Dos Santos Marcelle Pereira, Leocadio Vitor Emanuel, de Sá Hayashide Lívia, Marques Mariana, Carvalho Clara Fernandes, Galina Antonio, Diniz Luan Pereira
Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-902, RJ, Brazil.
Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-902, RJ, Brazil.
Toxins (Basel). 2025 Jun 9;17(6):293. doi: 10.3390/toxins17060293.
Microglia, the resident immune cells of the central nervous system (CNS), play essential roles in maintaining brain homeostasis. While transient activation is protective, chronic microglial reactivity contributes to neuroinflammatory damage and neurodegeneration. The mitochondrial mechanisms underlying this shift remain poorly understood. Here, we investigated whether lipopolysaccharide (LPS) induces coordinated mitochondrial and metabolic alterations in BV-2 microglial cells. LPS stimulation (100 ng/mL, 24 h) induced a reactive phenotype, with increased Iba1 (+82%), F4/80 (+132%), and Cd68 (+44%), alongside elevated hydrogen peroxide (6-fold) and nitrite (45-fold). Cytotoxicity increased by 40% (LDH assay), and cell viability dropped to ~80% of the control (MTT). Extracellular lactate increased, indicating glycolytic reprogramming. However, LPS-primed cells showed greater ATP depletion under antimycin A challenge, reflecting impaired metabolic flexibility. Hoechst staining revealed a ~4-fold increase in pyknotic nuclei, indicating apoptosis. Mitochondrial dysfunction was confirmed by a 30-40% reduction in membrane potential (TMRE, JC-1), a ~30% loss of Tomm20, and changes in dynamics: phospho-Drp1 increased (+23%), while Mfn1/2 decreased (33%). Despite a ~70% rise in Lamp2 signal, Tomm20-Lamp2 colocalization decreased, suggesting impaired mitophagy. High-resolution respirometry revealed decreased basal (-22%), ATP-linked (24%), and spare respiratory capacity (41%), with increased non-mitochondrial oxygen consumption. These findings demonstrate that LPS induces mitochondrial dysfunction, loss of metabolic adaptability, and increased apoptotic susceptibility in microglia. Mitochondrial quality control and energy flexibility emerge as relevant targets to better understand and potentially modulate microglial responses in neuroinflammatory and neurodegenerative conditions.
小胶质细胞是中枢神经系统（CNS）中的常驻免疫细胞，在维持脑内稳态中发挥着重要作用。虽然短暂激活具有保护作用，但慢性小胶质细胞反应性会导致神经炎症损伤和神经退行性变。这种转变背后的线粒体机制仍知之甚少。在此，我们研究了脂多糖（LPS）是否会在BV-2小胶质细胞中诱导线粒体和代谢的协同改变。LPS刺激（100 ng/mL，24小时）诱导了一种反应性表型，Iba1（增加82%）、F4/80（增加132%）和Cd68（增加44%）增加，同时过氧化氢（约6倍）和亚硝酸盐（约45倍）升高。细胞毒性增加了40%（乳酸脱氢酶测定），细胞活力降至对照的约80%（MTT）。细胞外乳酸增加，表明糖酵解重编程。然而，LPS预处理的细胞在抗霉素A刺激下显示出更大的ATP消耗，反映出代谢灵活性受损。Hoechst染色显示固缩核增加约4倍，表明细胞凋亡。线粒体功能障碍通过膜电位降低30 - 40%（TMRE、JC-1）、Tomm20丢失约30%以及动力学变化得到证实：磷酸化Drp1增加（+23%），而Mfn1/2减少（33%）。尽管Lamp2信号增加约70%，但Tomm20-Lamp2共定位减少，表明线粒体自噬受损。高分辨率呼吸测定显示基础呼吸（-22%）、ATP相关呼吸（24%）和备用呼吸能力（41%）降低，非线粒体氧消耗增加。这些发现表明，LPS在小胶质细胞中诱导线粒体功能障碍以及代谢适应性丧失，并增加细胞凋亡易感性。线粒体质量控制和能量灵活性成为更好理解并潜在调节神经炎症和神经退行性疾病中小胶质细胞反应的相关靶点。
