性；发现的天然产物杂化和消旋化揭示了昆虫天然产物可能的重要特征。另外也是重要的一点是本项目为后续开展磷酸酯和吡啶生物碱杂合化合物的活性研究和氨基酸N-烷基化物的形成机制提供了新的生长点。