基于PI3K-Akt通路的参芎葡萄糖注射液抗氧化应激的药效物质基础研究

Oxidative stress may directly cause cardiovascular system injury, which is an important factor leading to cardiovascular diseases. Therefore, it is of great importance to prevent oxidative stress for the treatment of cardiovascular disease. Shengxiong glucose injection (SGI), a traditional Chinese medicine injection produced by Guizhou with sales of 1.2 billion per year, is clinically effective and widely used for the treatment of various cardiovascular diseases. Our previous investigations showed that SGI could exert a strong anti-oxidative stress effect by activating PI3K/Akt pathway. Up to now, the active components of anti-oxidative activity of SGI are still unclear, which retards the deep exploitation of SGI. In the current project, the mechanism of the regulation of PI3K/Akt pathway by the SGI will be further investigated to identify the target proteins, which are heterologously expressed afterwards. Then, by using affinity ultrafiltration coupled with liquid chromatography-mass spectrometry (LC-MS) technology, the chemicals bound to the heterologously expressed proteins will be isolated and identified. In addition, these chemicals will be evaluated and the formula of these chemicals will be optimized by using cell and animal models. Our results may provide a new approach for the improvement of efficacy and safety of SGI.

氧化应激可造成心血管组织损伤，是引发心血管疾病的原因之一。拮抗氧化应激对心血管疾病的治疗具有重要意义。贵州中药注射剂大品种参芎葡萄糖注射液（SGI）是临床常用的心血管疾病治疗药物，年销售额达12亿元。本课题组前期研究发现，SGI可激活PI3K/Akt通路产生较强的抗氧化应激作用。但迄今为止，SGI抗氧化应激的药效物质基础尚未明确，限制了该产品有效性和安全性的进一步提升。本课题拟使用磷酸化蛋白组学、小分子-蛋白相互作用组学、生物信息学和文献分析等手段，筛选SGI调控PI3K/Akt通路所作用的上游靶蛋白，并用细胞模型进行靶蛋白确证。体外表达已确证的靶蛋白，利用亲和超滤结合液相色谱-质谱技术，筛选鉴定SGI中与靶蛋白结合的小分子，最后用细胞和动物模型进行小分子活性验证和组方优化，由此阐明SGI抗氧化应激的药效物质基础，为进一步提升SGI的药效和安全性提供创新思路和实验依据。

本项目拟通过筛选参芎葡萄糖注射液（SGI）抗氧化应激的靶点，从而研究其抗氧化应激的药效物质基础。组学研究显示，SGI作用后，出现82个差异表达蛋白和180个差异修饰蛋白，并且高度富集在HSF1、PI3K/AKT、c-Jun和ERK等通路。PI3K/AKT通路筛选结果与前期实验一致。其他通路的验证实验表明，HSF1高表达能增强SGI抗氧化损伤作用，但敲低HSF1后，SGI抗氧化应激作用没有明显改变；抑制ERK可拮抗SGI 对H9c2细胞凋亡率、细胞存活率及凋亡相关蛋白Bcl-2/Bax和cleaved caspase 3的影响。因此我们也把ERK列为了SGI抗氧化应激的靶蛋白。此外，我们还发现SGI对细胞色素P450的丰度有影响，但细胞色素P450对SGI抗氧化应激作用无明显影响。.确定靶蛋白后，我们利用亲和超滤结合液质技术来筛选与PI3K、AKT和ERK结合的小分子，结果只筛选出与ERK结合的小分子：丹参素钠（SAAS）和盐酸川芎嗪（LZH）。活性验证和配比筛选实验发现，200 μmol/L的SAAS与100 μmol/L的LZH配伍对H2O2诱导H9c2细胞存活率、ROS水平和大鼠心肌缺血改善情况较SAAS、LZH单独给药更接近于空白组。进一步研究表明，SAAS、LZH和SAAS/LZH配伍均可通过上调PI3K、AKT和ERK的磷酸化水平，下调c-Jun磷酸化水平，从而上调Bcl-2/Bax水平，下调Cyt-c表达水平、caspase 3和caspase 9活化水平，进而抑制细胞凋亡，产生抗氧化应激作用。.综上所述，本项目发现SGI抗氧化应激作用与其激活PI3K/AKT、ERK和c-Jun通路有关，且SAAS和LZH是其活性成分。本项目对SGI抗氧化应激的药效物质基础进行了初步阐释，为其质量提升打下了一定的理论基础，不过SGI的药效物质基础仍需要进一步研究。