基于PI3K/Akt-SIRT1信号通路探讨补阳还五汤促进缺血性脑中风康复的药效物质基础及作用机制

Buyang Huanwu decoction (BYHWD) has significant efficacy in the rehabilitation of ischemic stroke. The research confirmed that BYHWD has the functions of “nourish qi and promot blood circulation and regeneration”, and it promotes the recovery of ischemic stroke through the brain microvessels and neuronal axons’ regeneration, but the effects of promoting neuron regeneration and its mechanism are not yet clear. So task group put forward scientific hypothesis that “BYHWD can promote proliferation of neuronal cells and inhibition apoptosis by regulating the PI3K / Akt and SIRT1 signal pathways in stroke state, it may be an important mechanism for promoting ischemic stroke rehabilitation”. The project intends to screen the affinity components of BYHWD by microdialysis combined with membrane-based solid-phase chromatography, and getting the molecular structure of the active substance through LC/MS and NMR technology; Western blotting and other methods were used to detect the effects of affinity components on the expression of related proteins and genes in ischemic lesions of animal models, and to verify their pharmacological activities; Combined with in vitro cell experiment, explore the mechanism of BYHWD promote proliferation of neuronal cells and inhibition apoptosis by regulating the PI3K / Akt and SIRT1 signal pathways. Clarify the substance basis and mechanism of BYHWD in the rehabilitation of ischemic stroke, explain the mechanism of group compatibility and provide a scientific basis for clinical rational use of drugs.

补阳还五汤在缺血性脑中风康复治疗中疗效显著。课题组前期研究证实补阳还五汤具有“益气行血生新”作用，通过脑微血管和神经元轴突的生新促进缺血性脑中风的康复，但其对神经元再生及作用机制尚不清楚。基于此，本课题提出“补阳还五汤通过调控脑中风状态下PI3K/Akt和SIRT1信号通路，促进神经元细胞增殖，抑制细胞凋亡可能是其促进缺血性脑中风康复的重要机制”的科学假说。拟运用微透析技术结合细胞膜固相色谱法筛选补阳还五汤亲和性成分，LC/MS、NMR技术在线检测活性物质的分子结构信息；免疫印迹等方法检测亲和性成分对模型动物缺血病灶区相关蛋白和基因表达的影响，验证其药理活性，结合体外细胞实验，探索补阳还五汤通过调控PI3K/Akt和SIRT1信号通路促进神经元细胞增殖及抑制细胞凋亡的作用机制，从而阐明补阳还五汤促进缺血性脑中风康复的药效物质基础及作用机制，阐释组方配伍机理，为临床合理用药提供科学依据。

课题组在“气旺则血行，血行则新生”的中医理论的指导下，研究证实补阳还五汤具有“益气行血生新”的作用，先探讨补阳还五汤神经元细胞亲和性成分的药理活性，将血清药理学方法、细胞膜固相色谱法及LC/MS联合，还原药物在体内表达的关键过程，从而高效、准确地筛选以效应细胞为靶点的活性物质16个。采用PI3K siRNA分别在神经元细胞水平及动物水平阻断PI3K/Akt信号通路，观察补阳还五汤亲和性成分调控OGD/R模型及大鼠缺血性脑损伤的影响，发现亲和性成分能够对MCAO大鼠的神经缺损、运动感知、脑梗死面积以及脑水肿均有缓解作用，同时上调PI3K、AKT、P-PI3K、P-AKT、Bcl-2蛋白的表达，下调BAX、GSK-3β蛋白表达的作用；亲和性成分对氧糖剥夺再灌注（OGD/R）模型细胞的活性有明显的促进作用，同时具有升高SOD水平、降低ROS、LDH和MDA，降低细胞凋亡率，升高p-PI3K/PI3K，p-AKT/AKT 和 Bcl-2/BAX的表达，降低GSK-3b表达的作用。用SIRT1 siRNA分别在神经元细胞水平及动物水平阻断SIRT1信号通路，观察补阳还五汤亲和性成分调控OGD模型及大鼠缺血性脑损伤的影响，研究发现SIRT1抑制剂EX527能够进一步降低OGD/R海马神经元细胞的活力及SOD 活力，提高LDH、MDA、ROS含量，提高细胞凋亡，下调SIRT1、FOXO1、Bcl-2和PGC-α蛋白表达，上调Bax蛋白表达，而亲核性成分能够明显逆转EX527抑制剂的作用，从而发挥对OGC/R损伤模型细胞起到保护作用；发现亲和性成分能够改善缺血性脑损伤大鼠的神经功能，降低mNSS评分及脑梗死体积，抑制氧化应激反应，降低血清LDH、MDA水平，改善脑缺血再灌注引起的神经元细胞损伤，增加尼氏体数量，减少脑组织细胞凋亡，下调BAX/Bcl-2蛋白表达比值，上调SIRT1蛋白表达，通过与SIRT1特异性抑制剂EX527的比较，进一步证明补阳还五汤亲和性成分对缺血性脑损伤大鼠的保护作用。