GPER/IGF-1R信号途径介导淫羊藿苷和淫羊藿素抗胶质细胞炎症反应的分子机制研究

Accumulating clinical and experimental evidence suggests that chronic neuroinflammation is associated with the progressive death of dopaminergic neurons in Parkinson’s disease (PD). Inhibition of microglia overactivation may be a possible therapeutic strategy of PD. In recent years, there is extensive literature focused on the beneficial effects of estrogens in multiple sclerosis, indicating a potent anti-inflammatory and neuroprotective effects of estrogen. These studies support the anti-inflammatory effects of estrogen are mediated by G protein-coupled estrogen receptor (GPER) and the cross-talk with growth factor receptor pathway. Our previous studies have clearly demonstrated that icariin and icaritin are phytoestrogens without side effect on the reproductive system. Icariin or icaritin treatment could protect against the neurotoxin MPTP-induced neurotoxicity via insulin-like growth factor-1 receptor (IGF-1R) signaling pathway. However, the protective effect of icariin or icaritin against LPS-induced inflammation in nigrostriatal system and the precise underlying mechanisms remains unclear. In the present study, we aim to evaluate the role of membrane estrogen receptor GPER and IGF-1R on the anti-inflammatory efficacy of icariin and icaritin. The pivotal signaling pathways will be investigated by using pharmacological blockade, molecular docking, lentivirus-mediated gene silence in primary microglia and astrocyte culture and sustantial nigra of rat as well as GPER-/- gene knockout mice. This newly discovered mechanism provides new insight to understand the biological effect of GPER and IGF-1R in nigrostriatal system and the anti-inflammatory molecular mechanism of icariin and icaritin. Our data will provide a potential new target for the treatment of PD and screening of new drugs.

临床发现帕金森病(PD)患者黑质含有大量的反应性小胶质细胞，其激活介导的炎性反应导致多巴胺(DA)能神经元损伤。雌激素可通过激活膜表面G蛋白耦联雌激素受体(GPER)发挥抗炎作用，保护DA能神经元。研究表明GPER可与生长因子受体信号通路交互对话，发挥生物学作用。本课题组首次报道具有类雌激素样作用的淫羊藿苷及其衍生物淫羊藿素的神经保护作用与胰岛素样生长因子-1受体(IGF-1R)途径有关，但二者是否通过GPER激活IGF-1R，对抗胶质细胞炎性反应，目前尚不清楚。本研究拟通过药理阻断、计算机分子对接技术、小RNA干扰技术沉默或过表达原代小胶、星胶和大鼠黑质GPER或IGF-1R基因及GPER-/-基因敲除小鼠，明确GPER/IGF-1R在胶质细胞炎性反应的作用，揭示淫羊藿苷和淫羊藿素抗炎作用的分子机制，并比较二者的药效。该研究将获得抗PD炎症反应的先导化合物，发掘具有潜在临床价值的前药。

小胶质细胞活化介导的神经炎症反应在帕金森病（PD）发病中发挥重要作用，通过抑制小胶质细胞的炎症反应保护中脑黑质多巴胺（DA）能神经元是一种有效的神经保护策略。细胞的炎性反应，本研究采用ELISA、HPLC、实时PCR、siRNA干扰技术、免疫印记、免疫荧光、分子对接及转基因动物等多种评价方法，系统探讨了淫羊藿苷及淫羊藿素抗炎神经保护作用的分子机制。结果显示：在细胞水平，淫羊藿苷及淫羊藿素能够对抗LPS诱导的小胶质细胞及星型胶质细胞炎症反应，减轻中脑神经元损伤，此作用可被GPER和IGF-1R特异性阻断剂G15和JB-1所阻断。淫羊藿苷及淫羊藿素单独作用于胶质细胞可诱导小胶质细胞向M2型转化及调节星形胶质细胞功能。在整体动物水平，淫羊藿苷及淫羊藿素能够减轻LPS导致的中脑黑质区炎症反应，增加纹状体内DA及其代谢产物的含量，减轻黑质DA能神经元的损伤，此作用亦可被G15和JB-1所阻断。应用分子对接及分子动力学分析，进一步明确淫羊藿苷和淫羊藿素与GPER构效关系。利用CRISPR/Cas9技术，构建了GPER基因敲除小鼠，验证GPER参与淫羊藿苷和淫羊藿素的抗炎作用，且淫羊藿素的抗炎神经保护作用优于淫羊藿苷。同时，研究还探讨了GPER与IGF-1R信号通路的交互串话在PD神经保护中的作用，证实GPER参与IGF-1的神经保护作用。综上所述，淫羊藿苷和淫羊藿素能够通过GPER和IGF-1R信号途径，对抗LPS诱导的胶质细胞过度激活，保护DA能神经元。本研究紧扣国际研究前沿课题，为获得抗PD炎症反应的先导化合物，发掘具有潜在临床价值的前药提供了理论和实验依据。