参远苷抑制脂多糖诱导的BV2小胶质细胞炎症机制研究

The therapeutic mechanism of Shenyuan Gan in lipopolysaccharide-induced neuroinflammation in BV2 microglial cells

  • 摘要:
    目的研究参远苷对脂多糖诱导的BV2小胶质细胞炎症反应的治疗作用。
    方法采用细胞增殖-毒性检测(CCK-8)法评价参远苷对BV2小胶质细胞的细胞毒性,并研究不同浓度参远苷对脂多糖(LPS)诱导的BV2小胶质细胞活力的影响;光学显微镜观察细胞形态变化;使用 Griess 试剂测定细胞培养上清液一氧化氮 (NO) 浓度;通过酶联免疫吸附试验(ELISA)测量细胞因子和炎症介质的表达;蛋白质印迹分析(Western blot)用于确定诱导型一氧化氮合酶 (iNOS)、核因子-κB p65 (NF-κB p65)、磷酸化-核因子 κB p65 (p-NF-κB p65)、核因子κB-α抑制蛋白 (IκB-α)和磷酸化核因子-κB-α抑制蛋白(p-IκB-α)、核苷酸结合寡聚化结构域样受体蛋白3 (NLRP3)以及caspase-1表达;免疫荧光染色观察iNOS、NLRP3和离子钙结合衔接分子1 (Iba1)的表达。
    结果参远苷对BV2小胶质细胞具有较低的细胞毒性作用,并能显著降低LPS诱导的BV2小胶质细胞形态变化(P < 0.05)。ELISA结果表明参远苷能显著抑制LPS诱导的BV2小胶质细胞中白细胞介素-1β(IL-1β)和白细胞介素-6(IL-6)表达量的增加(P <0.05)。Western blot结果显示,参远苷处理后的 iNOS、NF-κB p65和IκB-α磷酸化水平以及NLRP3和caspase-1的表达明显降低,IκB-α表达升高(P < 0. 05,与LPS处理组相比)。免疫荧光结果与Western blot结果一致,并且Iba1染色表明细胞形态趋于静息状态。这些结果表明,参远苷对LPS诱导的BV2小胶质细胞的炎症有一定的抑制作用。
    结论参远苷可能通过影响 NF-κB p65 和 IκB-α 的磷酸化水平来抑制 LPS 诱导的 BV2 小胶质细胞炎症因子释放。参远苷是治疗神经炎症相关疾病的有价值的候选药物。

     

    Abstract:
    ObjectiveTo study the therapeutic effects of Shenyuan Gan (参远苷, SYG) on the inflammatory response in BV2 microglial cells induced by lipopolysaccharide (LPS).
    MethodsThe cytotoxicity of SYG to BV2 microglial cells was evaluated using a Cell Counting Kit-8 (CCK-8) assay, and the effect of SYG concentrations on LPS-induced BV2 microglial cells was studied. The morphological changes were observed using an optical microscope. The nitric oxide (NO) concentration in cell culture supernatant was determined using Griess reagent. The expression of cytokines and inflammatory mediators were also measured by an enzyme-linked immunosorbent assay (ELISA). Western blot analysis was used to determine the levels of inducible NO synthase (iNOS), nuclear factor-kappa B (NF-κB) p65, alpha inhibitor of NF-κB (IκB-α), phosphorylation-IκB-α (p-IκB-α), NOD-like receptor 3 (NLRP3), and caspase-1 expression. Moreover, the expression of iNOS, NLRP3, and ionized calcium binding adapter molecule 1 (Iba1) was also observed using immunofluorescent staining.
    ResultsSYG had a low cytotoxic effect on BV2 microglial cells and could significantly decr-ease LPS-induced morphological changes of BV2 microglial cells (P < 0.05). ELISA results showed that SYG significantly inhibited the LPS-induced increase in interleukin (IL)-1β and IL-6 in BV2 microglia cells (P < 0.05), and Western blot analysis showed that the phosphorylation levels of iNOS, NF-κB p65, and IκB-α as well as NLRP3 and caspase-1 expression were also significantly decreased, and IκB-α expression was increased after SYG treatment (P < 0.05, compared with the LPS-treated group). The immunofluorescence results were consistent with the Western blot results, and Iba1 staining indicated that the cell morphology tended to be resting. These results indicate that SYG has a certain inhibitory effect on LPS-induced inflammation in BV2 microglial cells.
    ConclusionSYG can inhibit LPS-induced release of inflammatory factors in BV2 microglial cells by affecting the phosphorylation levels of NF-κB p65 and IκB. SYG is a valuable candidate for treating neuroinflammation-related diseases.

     

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