苯磷硫胺通过调控RAGE介导小胶质细胞活化干预阿尔茨海默病的作用研究

Our previous studies have demonstrated that benfotiamine ameliorated cognitive function and alleviated cerebral Aβ deposition and tau hyperphosphorylation in APP/PS1 transgenic mice. We also found beneficial effect of benfotiamine on cognitive ability in patients with Alzheimer’s disease (AD) over a period of 18 months administration in a small sample-sized clinical exploration study. However, the underlying mechanism needs to be clarified. Neuroinflammation induced by mal-activated microglia has been demonstrated to play an important role in pathology in AD. Receptor for advanced glycation end-products(RAGE) signaling acts as a critical inducer of inflammation impairment caused by microglia activation. Our preliminary studies revealed that benfotiamine inhibited Aβ-induced RAGE expression and mal-activation of microglia, suggesting that inhibition of microglial RAGE signaling might be involved in the beneficial role of benfotiamine in AD. In the current work, both in vitro and in vivo studies will be employed using microglial culture and APP/PS1 mice to clarify the effect of benfotiamine on neuroinflammation and microglia activation in AD. The present work will provide theoretical basis for the clinical application of benfotiamine in AD.

我们前期研究发现苯磷硫胺能改善APP/PS1转基因AD小鼠学习记忆并减少β-淀粉样蛋白(Aβ)沉积和Tau蛋白过度磷酸化等病理损害，小样本探索性临床研究也显示苯磷硫胺能长时间(超过18个月)改善AD患者认知功能，作用机制仍有待进一步阐明。小胶质细胞介导的神经炎症损害在AD发病机制中的重要性越来越受到重视。晚期糖化终末产物受体（RAGE）被认为是介导Aβ活化小胶质细胞引起炎症损伤的关键通路。我们预实验研究发现，苯磷硫胺可抑制Aβ引起的小胶质细胞活化和RAGE表达，提示RAGE信号通路可能是苯磷硫胺抑制AD小胶质炎症损害的调控靶点。本课题将通过体外细胞实验及在体动物模型研究，明确苯磷硫胺抑制Aβ活化小胶质细胞及其炎症损害的作用，并通过过表达和RNA干扰RAGE等方法研究RAGE信号通路在苯磷硫胺改善AD认知和减少病理损害的作用机制，为苯磷硫胺治疗AD的临床应用提供新的理论依据。

在体研究结果表明：与野生型小鼠相比，APP/PS1转基因AD模型小鼠大脑皮层和海马中大量小胶质细胞激活并围绕于Aβ斑块周围，脑内促炎症因子如IL-1β、TNF-α和iNOS表达明显增加；免疫荧光染色和Western blot结果显示：APP/PS1小鼠脑内RAGE表达明显上调；与小胶质细胞的双标染色显示，增多的RAGE与小胶质细胞存在共定位；且APP/PS1小鼠脑内的RAGE下游MAPK信号通路蛋白ERK、p38和JNK的磷酸化水平均较野生型小鼠组有明显增高。给予苯磷硫胺灌胃8周后，APP/PS1小鼠脑内炎症得到有效抑制、Aβ斑块沉积和小胶质细胞激活减少、RAGE表达下调，JNK磷酸化下降。体外结果显示BV2小胶质细胞经Aβ1-42预处理后，促炎因子IL-1β、TNF-α和iNOS的mRNA表达增多、RAGE表达及其下游信号通路ERK，p38及JNK的磷酸化水平增高。苯磷硫胺能够通过抑制BV2细胞的RAGE表达及其下游信号通路JNK磷酸化，显著减少促炎症因子的分泌。RAGE抑制剂能够模拟苯磷硫胺减少小胶质细胞促炎症因子的表达。综合体内和体外实验提示：苯磷硫胺在AD中发挥作用的机制可能与靶向调制小胶质细胞，减轻炎症反应相关。