秦巴太白楤木皂苷调控Apelin13/Akt/Sox2通路促神经血管新生治疗缺血性脑卒中的机制研究

The promotion of neuroangiogenesis is the core strategy for the recovery of ischemic stroke, which is consistent with the theory of “Reinforcing qi to enrich blood, Dispelling stasis to promote regeneration” in Chinese Medicine. Aralia taibaiensis is specially produced in Qinba mountain, which is beneficial to qi and blood circulation. Our previous studies have found that the total saponin extract from Aralia taibaiensis (sAT) could increase brain microvessel density, improve neurological damage caused by cerebral ischemia/reperfusion. Meanwhile, we found that sAT could promote neural stem cell (NSCs) and endothelial progenitor cells (EPCs) proliferation and differentiation showing that it has a potential faculty to promote neurogenesis and angiogenesis. However, the correlation between improving cerebral ischemia and promoting angiogenesis and the possible mechanism are still unclear. Apelin 13/Akt/Sox2 pathway plays an important regulatory role in neuroangiogenesis. In pre-experiments, sAT upregulated the expression of Apelin 13 in NSCs and EPCs, suggesting this pathway may contribute the effects of sAT. This study aims to investigate whether sAT improves the brain ischemia injuries through promoting the proliferation and differentiation of NSCs and EPCs, and neuroangiogenesis, and further investigate its molecular mechanism, especially the key role of Apelin 13/Akt/Sox2, illuminating the scientific connotation of “Reinforcing qi to enrich blood, Dispelling stasis to promote regeneration”.

促神经血管新生是缺血性脑卒中恢复的核心策略，与中医“益气生血、祛瘀生新”理论相符。太白楤木特产于秦巴山区，有益气活血、祛瘀除痹等功效。我们发现太白楤木皂苷（sAT）可增加脑缺血小鼠脑微血管密度，有明确的脑保护作用，同时sAT能诱导神经干细胞（NSCs）和内皮祖细胞（EPCs）增殖、分化，具有潜在促神经血管新生能力，但其改善脑缺血与促神经血管新生的相关性及作用机制尚不明确。Apelin13/Akt/Sox2通路在神经血管新生中发挥重要的调控作用。预实验发现，sAT可上调NSCs和EPCs的Apelin13表达，提示该通路可能参与了sAT的促神经血管新生作用。本课题拟在前期研究基础上，明确sAT通过诱导NSCs和EPCs的增殖、分化，促神经血管新生，改善脑缺血损伤，并揭示其具体分子机制，即Apelin13/Akt/Sox2通路在其中的关键作用，阐明sAT“益气生血、祛瘀生新”的科学内涵。

“益气生血、祛瘀生新”是中医药治疗脑血管疾病的重要法则，二者相辅相成，共同促进损伤部位的组织修复。太白楤木以根入药，具有益气活血、祛瘀除痹等功效，对缺血性脑梗死（IS）具有治疗作用，但机制不清。本研究从抑制IS引起的神经损伤和促进神经血管再生等角度深入探讨了太白楤木皂苷（sAT）治疗IS的作用机制及药效物质。在大脑中动脉结扎/再灌注（MCAO/R）和细胞缺氧再复氧（OGD/R）模型中，我们发现sAT能够有效抑制神经细胞损伤，抑制氧化应激和线粒体功能失常，采用特异性抑制剂或siRNA干扰表达，发现sAT通过Apelin 13调控AMPK/Akt及其下游蛋白表达发挥脑保护作用。OGD/R可通过促进P38 MAPK磷酸化，上调ATF4，抑制Apelin 13表达。sAT则可以抑制P38 MAPK磷酸化，下调ATF4，从而促进Apelin 13表达。同时，还发现sAT可促进HIF-1a表达，上调Apelin 13。楤木皂苷能够促进血管标志物CD31和VEGF表达，表明具有促进血管再生作用，而敲除Apelin受体AR后，楤木皂苷的促血管新生作用丧失，表明楤木皂苷通过Apelin/AR促进血管新生。楤木皂苷能够有效促进脑组织中SOX2、DCX和TUJ1蛋白表达，表明具有促进神经再生作用，而siRNA干扰AR后，楤木皂苷失去对神经再生的调控作用，表明楤木皂苷通过Apelin/AR促进神经再生。为进一步研究sAT中是哪种皂苷单体对Apelin具有调控作用，我们收集了22种已知皂苷单体成分，并通过RT-PCR、荧光素酶报告系统、基因干扰、分子生物学等手段发现楤木皂苷A，去葡萄糖竹节参皂苷IVa和竹节参皂苷IVa可同时影响P38 MAPK/ATF4和HIF-1a信号通路，促进Apelin表达。竹节参皂苷Ib，刺嫩芽皂苷F和拟人参皂苷RT1可通过影响HIF-1a信号通路，促进Apelin表达。通过本研究，我们确定了太白楤木治疗IS的作用机制，并筛选了其主要药效物质，为下一步开发和应用奠定基础。