Nicotinamide mononucleotide mitigates neuroinflammation by enhancing GPX4-mediated ferroptosis defense in microglia. Nicotinamide mononucleotide mitigates neuroinflammation by enhancing GPX4-mediated ferroptosis defense in microglia

Numerous neurological diseases involve neuroinflammation, a process in which immune cells, particularly microglia, contribute to neuronal death. Ferroptosis, a recently identified form of regulated cell death, is implicated in various diseases characterized by neuronal injury. Nicotinamide mononucleotide (NMN), a potent NAD+ precursor supplement, has been found to inhibit neuroinflammation and ferroptosis. However, the mechanisms of NMN in both ferroptosis and neuroinflammation remains unclear. The present study aimed to investigate the impact of NMN on neuroinflammation and the susceptibility of microglia to ferroptosis. Ferroptosis markers in microglia exposed to lipopolysaccharide (LPS) were analyzed using CCK8, flow cytometry, ELISA, and RT-qPCR. The effects of NMN on LPS-induced ferroptosis in microglia were evaluated through flow cytometry, western blotting, and immunofluorescence staining. RT-qPCR analysis assessed the inflammatory cytokine production of microglia subjected to ferrostatin-1-regulated ferroptosis. RNA sequencing elucidated the underlying mechanisms of NMN-associated microglia ferroptosis under LPS induction. In BV2 microglia, an inhibitor of Glutathione Peroxidase 4(GPX4), RSL3, was employed to suppress GPX4 expression. Intracerebroventricular injection of LPS was performed to evaluate neuroinflammation and microglia activation in vivo. LPS treatment resulted in decreased cell viability, accompanied by upregulation of ferroptosis markers SLC7A11 and GPX4, and elevated levels of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and total iron in a dose-dependent manner. NMN effectively rescued LPS-induced ferroptosis and improved cell viability in microglia. Co-administration of NMN and ferrostatin-1 significantly reduced proinflammatory cytokine production in microglia following the introduction of LPS stimuli. Mechanistically, NMN facilitated glutathione (GSH) production, and promoted resistance to lipid peroxidation in a GPX4-dependent manner, repressing cytokine transcription and protecting cells from ferroptosis. RNA sequencing elucidated the underlying mechanism of NMN-associated microglia ferroptosis under LPS induction. Furthermore, simultaneous injection of NMN ameliorated LPS-induced ferroptosis and neuroinflammation in mouse brains. The data from the present study indicated that NMN enhances GPX4-mediated ferroptosis defense against LPS-induced ferroptosis in microglia by recruiting GSH, thereby inhibiting neuroinflammation. Therefore, therapeutic approaches targeting ferroptosis in diseases using NMN should consider both its anti-ferroptosis and anti-inflammatory effects to achieve optimal outcomes, presenting promising strategies for treating neuroinflammation-related diseases or disorders. Overall design: To unravel the molecular mechanism underlying NMN-mediated rescue of LPS-induced ferroptosis in BV2 cells, RNA sequencing was conducted to identify differentially expressed genes between the LPS and LPS + NMN groups

众多神经系统疾病均涉及神经炎症(neuroinflammation)——该过程中免疫细胞（尤其是小胶质细胞(microglia)）介导神经元死亡。铁死亡(Ferroptosis)是近年发现的一种调控性细胞死亡形式，与多种以神经元损伤为特征的疾病密切相关。烟酰胺单核苷酸(Nicotinamide mononucleotide, NMN)是一种高效的烟酰胺腺嘌呤二核苷酸(NAD+)前体补充剂，被证实可抑制神经炎症与铁死亡，但NMN在铁死亡及神经炎症中的具体作用机制仍不明确。本研究旨在探究NMN对神经炎症以及小胶质细胞铁死亡易感性的影响。本研究通过细胞计数试剂盒-8(CCK8)、流式细胞术(flow cytometry)、酶联免疫吸附实验(ELISA)及实时定量聚合酶链式反应(RT-qPCR)，分析了脂多糖(lipopolysaccharide, LPS)刺激后小胶质细胞的铁死亡标志物表达水平；采用流式细胞术、蛋白质印迹(western blotting)及免疫荧光染色(immunofluorescence staining)，评估了NMN对脂多糖诱导的小胶质细胞铁死亡的干预效果；通过RT-qPCR检测了经铁抑素-1(ferrostatin-1)调控铁死亡的小胶质细胞的炎症因子分泌情况；借助RNA测序(RNA sequencing)阐明了脂多糖诱导下NMN调控小胶质细胞铁死亡的潜在分子机制。本研究选用谷胱甘肽过氧化物酶4(Glutathione Peroxidase 4, GPX4)抑制剂RSL3以抑制BV2小胶质细胞(BV2 microglia)中的GPX4表达；通过脑室内注射(intracerebroventricular injection)脂多糖构建体内神经炎症与小胶质细胞活化模型，以评估NMN的在体干预效果。研究发现，脂多糖处理可呈剂量依赖性降低细胞活力，同时上调铁死亡标志物SLC7A11与GPX4的表达，并升高丙二醛(malondialdehyde, MDA)、4-羟基壬烯醛(4-hydroxynonenal, 4-HNE)及总铁含量。NMN可有效逆转脂多糖诱导的小胶质细胞铁死亡，并改善细胞活力。联合使用NMN与铁抑素-1，可显著降低脂多糖刺激后小胶质细胞的促炎细胞因子分泌水平。从机制上看，NMN可促进谷胱甘肽(glutathione, GSH)合成，并以GPX4依赖的方式增强细胞对脂质过氧化的抵抗能力，进而抑制细胞因子转录、保护细胞免受铁死亡损伤。RNA测序进一步阐明了脂多糖诱导下NMN调控小胶质细胞铁死亡的潜在机制。体内实验显示，同时注射NMN可改善小鼠脑内脂多糖诱导的铁死亡与神经炎症。本研究数据表明，NMN通过招募GSH增强GPX4介导的铁死亡防御通路，从而拮抗脂多糖诱导的小胶质细胞铁死亡，进而抑制神经炎症。因此，采用NMN靶向铁死亡治疗相关疾病时，需同时兼顾其抗铁死亡与抗炎效应以实现最优疗效，这为治疗神经炎症相关疾病或紊乱提供了极具前景的策略。总体实验设计：为阐明NMN逆转脂多糖诱导的BV2细胞铁死亡的分子机制，本研究通过RNA测序筛选了脂多糖组与脂多糖+NMN组之间的差异表达基因。