以肠道菌群为始动因素探讨知母黄柏药对防治糖尿病脑病从“毒”论治的生物学机制

Based on the pathogenesis theroy of "the injury of brain collaterals by toxins" in Traditional Chinese Medicine (TCM), our previous study have confirmed that Zhimu and Huangbai herb pair (ZBHP) improve diabetes cognitive impairment. However, there is a paradox that the active components of saponins and alkaloids components in ZHHP with low bioavailability, it seemed difficult to explain the pharmacological mechanism. Given the impact of intestinal flora on brain function, this study hypothesis that "the anomaly of gut microbiota is the initial factor, ZHHP improves the intestinal microbiota by regulating intestinal microflora, relieves chronic inflammation, increases the stability of BBB and reduces neurotoxic substances, to play the role of improvement of cognitive dysfunction". The low bioavailability components of ZHHP would be selected, DNA sequencing technology, RT-PCR, immunohistochemistry, Western Blot methods would be employed. The effect of the drugs on the intestinal microbiota, intestinal barrier, BBB integrity and "toxins" levels and other key aspects would be evaluated. To reveal the biological mechanism and targets of ZHHP on treating diabetic cognitive impairment, further enrich modern scientific connotation of TCM on treating diabetes encephalopathy based on the "detoxifying" theropy, and provide a basis for the research on innovative TCM for treating diabetic encephalopathy.

基于中医“毒损脑络”病机，我们前期研究证实了“知母黄柏药对”具有改善糖尿病认知损伤的作用。但该“药对”中皂苷及生物碱类有效成分生物利用度低，难以全面解释其发挥药效的作用途径与机制。鉴于肠道菌群对脑功能的调控作用，我们提出以肠道菌群异常为始动因素，“知母黄柏药对”通过调控肠道菌群改善肠道微生态，缓解机体慢性炎症反应，改善肠道及BBB屏障功能以及减少神经毒性物质等环节，发挥改善糖尿病认知功能障碍作用的工作假说，选择“知母黄柏药对”中生物利用度低的有效成分为代表，采用DNA测序、RT-PCR、免疫组化、Western Blot等方法，考察“知母黄柏药对”基于肠道菌群异常的始动因素，对糖尿病肠道微生态、肠粘膜屏障、BBB完整性、神经毒性物质水平等关键环节的调控作用，揭示其治疗糖尿病认知损伤的作用靶点及机制，进一步丰富中医药从“毒”论治糖尿病脑病的现代科学内涵，为开发防治糖尿病脑病创新中药提供思路。

认知功能障碍是近年来越来越受到人们关注的糖尿病中枢神经系统并发症，严重影响患者的生活质量。鉴于其复杂的发病机制，临床仍没有有效的治疗有段。传统医学认为“毒损脑络”是糖尿病认知障碍的关键病机之一，“泻火解毒”为主要治疗原则。另外，近年大量研究表明肠道菌群对中枢神经系统病变具有重要的影响，并且糖尿病患者存在肠道菌群的异常。针对部分中药成分生物利用度低但又有明确药效的矛盾，本研究以肠道菌群为始动因素，选择清热药代表药物“知母黄柏药对”为治疗药物。在明确其通过维持肠道微生态平衡发挥治疗糖尿病脑病的基础上，研究再次针对机体慢性炎症反应、血脑屏障完整性破坏以及脑内微环境改变等糖尿病认知障碍的关键环节，尝试部分阐明知母黄柏药对间接发挥治疗糖尿病脑病的作用途径与机制。研究实施后发现知母黄柏药对可以通过维持糖尿病脑病小鼠的肠道微生物稳态、保护肠黏膜屏障完整性以及调节肠道短链脂肪酸水平发挥改善认知障碍的作用。具体体现在知母黄柏药对治疗可降低糖尿病脑病小鼠肠道Fimicutes与Bacteroidetes的比例、尿液乳果糖-甘露醇比值，增加肠粘液SIgA、黏蛋白MUC2含量、杯状细胞数目以及粪便丙酸、乙酸的含量。进一步的研究也发现知母黄柏药对降低了血和脑炎症因子的水平、皮层IgG渗漏、脑组织犬尿氨酸水平，增加了紧密连接蛋白、血管稳定因子α-SMA、水通道蛋白AQP4的表达以及神经保护因子犬尿酸的水平，表明知母黄柏药对通过维持肠道微生态平衡可以缓解慢性炎症反应、保护血脑屏障完整性、改善脑微环境以及调节犬尿氨酸代谢等糖尿病认知障碍的关键病理环节，部分阐明了知母黄柏药对间接发挥治疗糖尿病脑病的作用途径与机制。进一步丰富中医药从“毒”论治糖尿病脑病的现代科学内涵，为开发防治糖尿病脑病创新中药提供思路