Silencing of activating transcription factor 4 inhibits neuronal necroptosis after intracerebral hemorrhage in vitro

doi:10.12307/2024.147

Abstract

Abstract: BACKGROUND: Neuronal necroptosis induced by intracerebral hemorrhage is an important cause of secondary brain injury. Activating transcription factor 4 (ATF4) is a member of the transcription activator family, which plays an important role in secondary brain injury after intracerebral hemorrhage. However, the mechanism of ATF4 in neuronal necroptosis after intracerebral hemorrhage remains unclear.
OBJECTIVE: To explore the effect of ATF4 silencing (ATF4 small interfering RNA, ATF4 siRNA) on neuronal necroptosis after intracerebral hemorrhage.
METHODS: The HT-22 mouse hippocampal neuron cell line and the BV-2 mouse microglial cell line were co-cultured, and hemin was used to mimic an in vitro model of intracerebral hemorrhage. A gradient concentration of hemin was used to treat cells and was set in the interval of 0-100 μmol/L, and the cell viability was evaluated by MTT assay after 24 hours of administration of hemin. The cells were divided into four groups: the blank control group without any intervention; the control group was treated with hemin (50 μmol/L), and the other two groups were treated with negative control small interfering RNA (NC siRNA) and ATF4 small interfering RNA (ATF4 siRNA) 48 hours before administration of hemin. After the cells were treated with hemin (50 μmol/L) for 24 hours, PI/Hoechst staining was used to detect neuronal necroptosis. Western blot assay was used to detect the protein expression of ATF4, receptor-interacting protein 3 (RIP3), and mixed lineage kinase domain-like protein (MLKL), and double immunofluorescent staining was located in neurons to observe the level of neuronal necroptosis and the regulatory effect of ATF4 on it.
RESULTS AND CONCLUSION: (1) 50 μmol/L of hemin could induce neuronal necroptosis to a greater extent. (2) The number of PI+/Hoechst+ cells in the control group and NC siRNA group was higher than that in the blank control group (P < 0.000 1). The number of PI+/Hoechst+ cells in the ATF4 siRNA group was lower than that in the control group (P < 0.000 1). (3) Compared with the control group, the ATF4 siRNA group not only inhibited the expression of ATF4 protein (P < 0.001), but also inhibited the expression of RIP3 and MLKL protein (P < 0.001). (4) Through double immunofluorescent staining, compared with the control group, the protein expression of RIP3 and MLKL was significantly reduced in the ATF4 siRNA group (P < 0.000 1). (5) The results show that the silencing of the ATF4 gene can directly or indirectly inhibit the expression of genes related to neuronal necroptosis after intracerebral hemorrhage, and play a vital role in alleviating secondary brain injury.

Key words: intracerebral hemorrhage, secondary brain injury, neuron, activating transcription factor 4, necroptosis, small interfering RNA

CLC Number:

Feng Dengfeng, Wei Min, Zhang Hengzhu. Silencing of activating transcription factor 4 inhibits neuronal necroptosis after intracerebral hemorrhage in vitro[J]. Chinese Journal of Tissue Engineering Research, 2024, 28(13): 2030-2035.

Figures/Tables 5

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