School of Clinical Medicine, Chengdu University of TCM, Chengdu, Sichuan, China.
Department of Neurology, Hospital of Chengdu University of TCM, Chengdu, Sichuan, China.
Chem Biol Drug Des. 2024 Mar;103(3):e14481. doi: 10.1111/cbdd.14481.
Studies have shown that saikosaponin D (SSD) has favorable neurotherapeutic effects. Therefore, the objective of this study was to explore the efficacy and possible molecular mechanisms of SSD on pilocarpine (PP)-induced astrocyte injury. Primary astrocytes were isolated from juvenile rats and identified using immunofluorescence. The cells were treated with PP and/or SSD for 6 h and 12 h, respectively, followed by measurement of their viability through 3-(4,5-dimethylthiazol)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Next, quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the expression levels of Glial fibrillary acidic protein (GFAP), C3, S100 calcium binding protein A10 (S100a10), pentraxin 3 (Ptx3), toll-like receptor 4 (TLR4), and RAG in astrocytes after different treatments. Enzyme-linked immunosorbent assay and biochemical tests were utilized to evaluate the level of inflammatory factors [interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α)] secreted by cells and the content of oxidative stress-related factors (malondialdehyde [MDA] and glutathione [GSH]) or enzyme activity (catalase [CAT] and glutathione peroxidase [GPX]) in cells. The JC-1 mitochondrial membrane potential (MMP) fluorescence probe was used to measure the MMP in astrocytes. Additionally, western blot was applied to test the expression of proteins related to the nod-like receptor protein 3 (NLRP3)/caspase-1 signaling pathway. PP treatment (1 mM) induced cell injury by significantly reducing the viability of astrocytes and expression of cellular markers. SSD treatment (4 μM) had no toxicity to astrocytes. Besides, SSD (4 μM) treatment could significantly up-regulate the cell viability and marker expression of PP-induced astrocytes. Furthermore, SSD could be employed to inhibit inflammation (reduce IL-1β, IL-6, and TNF-α levels) and oxidative stress (decrease MDA level, elevate GSH level, the activity of CAT and GPX), and ameliorate mitochondrial dysfunction (upregulate JC-1 ratio) in PP-induced astrocytes. Moreover, further mechanism exploration revealed that SSD treatment significantly reduced the activity of the NLRP3/caspase-1 signaling pathway activated by PP induction. SSD increased cell viability, inhibited inflammation and oxidative stress response, and ameliorated mitochondrial dysfunction in PP-induced astrocyte injury model, thus playing a neuroprotective role. The mechanism of SSD may be related to the inhibition of the NLRP3/caspase-1 inflammasome.
研究表明，柴胡皂苷 D（SSD）具有良好的神经治疗作用。因此，本研究旨在探讨 SSD 对匹罗卡品（PP）诱导的星形胶质细胞损伤的疗效及其可能的分子机制。从幼年大鼠中分离出原代星形胶质细胞，并通过免疫荧光进行鉴定。用 PP 和/或 SSD 分别处理细胞 6 h 和 12 h，然后通过 3-(4,5-二甲基噻唑-2,5-二苯基四唑溴盐（MTT）测定细胞活力。接下来，采用定量实时聚合酶链反应（qRT-PCR）测定星形胶质细胞中神经胶质纤维酸性蛋白（GFAP）、C3、S100 钙结合蛋白 A10（S100a10）、五聚素 3（Ptx3）、Toll 样受体 4（TLR4）和 RAG 的表达水平。酶联免疫吸附试验和生化试验用于评估细胞分泌的炎症因子[白细胞介素（IL）-1β、IL-6 和肿瘤坏死因子α（TNF-α）]水平和细胞内氧化应激相关因子（丙二醛[MDA]和谷胱甘肽[GSH]）或酶活性（过氧化氢酶[CAT]和谷胱甘肽过氧化物酶[GPX]）含量。使用 JC-1 线粒体膜电位（MMP）荧光探针测定星形胶质细胞的 MMP。此外，采用 Western blot 检测与 NOD 样受体蛋白 3（NLRP3）/半胱天冬酶-1 信号通路相关的蛋白表达。PP 处理（1 mM）通过显著降低星形胶质细胞活力和细胞标志物表达来诱导细胞损伤。SSD 处理（4 μM）对星形胶质细胞没有毒性。此外，SSD（4 μM）处理可显著上调 PP 诱导的星形胶质细胞活力和标志物表达。此外，SSD 可抑制 PP 诱导的星形胶质细胞炎症（降低 IL-1β、IL-6 和 TNF-α 水平）和氧化应激（降低 MDA 水平，提高 GSH 水平，CAT 和 GPX 活性），改善线粒体功能障碍（上调 JC-1 比值）。此外，进一步的机制探索表明，SSD 处理可显著降低 PP 诱导激活的 NLRP3/半胱天冬酶-1 信号通路的活性。SSD 增加了细胞活力，抑制了炎症和氧化应激反应，改善了 PP 诱导的星形胶质细胞损伤模型中的线粒体功能障碍，从而发挥神经保护作用。SSD 的作用机制可能与抑制 NLRP3/半胱天冬酶-1 炎性小体有关。
