基于内质网应激IRE1/XBP1信号通路探讨温肾通督法保护脊髓损伤神经元的作用及机制

Traditional Chinese medicine has been confirmed to be effective on SCI while there is short of further study on the theory and mechanism of TCM. We established “Jisuikang” which had good effect on neural plasticity based on “Kidney-Du deficiency and blood stasis in Du” theory in early stage. Clinical and experimental study have confirmed that the warming kidney and unblocking Du Channel prescription-“Jisuikang” had exactly effect on promoting neural restoration of SCI, and was likely to promote XBP1 splicing activation after endoplasmic reticulum stress(ERS) to protect damaged neurons. To explore the best decomposed recipes of “Jisuikang” which can reduce ERS and anti-neuron apoptosis is great significance for revealing the TCM treatment rules of SCI. In this study, we will apply the targeted screening platform and high-throughput screening technology to explore the most optimal prescription of “Jisuikang” via IRE1/XBP1 pathway according to the pathogenesis of “qi,blood, meridians,cold and dampness”, and explore the mechanism of “Jisuikang” on anti-neuro apoptosis and reducing endoplasmic reticulum stress. We will try to reveal the deep mechanism of “Jisuikang” on SCI and elucidate that the “Warming kidney and unblocking Du Channel” is a vital therapy on SCI.

已有研究表明中医药防治脊髓损伤（SCI）临床疗效确切，但缺乏相关中医理论探索及作用机制的深入研究。课题组前期以“肾督虚损，瘀阻督脉”病机为指导，建立组方促进神经功能重塑，临床及实验研究均证实以此病机为核心的温肾通督方--“脊髓康”促进 SCI 修复作用确切，并且很可能是通过特异性的促进内质网应激（ERS）后 XBP1的剪接活化而保护受损的神经元。探索减轻ERS、抗神经元凋亡的温肾通督方-“脊髓康”的最佳拆方组份，对于揭示中医药治疗SCI的规律具有重要意义。本项目拟立足于SCI“气、血、经络、寒、湿”等病机要素，应用靶向筛选平台及高通量筛选技术，基于ERS的重要枢纽IRE1/XBP1通路对“脊髓康”进行拆方筛选，优化组方并探索“脊髓康”及功效性拆方减轻ERS、抗神经元凋亡的作用及机制，揭示“脊髓康”抗 SCI 的深层次作用机理，进一步阐明温肾通督是治疗脊髓损伤的重要治法。

目前针对脊髓损伤（SCI）尚缺乏有效的治疗方法，中医药具有多因素和多靶点的作用优势，治疗SCI作用确切。温肾通督方——“脊髓康”是基于SCI“肾虚督损，督脉瘀阻，枢机统率失职”病机而构建的具有“温肾通督，祛瘀利湿，调和气血”之功效的临床经验方。为了深入剖析该方药物组成之间的潜在联系与相互作用，课题组建立了本方水提物的化合物图谱并进行定量分析，检测出16种成分化合物，分别是阿魏酸、没食子酸、淫羊藿苷、大黄素、大黄酚、芦荟大黄素、大车前苷、丹酚酸A、原儿茶醛、松果菊苷、木兰花碱、槲皮素、大黄酸、丹酚酸B、橙皮苷、芍药苷；运用双荧光素酶报告基因系统的靶向筛选平台，基于IRE1/XBP1通路筛选出“脊髓康”有效性拆方组份，发现丹酚酸B、松果菊苷、大车前苷、橙皮苷、淫羊藿苷、芍药苷和芦荟大黄素可高效促进XBP1剪接活化，特异性针对IRE1/XBP1通路对内质网应激进行处理。同时运用流式细胞术、Western blot、q-PCR及透射电镜等技术，验证了“脊髓康”全方及有效性拆方调控内质网应激IRE1/XBP1通路抗神经元凋亡的有效性，结果显示有效拆方组、脊髓康组、淫羊藿苷组方中药物可改善毒胡萝卜素诱发的内质网应激，减少神经元样PC12细胞凋亡，且可降低Grp78表达，上调XBP1、IRE1蛋白和mRNA表达，从而有效保护受损神经元。因受损的神经元再生和修复能力有限，小胶质细胞担当着中枢神经系统内“巨噬细胞”的角色，在SCI的发展、恢复中意义重大，课题组又从调控小胶质细胞吞噬作用角度进行了研究，结果显示有效拆方组、脊髓康组药物均可不同程度的促进小胶质细胞发挥其吞噬作用，改善神经损伤再生微环境。研究结果从多角度阐明了“脊髓康”及有效组份抗神经元凋亡的作用及可能分子机制，为“脊髓康”抗SCI的深入研究提供了实验依据，为中医药促进神经功能恢复的治疗策略提供了重要依据。