灯盏花素抑制神经细胞坏死性凋亡的机制

LIU  Yuying1; XIAO  Meng1; WANG  Fang1; WANG  Yaozhen2; LIU  Peng1*

doi:10.16098/j.issn.0529-1356.2026.01.012

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解剖学报 ›› 2026, Vol. 57 ›› Issue (1) : 77-83. DOI: 10.16098/j.issn.0529-1356.2026.01.012

神经生物学

灯盏花素抑制神经细胞坏死性凋亡的机制

刘玉莹¹肖萌¹王芳¹王耀振²刘鹏^1*

作者信息 +

Mechanism of inhibiting neuronal necroptosis by breviscapine

LIU Yu-ying¹, XIAO Meng¹, WANG Fang¹, WANG Yao-zhen², LIU Peng^1*

Author information +

文章历史 +

摘要

目的探讨灯盏花素（BRE）调节受体相互作用蛋白激酶（RIP）1/RIP3/混合谱系激酶结构域样蛋白（MLKL）信号通路对氧糖剥夺/复氧（OGD/R）诱导的神经细胞坏死性凋亡的影响。方法将HT22细胞随机分为对照（Ctrl）组、模型（Mod）组、BRE组（20 μmol/L BRE）、RIP1/RIP3/MLKL信号通路抑制剂Necrostatin-1（Nec-1）组、RIP1/RIP3/MLKL信号通路激活剂Z-VAD-FMK组及BRE+Z-VAD-FMK组。除Ctrl组正常培养外，其余各组进行OGD/R诱导细胞模型。CCK-8和集落形成实验检测细胞增殖；ELISA检测白细胞介素（IL）-1β、肿瘤坏死因子（TNF）-α水平；流式细胞术检测细胞程序性坏死细胞率；免疫细胞化学染色检测p-MLKL表达；Western blotting检测p-RIP1/RIP1、p-RIP3/RIP3、p-MLKL/MLKL、Caspase-8蛋白表达。结果Mod组与Ctrl组相比，细胞的存活率、集落数及Caspase-8表达降低，程序性坏死细胞率、IL-1β、TNF-α、p-RIP1/RIP1、p-RIP3/RIP3、p-MLKL/MLKL升高（P<0.05），p-MLKL阳性信号在细胞膜/胞质呈点状或团块状聚集，p-MLKL⁺细胞百分比增加（P<0.05）；与Mod组相比，BRE组、Nec-1组细胞存活率、集落数及Caspase-8表达升高，程序性坏死细胞率、IL-1β、TNF-α、p-RIP1/RIP1、p-RIP3/RIP3、p-MLKL/MLKL降低（P<0.05），p-MLKL阳性信号减弱，p-MLKL⁺细胞百分比下降（P<0.05）；而Z-VAD-FMK组各指标与之相反（P<0.05）；BRE+Z-VAD-FMK组与BRE组相比，细胞的存活率、集落数及Caspase-8表达降低，程序性坏死细胞率、IL-1β、TNF-α、p-RIP1/RIP1、p-RIP3/RIP3、p-MLKL/MLKL升高（P<0.05），p-MLKL阳性信号增强，p-MLKL⁺细胞百分比增加（P<0.05）；但BRE+Z-VAD-FMK组与Z-VAD-FMK组相比，细胞的存活率、集落数及Caspase-8表达升高，程序性坏死细胞率、IL-1β、TNF-α、p-RIP1/RIP1、p-RIP3/RIP3、p-MLKL/MLKL降低（P<0.05），p-MLKL阳性信号减弱，p-MLKL⁺细胞百分比下降（P<0.05）。结论BRE可能通过阻断RIP1/RIP3/MLKL信号通路，对OGD/R诱导的神经细胞的坏死性凋亡起到保护作用。

Abstract

Objective To investigate the effect of breviscapine (BRE) on oxygen-glucose deprivation/reoxygenation (OGD/R)-induced necroptosis of neural cells by regulating the receptor-interacting protein (RIP) 1/RIP3/mixed-lineage kinase-like protein (MLKL) signaling pathway. Methods HT22 cells were randomly grouped into control (Ctrl) group, model (Mod) group, BRE group (20 μmol/L BRE), RIP1/RIP3/MLKL signaling pathway inhibitor Necrostatin-1 (Nec-1) group, RIP1/RIP3/MLKL signaling pathway activator Z-VAD-FMK group, and BRE+Z-VAD-FMK group. Except for the Ctrl group, the remaining groups were subjected to OGD/R induced cell models. Cell counting kit-8 and colony formation assay were applied to detect the proliferation. ELISA was applied to detect the levels of interleukin (IL)-1β and tumor necrosis factor (TNF)-α. Flow cytometry was applied to detect the programmed cell death rate. Immunocytochemical staining was used to detect the expression of p-MLKL. Western blotting was applied to detect the expression of p-RIP1/RIP1, p-RIP3/RIP3, p-MLKL/MLKL and Caspase-8. Results Compared with the Ctrl group, the survival rate and colony number of cells, and the expression of Caspase-8 in the Mod group were lower, while the programmed necrosis, IL-1β, TNF-α, p-RIP1/RIP1, p-RIP3/RIP3, and p-MLKL/MLKL were higher (P<0.05), and the p-MLKL positive signal was aggregated in punctate or clumped form on the cell membrane/cytoplasm, and the percentage of p-MLKL⁺ cells increased (P<0.05). Compared with the Mod group, the survival rate and colony number, and the expression of Caspase-8 of cells in the BRE group and Nec-1 group were higher, while the programmed cell death rate, IL-1β, TNF-α, p-RIP1/RIP1, p-RIP3/RIP3, and p-MLKL/MLKL were lower (P<0.05), the positive signal of p-MLKL weakened, and the percentage of p-MLKL⁺ cells decreased (P<0.05); while in the Z-VAD-FMK group, all the indicators were opposite (P<0.05). Compared with the BRE group, the cell survival rate, the number of colony, and the expression of Caspase-8 in the BRE+Z-VAD-FMK group decreased, while the rates of programmed necrosis cells, IL-1β, TNF-α, p-RIP1/RIP1, p-RIP3/RIP3, and p-MLKL/MLKL increased (P<0.05), and the positive signal of p-MLKL was enhanced, and the percentage of p-MLKL⁺ cells increased (P<0.05). However, compared with the Z-VAD-FMK group, the survival rate and colony number, and the expression of Caspase-8 of cells in the BRE+Z-VAD-FMK group were higher, while the programmed cell death rate, IL-1β, TNF-α, p-RIP1/RIP1, p-RIP3/RIP3, and p-MLKL/MLKL were lower (P<0.05), and the positive signal of p-MLKL was weakened, and the percentage of p-MLKL⁺cells decreased (P<0.05). Conclusion BRE may protect against OGD/R-induced necroptosis of neural cells by blocking the RIP1/RIP3/MLKL signaling pathway.

导出引用

刘玉莹肖萌王芳王耀振刘鹏. 灯盏花素抑制神经细胞坏死性凋亡的机制[J]. 解剖学报. 2026, 57(1): 77-83 https://doi.org/10.16098/j.issn.0529-1356.2026.01.012

LIU Yu-ying, XIAO Meng, WANG Fang, WANG Yao-zhen, LIU Peng. Mechanism of inhibiting neuronal necroptosis by breviscapine[J]. Acta Anatomica Sinica. 2026, 57(1): 77-83 https://doi.org/10.16098/j.issn.0529-1356.2026.01.012

中图分类号： R322.8 R744.9

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