基于AGEs/RAGE信号阐释糖尿病脑病“毒损脑络”病机的生物学机制及知母黄柏药对的干预作用

Diabetic encephalopathy is one of a serious complication of diabetes, cognitive dysfunction is the main symptom. It’s believed that "the injury of brain collaterals by toxins" is the key pathogenesis in Traditional Chinese Medicine (TCM). Modern medicines have shown that diabetes is accompanied by microvascular injury, blood-brain barrier (BBB) integrity and transshipment dysfunction, and then lead to increased inflammatory cytokines, Aβ and other neurotoxin substances in the brain. Based on this, we hypothesis that the disturbed of brain microenvironment mediated by the BBB integrity and transshipment dysfunction is the most important biological mechanisms of "the injury of brain collaterals by toxins" pathogenesis in diabetic encephalopathy. The therapies and formulas of nourishing Yin, purging Fire and detoxification could treat diabetic encephalopathy by inhibiting AGEs/RAGE signal, stabilizing BBB and then improving brain microenvironments. Zhimu and Huangbai herb pair would be selected in the present study, the effect of the drugs on the BBB integrity and transshipment function, brain microenvironment, neuroprotection and neurogenesis would be evaluated in vitro and in vivo based on AGEs/RAGE signal. To explain the modern biological mechanisms of "the injury of brain collaterals by toxins" pathogenesis, and clarify that the therapies and formulas of nourishing Yin, purging Fire and detoxification could intervene brain microenvironment during treatment on diabetic encephalopathy.

糖尿病脑病主要表现为认知损伤及痴呆，传统医学认为“毒损脑络”是其关键病机，滋阴清热解毒是其基本治法之一，但该病机的生物学机制尚不明确。现代医学研究表明糖尿病患者存在体内AGEs水平升高，血脑屏障（BBB）完整性及Aβ转运功能异常，脑内小胶质细胞过度活化的现象。基于此，我们推测BBB完整性及转运功能异常导致的脑组织微环境紊乱可能是糖尿病脑病“毒损脑络”病机的关键生物学基础，滋阴清热解毒治法方药可以通过抑制AGEs/RAGE信号，干预BBB完整性，调节脑内微环境而发挥治疗糖尿病脑病的作用。课题拟基于AGEs/RAGE信号，选择知母黄柏药对为代表药物，以AGEs抑制剂及RAGE激动剂为工具药，体内外研究相结合，考察药物通过干预BBB稳定性及Aβ转运功能，抑制小胶质细胞活化，进而改善脑内微环境发挥神经保护的作用及机制，以期阐释“毒损脑络”病机的现代生物学机制，并明确滋阴清热解毒治法方药的干预作用。

近年来糖尿病轻度认知功能障碍等新的糖尿病并发症越来越受到人们的关注，但目前尚缺乏很好的治疗策略。本研究基于糖尿病轻度认知功能障碍的“毒损脑络”病机，选择滋阴清热解毒代表方药知母黄柏药对（知柏）进行干预，基于微环境相关细胞因子的调节，考察了知柏防治糖尿病脑病的作用及机制。.采用链脲霉素复合高脂饮食方法复制糖尿病模型，饲养6周使其出现自发性轻度认知功能障碍。设置正常对照组、模型组，知母黄柏药对组，灌胃给药6周，Morris水迷宫实验明确了知柏具有改善糖尿病认知功能障碍的作用。Elisa实验结果发现知柏能显著降低血清AGEs水平，明显升高BDNF等生长因子水平，提示知柏能够改善小鼠机体微环境。离体实验也证实了知母黄柏药对有效成分的神经、内皮保护作用。.脑组织病理结果显示知柏可改善实验小鼠海马组织病变；Brdu掺入及免疫组织化学染色结果显示，知柏可以增加实验小鼠海马BrdU、DCX、SYN阳性细胞数；Western Blot及PCR-Array结果显示，知柏可上调实验小鼠海马p-Akt、p-mTOR、p-Raptor、p-S6K1蛋白表达，下调RAGE、FoxO3、Beclin-1、LC3B蛋白表达。提示知柏可能通过抑制AGEs/RAGE、调节自噬等信号促进糖尿病脑病脑病小鼠海马神经再生。.鉴于骨骼肌分泌功能对维持机体微环境的重要作用，本研究考察了知柏对实验小鼠骨骼肌功能的影响。小鼠转棒仪和抓力测定结果显示，知柏能明显增强糖尿病小鼠抓力；蛋白定量及骨骼肌病理组织切片结果显示，知柏能增加糖尿病小鼠股四头肌重量及蛋白含量，增加骨骼肌肌纤维直径；Western Blot结果发现，知柏能上调骨骼肌p-Akt、p-mTOR、p-Raptor、p-S6K1蛋白表达。提示知柏改善机体微环境可能与抑制糖尿病小鼠骨骼肌萎缩有关；.本研究进一步证实了知母黄柏药对具有改善糖尿病脑病小鼠机体微环境，改善认知功能障碍的作用。其机制可能与抑制AGEs/RAGE信号，调控Akt/mTOR信号等有关。本研究为中药防治糖尿病中枢神经系统并发症提供了新的策略与依据，为相关创新中药的研发提供了一定实验依据。