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Oxymatrine inhibits neuroinflammation by Regulating M1/M2 polarization in N9 microglia through the TLR4/NF-κB pathway
International Immunopharmacology ( IF 4.8 ) Pub Date : 2021-09-10 , DOI: 10.1016/j.intimp.2021.108139
Xiao-Long Wang 1 , Fei Chen 1 , Hui Shi 1 , Man Zhang 1 , Lin Yan 2 , Xiu-Ying Pei 3 , Xiao-Dong Peng 4
Affiliation  

Microglia are the primary immune cells involved in the immune response, inflammation, and injury repair in the central nervous system. Under different stimuli, the dual polarization of classically-activated M1 microglia and anti-inflammatory selectively-activated M2 microglia is observed. Oxymatrine (OMT) exerts various anti-inflammatory and neuroprotective effects, but the mechanism underlying its action remains unclear. In the present study, we investigated the effects of OMT on the polarization of M1/M2 microglia in a lipopolysaccharide (LPS)-induced inflammation model in order to elucidate the potential molecular mechanism of action of OMT in vitro. We first used a Cell Counting Kit-8 (CCK-8) to evaluate the effects of different concentrations OMT on the viability of N9 microglia to determine the appropriate concentration for follow-up experiments. Next, Griess reagent and enzyme-linked immunosorbent assay (ELISA) kits were used to detect the expression of the inflammation-related factors nitric oxide (NO), tumour necrosis factor-alpha (TNF-α), and interleukin (IL)-6, −1β, and −10. To evaluate the protective effects of OMT, the ultrastructure of the cells was observed using electron microscopy. Immunofluorescence, flow cytometry, and western blotting were performed to evaluate the effects of OMT on the following markers of M1 and M2 microglia: CD16/32, CD206, Arginase-10 (Arg-1), and inducible nitric oxide synthase (iNOS). Lastly, western blotting and quantitative polymerase chain reaction (qPCR) were used to detect factors associated with the Toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB) signalling pathway in order to explore the potential mechanism by which OMT regulates microglial polarization. The viability of N9 cells did not decrease when treated with a concentration of 1000 μg/mL OMT. Electron microscopy revealed that a concentration of 100 μg/mL OMT exerted a protective effect on N9 cells stimulated by LPS. The results of the present study indicated that OMT inhibited the over-activation of microglia, increased the levels of the M2 marker IL-10, decreased the levels of the M1 markers NO, TNF-α, IL-6, and IL-1β, promoted the polarization of N9 microglia to the M2 phenotype, and regulated M1/M2 polarization in the microglia by inhibiting TLR4/NF-κB signalling, which effectively attenuated the LPS-induced inflammatory response.



中文翻译:

氧化苦参碱通过 TLR4/NF-κB 通路调节 N9 小胶质细胞中的 M1/M2 极化来抑制神经炎症

小胶质细胞是参与中枢神经系统免疫反应、炎症和损伤修复的主要免疫细胞。在不同的刺激下,观察到经典激活的M1小胶质细胞和抗炎选择性激活的M2小胶质细胞的双重极化。氧化苦参碱 (OMT) 具有多种抗炎和神经保护作用,但其作用机制仍不清楚。在本研究中,我们在脂多糖 (LPS) 诱导的炎症模型中研究了 OMT 对 M1/M2 小胶质细胞极化的影响,以阐明 OMT在体外的潜在作用分子机制。. 我们首先使用 Cell Counting Kit-8 (CCK-8) 来评估不同浓度 OMT 对 N9 小胶质细胞活力的影响,以确定后续实验的合适浓度。接下来,使用Griess试剂和酶联免疫吸附试验(ELISA)试剂盒检测炎症相关因子一氧化氮(NO)、肿瘤坏死因子-α(TNF-α)和白细胞介素(IL)-6的表达。 、-1β 和 -10。为了评估 OMT 的保护作用,使用电子显微镜观察细胞的超微结构。进行免疫荧光、流式细胞术和蛋白质印迹以评估 OMT 对以下 M1 和 M2 小胶质细胞标志物的影响:CD16/32、CD206、Arginase-10 (Arg-1) 和诱导型一氧化氮合酶 (iNOS)。最后,采用蛋白质印迹和定量聚合酶链反应(qPCR)检测与Toll样受体4/核因子-κB(TLR4/NF-κB)信号通路相关的因子,以探索OMT调节小胶质细胞极化的潜在机制. 当用 1000 μg/mL OMT 处理时,N9 细胞的活力没有降低。电镜显示,100 μg/mL OMT 对 LPS 刺激的 N9 细胞具有保护作用。本研究结果表明 OMT 抑制小胶质细胞的过度活化,增加 M2 标志物 IL-10 的水平,降低 M1 标志物 NO、TNF-α、IL-6 和 IL-1β 的水平,促进 N9 小胶质细胞向 M2 表型的极化,并通过抑制 TLR4/NF-κB 信号传导调节小胶质细胞中的 M1/M2 极化,

更新日期:2021-09-10
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