Objective: To explore the effects of sulforaphane (SFN) and Aβ25-35 fibers (fAβ25-35) on M1/M2 polarization of BV-2 cells and neuroinflammation-mediated programmed necrosis of neural stem cells.
Methods: BV-2 cells treated with different concentrations of fAβ25-35 and SFN were examined for changes in cell viability using the CCK-8 kit. The effect of fAβ25-35 alone or in combination with SFN or SB203580 on expressions of IL-6 and TNF-α mRNA and proteins were assessed in BV-2 cells using RT-qPCR and ELISA. CD16/32 and CD206 in the treated cells were analyzed with flow cytometry, and the cellular expressions of p-p38 and p-p65 protein were detected with Western blotting. C17.2 cells co-cultured with BV-2 cells for 24 h were examined for p-mlkl protein expression using Western blotting.
Results: fAβ25-35 at the concentration of 6.25 μmol/L significantly increased the viability of BV-2 cells (P < 0.01) whereas fAβ25-35 beyond 50 μmol/L decreased the cell viability (P < 0.0001). Treatment of BV-2 cells with SFN below 10 μmol/L for 24 h did not significant affect the cell viability (P > 0.05). BV-2 cells treated with fAβ25-35 alone, as compared with the cells in the other 3 groups, showed significantly increased IL-6 and TNF-α mRNA and protein expressions (P < 0.001), enhanced CD16/32 expression (P < 0.05), lowered CD206 expression (P < 0.01), and increased protein expressions of p-p38 and p- p65 (P < 0.01). C17.2 cells co-cultured with BV-2 cells treated with fAβ25-35, compared with the combined treatments, showed a significant reduction in the protein expression of p-mlkl (P < 0.05).
Conclusion: SFN reverses M1 type microglia polarization and neuroinflammation-mediated programmed necrosis of neural stem cells by downregulating the MAPK/NF-κB signaling pathway in Aβ25-35-activated BV-2 cells.
目的: 探讨莱菔硫烷（SFN）和Aβ25-35纤维（fAβ25-35）对BV-2细胞M1/M2极化的影响，及其通过下调MAPK/NF-κB信号通路逆转M1型小胶质细胞极化逆转神经炎症介导的C17.2神经干细胞程序性坏死。
方法: 以不同浓度Aβ25-35和SFN对BV-2细胞作用后用CCk8试剂盒检测细胞活性。实验分组：溶媒对照组、Aβ组、Aβ+SFN组、Aβ+SB203580组。对BV-2细胞进行IL-6及TNF-α mRNA检测，对细胞上清进行IL-6及TNF-αElisa检测，对BV-2细胞进行CD16/32和CD206标记的流式细胞检测，提取BV-2蛋白后进行Western blotting检测p-p38和p-p65蛋白表达；BV-2与C17.2共培养24 h后提取C17.2蛋白进行进行Western blotting检测p-mlkl蛋白表达。
结果: CCK8结果显示，与溶媒对照组相比，6.25 μmol/L浓度fAβ25-35导致BV-2细胞活力增加（P < 0.01），≥ 50 μmol/L浓度fAβ25-35BV-2细胞活力降低（P < 0.0001）。SFN在0~10 μmol/L范围内，对BV-2细胞作用24 h后无明显差异（P > 0.05）。RT-qPCR、ELISA、流式细胞术以及Western blotting结果显示，在BV-2细胞中，Aβ组与其他三组相比，IL-6和TNF-α mRNA表达升高（P < 0.001）、上清中IL-6和TNF-α浓度升高（P < 0.001）、CD16/32表达升高（P < 0.05）、CD206表达降低（P < 0.05）以及p-p38和p-p65表达升高（P < 0.01）。Western blotting结果显示，在C17.2细胞中，Aβ组与其他三组相比p-mlkl表达升高（P < 0.05）。
结论: SFN通过在fAβ25-35激活的BV-2细胞中下调MAPK/NF-κB信号通路逆转M1型小胶质细胞极化逆转神经炎症介导的C17.2神经干细胞程序性坏死。
Keywords: Alzheimer's disease; M1; MAPK; NF-κB; microglia; neural stem cells; neuroinflammation; sulforaphane; β-amyloid.