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  

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

作　　者：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 ZHANG Jiafa;YANG Canhong;ZHANG Shufen;CAO Tingting;PENG Rui;GUOWeihong;YANYuping;XIE Shuting;PENG Xiaojia;LÜTianming;HUANG Tianrong(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)

机构地区：[1]南方医科大学第三附属医院神经内科,广东广州510630 [2]广州市南沙区第二人民医院,广东广州511455

出　　处：《南方医科大学学报》2023年第12期2132-2138,共7页Journal of Southern Medical University

基　　金：广东省基础与应用基础研究基金区域联合基金项目(2020A1515110506);广东省自然科学基金自由申请项目(2021A1515010006);南方医科大学第三附属医院院长基金(YM2021002);2022年度院长基金护理专项(YH202203);南方医科大学护理科研专项基金重点项目(Z2021002)。

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-αmRNA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,6.25μmol/L浓度fAβ25-35导致BV-2细胞活力增加(P<0.01),≥50μmol/L浓度fAβ25-35BV-2细胞活力降低(P<0.0001)。SFN在0~10μmol/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-αmRNA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。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-αmRNA 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 6.25μmol/L significantly increased the viability of BV-2 cells(P<0.01)whereas fAβ25-35 beyond 50μmol/L decreased the cell viability(P<0.0001).Treatment of BV-2 cells with SFN below 10μmol/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-αmRNA 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.

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 

分 类 号：R96[医药卫生—药理学]