补肾活血法防治糖尿病脑微血管病变的作用及机制研究

Diabetic cerebral microangiopathy is a common vascular complication of type 2 diabetes mellitus(T2DM). In Traditional Chinese Medicine, kidney deficiency and damage of essence syndrome and syndrome of static blood obstructing the collateral are regarded to be basic pathogenesis of cognitive impairment caused by diabetic cerebral microangiopathy. Nourishing kidney and activating blood method is commonly used in clinical application, while there is a lack of evidence to prove the efficacy and pharmacological mechanism so far. It is reported that LRP-1/RAGE mediates the internal and external transport of Aβ across blood-brain barrier. Because of its abnormal transport , Aβ deposit at the vessel wall, and attributes to diabetic cerebral microangiopathy, which is a main risk factor for cognitive impairment. Previous researches confirm that modified Wu-Zi-Yan-Zong prescription(MWP), a prescription aiming at nourishing kidney, can improve cognitive impairment and protect neurons by reducing toxicity of Aβ. And further studies show that, Bushen Huoxue prescription(BSHX), in which leech is added based on MWP, can improve T2DM’s memory deterioration syndrome by nourishing kidney and activating blood. In this study, Aβ and cerebral microangiopathy are focused as breakthrough points, and in vivo and in vitro experiments are carried out to study the efficacy of nourishing kidney and activating blood method in improving cognitive impairment by preventing cerebral microangiopathy caused by T2DM. What’s more, the study will declare whether the efficacy is associated with the regulation of LRP-1/RAGE, so as to affect the internal and external transport of Aβ across blood-brain barrier. The study will not only provide theoretical basis for the clinical trial of nourishing kidney and activating blood method, but also pave a way for the basic research on the prevention and treatment of diabetic vascular complications.

脑微血管病变是2型糖尿病（T2DM）常见血管并发症，可导致认知功能障碍。中医认为肾精亏虚、瘀血阻络是该病的核心病机，临床多采用补肾、活血治法，但在药效及药理机制方面尚缺乏明确的实验依据。研究报道LRP-1/RAGE途径介导β淀粉样蛋白(Aβ)在血脑屏障内外转运。Aβ转运异常导致Aβ在脑微血管壁上沉积是诱发DM脑微血管损伤，出现认知功能障碍的主要病理机制之一。前期研究证实补肾法（加味五子衍宗方）可通过抑制Aβ毒性改善认知功能，补肾活血法（加味五子衍宗方加味水蛭）在降血糖同时改善T2DM患者记忆力。本研究以Aβ与脑微血管损伤间的联系为切入点，以整体离体实验研究补肾活血法防治T2DM脑微血管病变，改善认知功能的药效作用；并阐明该作用是否与通过LRP-1/RAGE途径调控Aβ在脑内外的转运有关。本研究将为指导补肾活血治法临床应用提供理论依据，同时为中医药防治糖尿病血管并发症的基础研究开启新的思路。

前期研究证实加味五子衍宗方可通过抑制A毒性改善MCI患者的认知功能，挽救损伤的神经元。临床中发现在该方基础上加味水蛭组方而成的补肾活血方（BSHX）可在控制血糖的基础上有效改善2型糖尿病（T2DM）患者记忆力减退的症状。为明确该方防治T2DM脑血管并发症的作用，本课题拟基于体内外实验，研究BSHX改善T2DM合并认知障碍的作用及机制。体内研究证实BSHX在调节糖脂代谢的基础上，能基于LRP-1/RAGE途径，拮抗糖基化终末产物（AGEs）损伤海马神经元，降低Aβ在脑内外的表达，上调ZO-1等血脑屏障紧密连接蛋白，改善脑微循环，进而提高KK-Ay小鼠的认知功能。体外研究证实BSHX及其拆方能拮抗氧化应激、炎症和内质网应激损伤，提高LRP-1等蛋白的表达，抑制RAGE的表达，从而发挥神经保护作用，且全方效果优于拆方。对全方的进一步机制研究显示全方具有明显的抗AGEs诱导Bend.3脑微血管内皮细胞损伤作用。该方可以通过下调FoxO1/3蛋白的表达并抑制其向核转移，进而抑制下游凋亡蛋白Bim的表达和caspase的激活来实现脑细胞保护作用。以PC12细胞为模型，我们进一步证实全方具有明显的抗高糖诱导PC12细胞损伤作用，该方可通过调节caspase-3 及JNK/p38 MAPK信号通路来保护PC12细胞免受高糖的毒性损伤。全方的有效成分之一淫羊藿苷（ICA）和五味子醇甲（SCH）也具有明显的抗AGEs诱导PC12细胞损伤作用。其中ICA是通过特异性的靶向Bax蛋白调节其生物功能，最终实现抗AGEs诱导的神经凋亡作用；而SCH是通过特异性的靶向溶酶体ATP6V0d1蛋白，调节溶酶体酸度，进而诱导Bim蛋白自噬性降解，进而实现神经保护作用的。临床试验证实BSHX能不同程度的改善2型糖尿病合并认知功能障碍患者的血糖，提高患者的认知功能，其作用机制与降低血清中的HCY及hs-CRP含量密切相关。本研究为BSHX方改善糖尿病认知功能障碍提供了现代科学诠释，也为阐述补肾活血中医药理论提供了实验依据。