玄参环烯醚萜苷类成分调控钙平衡负反馈调节内质网应激介导脑损伤保护作用的药效物质基础及分子机制研究

Calcium homeostasis unbalance is the the important causes of endoplasmic reticulum stress (ERS). Regulating calcium homeostasis unbalance and inhibiting the cell apoptosis mediated by ERS became a new way of drug resistance to CRI. Radix scrophulariae is the dry root for scrophulariaceae, and it is one of the famous eight flavors in Zhejiang. During previous study of radix scrophulariae, our team found that its new uses for treatment of cerebral ischemia reperfusion injury, and we applied for a patent. Recently, for the first time, our group found the iridoid glycosides could obviously improve the activity of SERCA in cerebral ischemia-reperfusion injury in the rats , at the same time, it could reduce the protein expression of calmodulin PLB and NCX. Besides, iridoid glycosides could reduce the calcium overload induced by CRI and suppress the apoptosis induced by endoplasmic reticulum stress. On the basis of preliminary studies, this project intends to explore iridoid glycoside of radix scrophulariae and its main active ingredients based on the regulation of calcium homeostasis unbalance and inhibition of cerebral ischemia reperfusion injury mediated endoplasmic reticulum stress. The research intends to demonstrate that the protective effects against cerebral ischemia reperfusion injury of iridoid glycoside of radix scrophulariae may be related to direct intervention of the expression of calcium signaling protein like SERCA, inhibition of endoplasmic reticulum stress, reducing the nerve cell apoptosis, affecting the interaction of calcium and the endoplasmic reticulum, and reaching the steady state equilibrium in the ischemia reperfusion rats. In addition, this project aims to confirm the signaling pathways and molecular targets of the iridoid glycoside of radix scrophulariae, and to explore the molecular mechanism of protection of cerebral ischemia reperfusion injury.

钙稳态失衡是诱导内质网应激（ERS）发生的重要原因。通过调控钙稳态失衡进而抑制ERS介导的细胞凋亡成为药物抗CRI新途径。玄参为玄参科玄参的干燥根，是著名的浙八味之一。课题组前期对玄参研究，发现其治疗脑缺血再灌注损伤的新用途，申请了1项专利。近期课题组首次发现环烯醚萜苷类成分可明显提高脑缺血再灌注损伤大鼠脑组织中SERCA活性，同时降低钙调蛋白PLB和NCX蛋白表达。减少CRI所致钙超载，抑制内质网应激诱导的凋亡。本课题拟在此基础上对玄参环烯醚萜苷及其主要活性成分进行基于调控钙稳态失衡抑制内质网应激介导脑缺血再灌注损伤保护机理的研究，论证玄参环烯醚萜苷抗脑缺血再灌注损伤的保护作用可能与直接干预SERCA等钙信号传导蛋白的表达，抑制内质网应激，减少神经细胞凋亡，影响脑缺血再灌注时钙及内质网的交互作用，达到二者的稳态平衡有关，并确证其作用信号通路与靶分子，探索其保护脑缺血再灌注损伤的分子机制。

钙稳态失衡是诱导内质网应激（ERS）发生的重要原因。通过调控钙稳态失衡进而抑制ERS介导的细胞凋亡成为药物抗CRI新途径。玄参为玄参科玄参的干燥根，是著名的浙八味之一。课题组首次发现环烯醚萜苷类成分可明显提高脑缺血再灌注损伤大鼠脑组织中SERCA活性，同时降低钙调蛋白PLB和NCX蛋白表达。减少CRI所致钙超载，抑制内质网应激诱导的凋亡。在此基础上，本课题组对玄参环烯醚萜苷及其主要活性成分进行基于调控钙稳态失衡抑制内质网应激介导脑缺血再灌注损伤保护机理的研究。.在细胞实验中，通过建立神经细胞再灌注模型，神经细胞氧糖剥夺再灌注模型，原代皮层细胞与PC12神经元细胞的氧糖剥夺/复氧复糖模型。通过构建慢病毒干扰体，感染PC12细胞，建立稳定细胞株，构建SERCA shRNA表达载体。研究表明，玄参环烯醚萜苷主要成分哈巴苷通过抑制ERS介导的神经细胞凋亡，从而对脑缺血再灌注损伤起到保护作用，其作用靶点为SERCA；.在动物实验中，采用线栓法制备大鼠脑缺血90min再灌注24h模型（CIRI模型），实验结果证实，玄参环烯醚萜苷对大鼠脑缺血再灌注具有显著的保护作用，其作用机制可能与其抑制内质网应激有关；通过其对CIRI大鼠海马神经细胞内质网超微结构的影响，提示玄参环烯醚萜苷可能通过抑制GRP78，Caspase-12及CHOP的表达，拮抗ERS诱导的细胞凋亡途径，从而对脑缺血再灌注损伤起到保护作用。.在环烯醚萜苷的物质基础研究方面，本课题组从玄参中分离得到7个化合物，并得首分化合物Harpagoside，对分离得到的化合物和从TCMSP数据库及文献中已报导的玄参环烯醚萜苷类化合物进行活性靶点预测玄参环烯醚萜苷类化合物主要通过作用于酸中毒、炎症、氧化应激、细胞凋亡等通路来治疗脑缺血。.本课题论证了玄参环烯醚萜苷抗脑缺血再灌注损伤的保护作用与直接干预SERCA等钙信号传导蛋白的表达，抑制内质网应激，减少神经细胞凋亡，影响脑缺血再灌注时钙及内质网的交互作用，达到二者的稳态平衡有关，并确证其作用信号通路与靶分子，探索了其保护脑缺血再灌注损伤的分子机制。