羟基积雪草苷通过调节TLR4受体介导的神经损伤/保护通路发挥抗脑缺血再灌注损伤作用机制研究

MyD88-dependent NF-KB pathways, mediated by TLR4, play an important role in the induction of I/R injury and secondary neuroinflammation. Madecassoside, a triterpenoid derivative isolated from Centella asiatica, exhibits low toxicity effect. Our previous research has indicated that madecassoside had remarkable neuroprotective effect against cerebral ischemia reperfusion, and the potential mechanism might be relevant to anti-inflammation effect, via inhibiting TLR4/MyD88/NF-κB signal pathway,and probably as well as as well as promoting PI3K/Akt pathway. Based on the previous studies, We will further study the following questions: 1. Whether MA can promote other TLR4-mediated pathways, such as PI3K/Akt pathway, by disrupting the interaction between TLR4 and MyD88. 2. whether TLR4 is a critical modulator of cell suvival and ischemic injury in the brain; This study will provide new perspectives for the treatment of ischemic brain injury with madecassoside.

Toll样受体4(TLR4)介导的MyD88依赖性NF-κB信号通路对脑缺血再灌注(I/R)损伤及卒中后炎症反应至关重要。羟基积雪草苷(MA) 是从中草药积雪草中提取的毒性极低的单体，我们在前期研究发现，MA对大鼠脑I/R损伤具显著脑保护作用，其作用机制不仅与其抑制TLR4/MyD88/NF-κB损伤通路、发挥显著抗炎等作用有关，与PI3K/Akt神经保护通路的激活可能也相关。本项目拟立项于MA的多靶点抗I/R损伤前期工作基础，对TLR4在脑缺血再灌注损伤中的双重作用进行探索性研究，考察MA对缺血再灌注损伤中TLR4介导的TLR4/MyD88/NF-κB、PI3K/Akt两种不同信号的调控机制，阐明MA抗I/R损伤的药理学功能及分子机制，为开发以TLR4为靶点的新型脑缺血治疗药物提供科学依据。

如何减轻脑缺血再灌注损伤，是治疗脑卒中的关键，也是研究有效的脑卒中治疗药物的热点和难点，目前缺少安全、有效的神经保护剂。中药提取物羟基积雪草苷（MA）具有抗炎、抗氧化、促进伤口愈合等广泛的药理学活性，我们首次发现MA可以减小脑缺血/再灌损伤大鼠的皮层梗死面积，减少神经元凋亡，减轻中枢炎症反应，改善神经功能损伤；同样，MA可以减轻OGD/R以及LPS诱导的BV2损伤以及炎症反应。在大鼠MCAO模型和OGD/R损伤的BV2细胞模型上，进一步揭示了TLR4 是调节缺血再灌注损伤和细胞存活的关键分子，TLR4 被激活后会同时触发MyD88/NF-κB依赖性神经损伤通路和TRIF 依赖的PI3K/Akt 保护性通路。我们通过免疫共定位、pull-down、SPR等技术进一步证明了MA可以与TLR4直接结合，通过抑制TLR4/MyD88/NF-κB 通路、激活PI3K/Akt 通路发挥其显著的抗炎、抗凋亡等作用，从而具有显著的抗脑缺血再灌注损伤效果。本研究阐明了MA 抗脑缺血损伤药理学分子机制，为开发以TLR4 为靶点的新型脑缺血再灌注损伤治疗药物提供理论依据。