Kim Do-Yeon, Leem Yea-Hyun, Park Jin-Sun, Kim Seong-Eun, Choi Youn-Hee, Kang Jihee Lee, Kim Hee-Sun
Department of Molecular Medicine and Inflammation-Cancer Microenvironment Research Center, School of Medicine, Ewha Womans University, Seoul, South Korea.
Department of Physiology and Inflammation-Cancer Microenvironment Research Center, School of Medicine, Ewha Womans University, Seoul, South Korea.
Biochem Pharmacol. 2025 Sep;239:117021. doi: 10.1016/j.bcp.2025.117021. Epub 2025 Jun 3.
Necroptosis is involved in neuronal cell death and inflammation, with the receptor-interacting protein kinase (RIPK) 1-RIPK3-mixed lineage kinase domain-like protein (MLKL) necrosome complex playing a significant role. Although MLKL is considered the final executor of necroptosis, its role in neuroinflammation remains unclear. In the present study, we explored the role of MLKL in lipopolysaccharide (LPS)- or polyinosinic-polycytidylic acid (poly(I:C))-induced neuroinflammation using the MLKL-specific inhibitor necrosulfonamide (NSA). NSA or MLKL siRNA reduced nitric oxide and proinflammatory cytokine production under LPS- or poly(I:C)-induced inflammation and LPS/QVD/BV6- or poly(I:C)/QVD/BV6-induced necroptosis in BV2 microglial cells; QVD is a pan-caspase inhibitor, and BV6 antagonizes inhibitor of apoptosis protein. Additionally, NSA suppressed the phosphorylation of RIPK1-RIPK3-MLKL and reduced the expression of damage-associated molecular patterns, including high mobility group box 1, in inflammatory/necroptotic BV2 cells. Subsequent mechanistic studies revealed that NSA reduces inflammation by upregulating nuclear factor-erythroid 2 (NF-E2)-related factor (Nrf2) signaling pathways and blocking reactive oxygen species, mitogen-activated protein kinases, and nuclear factor-κB. The anti-inflammatory effects of NSA were confirmed in the brains of mice with systemic inflammation caused by LPS or poly(I:C) injection. NSA suppressed microglial activation, proinflammatory gene expression, and disruption of blood-brain barrier integrity while upregulating Nrf2-mediated antioxidant enzymes in the brains of LPS- or poly(I:C)-injected mice. Furthermore, NSA suppressed the phosphorylation and expression of RIPK1-RIPK3-MLKL, and p-MLKL expressed in activated microglia, indicating that MLKL plays a crucial role in microglial activation in mice with systemic inflammation. Therefore, modulating MLKL expression may be an effective treatment for necroptosis-related neuroinflammatory disorders.
坏死性凋亡参与神经元细胞死亡和炎症反应，受体相互作用蛋白激酶（RIPK）1-RIPK3-混合谱系激酶结构域样蛋白（MLKL）坏死小体复合物发挥重要作用。尽管MLKL被认为是坏死性凋亡的最终执行者，但其在神经炎症中的作用仍不清楚。在本研究中，我们使用MLKL特异性抑制剂坏死磺酰胺（NSA）探讨了MLKL在脂多糖（LPS）或聚肌苷酸-聚胞苷酸（poly(I:C)）诱导的神经炎症中的作用。NSA或MLKL siRNA可减少LPS或poly(I:C)诱导的炎症以及LPS/QVD/BV6或poly(I:C)/QVD/BV6诱导的BV2小胶质细胞坏死性凋亡过程中一氧化氮和促炎细胞因子的产生；QVD是一种泛半胱天冬酶抑制剂，BV6可拮抗凋亡蛋白抑制剂。此外，NSA可抑制炎症/坏死性凋亡BV2细胞中RIPK1-RIPK3-MLKL的磷酸化，并降低包括高迁移率族蛋白B1在内的损伤相关分子模式的表达。随后的机制研究表明，NSA通过上调核因子红系2（NF-E2）相关因子（Nrf2）信号通路并阻断活性氧、丝裂原活化蛋白激酶和核因子κB来减轻炎症。NSA的抗炎作用在LPS或poly(I:C)注射引起全身炎症的小鼠脑中得到证实。NSA可抑制小胶质细胞活化、促炎基因表达以及血脑屏障完整性的破坏，同时上调LPS或poly(I:C)注射小鼠脑中Nrf2介导的抗氧化酶。此外，NSA可抑制RIPK1-RIPK3-MLKL的磷酸化和表达，以及活化小胶质细胞中表达的p-MLKL，表明MLKL在全身炎症小鼠的小胶质细胞活化中起关键作用。因此，调节MLKL表达可能是治疗坏死性凋亡相关神经炎症性疾病的有效方法。
