基于鞘脂组学与质谱成像技术的补阳还五汤脑缺血损伤保护的时空特性及作用机制研究

Ischemic cerebral apoplexy is a common clinical disease, and Buyang Huanwu Decoction is a classic prescription with definite curative effect. The spatiotemporal characteristics and action mechanism study on cerebral ischemia injury protection of Buyang Huanwu Decoction is the main contents of the project based on the closely relation between sphingolipids and cerebral ischemia injury.The rat focal cerebral ischemia model will be built with monofilament.The time variation characteristics of cerebral ischemia injury protection of Buyang Huanwu Decoction will be found by metabonomics study of microdialysis samples collected from rat hippocampal region and determined by UPLC-Q/TOF-MS.The brain sphingolipidomics study will be carried out on cerebral ischemia injury protection of Buyang Huanwu Decoction which is helpful to identify the sphingolipids biomarkers.The brain tissue level of S1PR1、SphK1、SphK2mRNA and expression of PI3K,Akt, phosphorylated Akt, also will be detected from brain SphK-S1P-S1PR1 and its downstream PI3K/Akt pathway.The two-dimensional mass spectrometry imaging experiments and three-dimensional reconstruction model of brain tissue sections will be established by using MALDI-TOF-MS to fully mine the sphingolipids spatial distribution characteristics in ischemic injury tissue.At last exploring the possible action mechanism and target of cerebral ischemia injury protection of Buyang Huanwu Decoction based on the spatiotemporal characteristics,sphingolipid metabolism profile and sphingolipid signaling pathway expression differences between groups.The study will contribute to the deepening of its clinical application.

缺血性脑中风是临床常见病，补阳还五汤是有确切疗效的经典名方。本课题基于鞘脂与脑缺血损伤的密切联系，拟进行补阳还五汤脑缺血损伤保护的时空特性及作用机制研究。线栓法复制脑缺血大鼠模型，海马区微透析探针实时采样，UPLC-Q/TOF-MS进行微透析样品的代谢组学研究，获取补阳还五汤脑缺血损伤保护的经时变化特征。进行补阳还五汤干预脑缺血损伤大鼠脑鞘脂组学研究，识别鞘脂标记物，检测脑SphK-S1P-S1PR1及其下游PI3K/Akt通路S1PR1、SphK1、SphK2mRNA水平及PI3K、Akt、磷酸化Akt表达。MALDI-TOF-MS进行脑组织切片二维质谱成像并进行三维重建，挖掘鞘脂在缺血损伤脑组织的空间分布特征。基于补阳还五汤脑缺血损伤保护的时空特性，根据鞘脂代谢轮廓及鞘脂信号通路表达组间差异性，探讨补阳还五汤逆转脑缺血损伤的作用机制及可能的作用靶点，有助于深化其临床应用。

缺血性脑卒中是临床常见病，补阳还五汤是疗效确切的经典名方。PI3K/Akt信号通路是经典的脑缺血损伤保护通路，鞘脂与脑缺血损伤存在密切联系，1-磷酸鞘氨醇（S1P）作用于S1P受体后可激活下游PI3K/Akt信号通路。在中药复方传统制法水煎法基础上，真空冷冻干燥法将补阳还五汤水煎液制备成性质稳定、易于储存、便于应用的冻干物。采用超高效液相色谱法建立补阳还五汤冻干物指纹图谱及11种成分同步检测方法，进行了成分准确定量和相似度评价。采用线栓法成功复制了稳定、可靠、临床症状相似度高的大鼠/小鼠永久性大脑中动脉阻塞（pMCAO）模型。脑微透析法结合液质联用技术进行补阳还五汤冻干物干预正常与pMCAO大鼠的脑海马区洋川芎内酯A、阿魏酸、芒柄花素、毛蕊异黄酮、芒柄花苷、毛蕊异黄酮苷、芍药苷药动学特征的比较研究。采用UPLC-Q-TOF/MS进行大鼠脑脊液、血液、全脑及海马、纹状体、前额叶等特征性脑区的鞘脂组学研究。采用改良神经功能缺损评分、基于TTC染色的脑相对梗死体积等指标充分评价补阳还五汤干预缺血性脑卒中的疗效。HE染色基础上，WB、IHC法等对前额叶、海马、纹状体SphK1、SphK2、S1PR1、PI3K、Akt、p-Akt等蛋白进行半定量和定位研究，测定S1P/S1PR1信号通路中SphK1、SphK2、S1PR1等关键靶点mRNA表达。微透析技术联用UPLC-Q-TOF/MS进行补阳还五汤干预pMCAO大鼠的空间特异性代谢组学。LC-MS/MS技术建立了正常和pMCAO大鼠血清及脑组织及OGD/R HT22细胞内源性鞘脂的绝对定量方法，对比缺血缺氧状态下内源性鞘脂变化。UPLC-Q-TOF/MS辨识补阳还五汤pMCAO大鼠入血、入脑成分。本研究从多角度阐明补阳还五汤基于S1P/S1PR1/PI3K/Akt信号通路干预缺血性脑卒中的时空特性及作用机制，有助于深化其临床应用。