南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (12): 2132-2138.doi: 10.12122/j.issn.1673-4254.2023.12.19

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莱菔硫烷通过下调MAPK/NF-κB信号通路逆转Aβ纤维介导的M1型小胶质细胞极化和神经炎症介导的神经干细胞程序性坏死

张嘉发，杨灿洪，张淑芬，曹婷婷，彭 瑞，郭蔚泓，严予苹，谢淑婷，彭晓佳，吕田明，黄添容   

  1. 南方医科大学第三附属医院神经内科，广东 广州 510630；广州市南沙区第二人民医院，广东 广州 511455
  • 出版日期:2023-12-20 发布日期:2023-12-29

Sulforaphane reverses Aβ fiber-mediated M1 type microglia polarization and neuroinflammation-mediated necroptosis of neural stem cells by downregulating the MAPK/NF-κB signaling pathways

ZHANG Jiafa, YANG Canhong, ZHANG Shufen, CAO Tingting, PENG Rui, GUO Weihong, YAN Yuping, XIE Shuting, PENG Xiaojia, LÜ Tianming, HUANG Tianrong   

  1. Department of Neurology, Third Affiliated Hospital of Southern Medical University, Guangzhou 510630, China; Department of Internal Medicine, Nansha District Second People's Hospital, Guangzhou 511455, China
  • Online:2023-12-20 Published:2023-12-29

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摘要： 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Westernblotting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。

关键词: 莱菔硫烷；阿尔兹海默病；小胶质细胞；神经干细胞；淀粉样蛋白β；MAPK；NF-κB；M1；神经炎症

Abstract: 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.

Key words: sulforaphane; Alzheimer's disease; microglia; neural stem cells; β-amyloid; MAPK; NF-κB; M1; neuroinflammation