人参皂苷Rg1通过抑制炎症反应保护多巴胺能神经元的分子机制研究

Substantial evidence has indicated that neuroinflammation participates in the pathogenesis of Parkinson's disease (PD). Microglial activation plays a pivotal role in the progressive neurodegeneration of PD by producing various pro-inflammatory cytokines and nitric oxid. Anti-inflammatory processes might be a promising therapeutic approach to slow the progression of PD. .In recent years, ginseng has become one of the most commonly used alternative herbal medicines in the West. Ginsenoside Rg1 is the major pharmacologically active compound of ginseng and has been identified as a potent phytoestrogen. Ginsenoside Rg1 has been reported to exert neurotropic and neuroprotective effects both in vivo and in vitro. However, the mechanism that transmits the neuroprotective effects of Rg1 is still unclear. .Our previous study has demonstrated that ginsenoside Rg1 is a new phytoestrogen that exerts estrogen-like activity in human breast cancer (MCF-7) cells. Rg1 could activate the insulin-like growth factor-I receptor (IGF-IR) signaling pathway through enhancing tyrosine phosphorylation of insulin receptor substrate-1 in MCF-7 cells. Moreover, Rg1 treatment could protect against the 6-OHDA-induced neurotoxicity both in PD rat model and cell model. The neuroprotective effects of Rg1 on dopaminergic neuron could be also completely abolished by ER antagonist ICI182,780 and IGF-IR antagonist JB-1. However, direct neuroprotective effects of Rg1 upon microglia cannot be excluded. .There is accumulating evidence showing an abundant co-expression of ER and IGF-IR in neurons and glia in the nigrostriatal system. Rg1 could inhibit the inflammation in both cerebral cortex and hippocampus in LPS-induced microglial activation in mice. Based on the information, we hypothesize that the anit-inflammatory effect of Rg1 might be mediated through the activation of ER and IGF-IR-mediated pathway. Furthermore, recent study has demonstrated that microglial glucocorticoid receptor (GR) plays a pivotal role in regulating dopaminergic neurodegeneration in parkinsonism. Rg1 has been shown to be a functional ligand of GR. So in the present study, we will also demonstrate if GR is involved in the anti-inflammatory effect of Rg1. .The present study will test the pivotal signaling pathways that involve in the anti-inflammatory effect of Rg1 by using LPS-induce PD rat model, co-culture of midbrain microglia and dopaminergic neuron as well as primary microglia. This newly discovered mechanism of action of Rg1 provides new insight to understand the pharmacological effects of ginsenoside Rg1 in the central nervous system.

小胶质细胞激活介导的炎症反应在PD的发病中发挥重要作用。类固醇激素受体介导的信号途径参与机体的免疫调控。在前期工作中，我们在国际上首次报道了人参皂苷Rg1对黑质多巴胺能神经元的保护作用与雌激素受体(ER)和胰岛素样生长因子I受体(IGF-IR)途径有关。由于ER与IGF-IR在胶质细胞上是广泛共存的，我们推测Rg1可能通过ER和IGF-IR信号途径发挥其抗炎作用。另有文献报道，小胶质细胞表达的糖皮质激素受体(GR)在多巴胺能神经元的死亡过程中发挥重要作用。作为GR的功能性配体，Rg1的抗炎作用是否与GR信号途径有关?本项目拟通过脂多糖诱导的大鼠PD模型、小胶质细胞-神经元共培养模型及原代培养的小胶质细胞，系统探讨Rg1对黑质小胶质细胞炎性反应的抑制作用是否与ER、IGF-IR及GR信号途径有关，明确Rg1抗炎作用的主要靶点，为Rg1开发为抗PD新药提供更完善的实验依据。

本研究采用ELISA、HPLC、实时PCR、siRNA干扰技术、免疫共沉淀、免疫印记及免疫荧光等多种评价方法，系统探讨了人参皂苷Rg1通过抑制炎症反应保护多巴胺能神经元的分子机制。在整体动物水平，本研究采用黑质注射脂多糖（LPS）制备帕金森病炎症大鼠模型，腹腔注射给予Rg1，同时侧脑室给予糖皮质激素受体阻断剂RU486、膜性雌激素受体GPER1阻断剂G15、胰岛素样生长因子I受体阻断剂JB1或GPER1激动剂G1，探讨Rg1的抗炎神经保护作用机制。结果显示：（1）Rg1或G1能够明显抑制LPS诱导的大鼠旋转行为。（2）Rgl或G1能够明显拮抗LPS诱导的患侧Str内多巴胺（DA）及其代谢产物含量的下降和SN致密带TH免疫反应阳性神经元数量及蛋白表达的下降。（3）免疫荧光结果显示，LPS诱导的SN区小胶质细胞激活，可以被Rg1或G1所拮抗。（4）Rg1或G1能够明显抑制LPS诱导的黑质炎性因子蛋白和基因表达及炎症信号通路（MAPKS及IB）的激活。（5）免疫共沉淀的结果显示Rg1能够促进GPER1与IGF-IR的相互作用。上述作用均可以被RU486、G15或JB-1所阻断。在离体细胞水平，本研究应用LPS诱导小胶质细胞的激活，探讨人参皂苷Rg1对小胶质细胞炎症反应的抑制作用及其机制。结果显示，（1）Rg1和G1能够明显抑制LPS诱导的小胶质细胞炎症因子的表达。（2）LPS能够明显增加 MAPKs及IκB的磷酸化水平，人参皂苷Rg1 和G1预保护能够对抗LPS引起的蛋白磷酸化。（3）小胶质细胞和神经元共培养结果显示，Rg1能够明显抑制炎症反应对多巴胺能神经元的损伤。上述作用均可以被可被G15、ICI182,780、JB1或RU486阻断。（4）应用Lv-siGPER干扰BV2小胶质细胞GPER的表达，Rg1和G1对LPS诱导的 iNOS和COX2表达的抑制作用明显减弱。（5）应用CAS9-Easy双载体慢病毒系统，沉默膜受体IGF-IR，Rg1对LPS诱导的COX-2基因表达的抑制作用明显降低，但iNOS基因表达的作用不明显。综上所述，人参皂苷Rg1能够通过GR、GPER1及IGF-IR，多靶点对抗LPS诱导的大鼠SN小胶质细胞的过度激活，抑制炎性因子对DA能神经元的损伤。本研究不仅为人参皂苷Rg1抗PD免疫炎症的分子机制提供实验依据，而且为PD的治疗提供了新的靶点。