LIU Yu-ying, XIAO Meng, WANG Fang, WANG Yao-zhen, LIU Peng
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.
