从内质网应激途径的细胞保护角度探讨栀子抗帕金森病的活性成分及作用机制

It has been reported that endoplasmic reticulum (ER) stress-induced apoptosis is involved in the pathology of PD. Recent studies show that iridoids, one of the major active constitutents in Gardenia jasminoides Ellis, reduced the expression of ER stress-associated protein and the cleavage of apoptosis-associated protein. Thus, the agent may be effective for treating neurodegenerative diseases including PD. We have confirmed that Huanglian jiedu decoction (HJD), a traditional Chinese medicine compound recipe, and Gardenia jasminoides Ellis could regulate dopamine (DA) metabolism and possess the neuroprotective effects on neuronal injury according to clinical trials , cell research and animal experiments. Therefore, we hypothesized that Gardenia jasminoides Ellis protects neurons from ER stress response-signaling pathway and may be valuable in treating PD. In our preliminary screening, we identified some of active constitutents that could inhibit MPP+-induced apoptosis in SH-SY5Y cells by the means of combined computer-virtual screening and in vitro biological activity evaluation. In this study, first, we predicted the compounds of Gardenia jasminoides Ellis based on computer-virtual screening and then evaluated the neuroprotective effect of active constitutents against ER stress-induced apoptosis in cell culture and animal models of PD. Finally, we investigated the cytoprotection mechanism via ER stress-induced survival and apoptosis. The study aims to provide scientific evidence and ideas for the development of drugs against PD.

内质网应激（ERS）介导的细胞凋亡参与帕金森病（PD）的发病过程。有报道栀子主要药效成分环烯醚萜类化合物可抑制ERS相关蛋白表达，降低凋亡相关蛋白裂解，有助于神经退行性疾病病变的改善。课题组经临床疗效评价及离体整体实验证实，黄连解毒汤及其组成生药栀子具有调节多巴胺代谢和神经保护作用。由此推测，栀子可能通过干预ERS的自身保护以及过度应激诱发的凋亡机制，发挥其神经保护作用，从而达到防治PD的目的。前期工作将计算机虚拟筛选与体外生物活性评价结合，初步筛选出可抑制MPP+诱导SH-SY5Y细胞凋亡的栀子活性化合物。本项目将以此为突破口，以ERS标志物为靶点，依托计算机虚拟筛选技术，结合药物筛选PD细胞模型和动物模型，以期确定栀子中抑制DA能神经元凋亡的活性成分；进而阐明其针对ERS生存和凋亡途径的细胞保护机制，为进一步开发抗PD的药物提供科学依据和研究思路。

内质网应激（ERS）介导的细胞凋亡参与帕金森病（PD）的发病过程。有报道栀子主要药效成分环烯醚萜类化合物可抑制ERS相关蛋白表达，降低凋亡相关蛋白裂解，有助于神经退行性疾病病变的改善。课题组经临床疗效评价及离体整体实验证实，黄连解毒汤及其组成生药栀子具有调节多巴胺（DA）代谢和神经保护作用。由此推测，栀子可能通过干预ERS的自身保护以及过度应激诱发的凋亡机制，发挥其神经保护作用，从而达到防治PD的目的。.本课题选取ERS信号通路靶向分子，将计算机虚拟筛选与体外生物活性评价相结合，确定抑制DA能神经元凋亡的栀子活性成分——山栀苷。进而基于ERS过度应激诱导的凋亡机制，观察山栀苷对MPTP诱导小鼠DA能神经元损伤的保护作用及机制。结果显示，山栀苷灌胃2周后，小鼠行为学有所改善；黑质致密部和纹状体DA能神经元的损伤减轻，酪氨酸羟化酶（TH）阳性细胞和蛋白表达显著增多；ERS凋亡传导通路标志分子p-JNK、JNK、Bcl-2、Bax以及c-Caspase 3等表达水平下调。同时，课题组基于PERK信号通路介导的ERS和自噬的交互效应，观察山栀苷对6-OHDA诱导SH-SY5Y细胞损伤的保护作用及机制。以300 μM 6-OHDA处理SH-SY5Y细胞24 h构建PD体外模型，结果显示，山栀苷预处理后细胞存活率显著升高而凋亡率显著降低，细胞核皱缩凋亡和内质网弥散化分布有所减轻；山栀苷可调控PERK/eIF2α磷酸化，下调ATF4和CHOP的表达，同时，阻抑P62和LC3的表达，提高Bcl-2蛋白合成水平，发挥其神经保护作用。由此可见，山栀苷可通过调控PERK/eIF2α途径，保护机体免受ERS过度激活的损伤，恢复自噬功能。.本课题将以计算机虚拟筛选为代表的新药研发技术与中药体系相结合，为天然药物的深入研究提供了有力的技术支持和借鉴。ERS与自噬的持续性、破坏性交互作用是PD发生、发展的重要因素，针对这一病理过程进行干预，对于缓解PD病程具有重要意义，亦为PD治疗药物的研发指明了方向。