Histone lactylation modulates apoptosis and stress response through PI3K/AKT signaling in GC-1 spermatogonial cells under hypoxia

La lactilación de histonas modula la apoptosis y la respuesta al estrés mediante la vía de señalización PI3K/AKT en células espermatogoniales GC-1 bajo hipoxia

Background: Histone lactylation is a recently identified epigenetic modification arising from lactate metabolism. This study aimed to determine whether lactate-induced histone lactylation contributes to the regulation of apoptosis in mouse Germ Cell-1 (GC-1) cells under hypoxic stress. Methods: A hypoxia-induced cell model was established to examine lactate accumulation, histone lactylation, and apoptosis in GC-1 cells. The experimental groups included the normoxia control (Con group), hypoxia (Hy group), hypoxia + sodium oxamate (Hy + Oxa group), and L-lactate treatment (Nala group). Lactate levels, histone lactylation, apoptosis-related gene and protein expression, and apoptosis rates were assessed by lactate assays, Western blot, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR), and Annexin V/propidium iodide (PI) flow cytometry. Results: After 36 h of hypoxia, cell viability decreased to (58.55% ± 6.20%) (p < 0.001). Hypoxia exposure caused increase in intracellular lactate concentration and induced histone lactylation (p < 0.01). Compared with the Con group, cells in the Hy group exhibited increased expression of pro-apoptotic markers Bax and caspase-3, reduced Bcl-2 expression, and higher apoptosis levels (p < 0.05). The Hy + Oxa group showed reduced histone lactylation and decreased apoptosis compared with the Hy group (p < 0.05). The Nala group exhibited patterns similar to the Hy group, including increased histone lactylation and enhanced apoptosis (p < 0.05), confirming that lactate regulates apoptosis through histone lactylation. Furthermore, both hypoxia and exogenous lactate significantly reduced the ratios of phosphorylated phosphatidylinositol 3-kinase (p-PI3K)/PI3K and phosphorylated protein kinase B (p-AKT)/AKT, whereas oxamate partially restored their activity (p < 0.05). Conclusions: Hypoxia-induced lactate accumulation promotes apoptosis of GC-1 cells by enhancing histone lactylation, particularly at the H3K18 site, partly through suppression of the PI3K/AKT signaling pathway. These findings provide new mechanistic insights into hypoxia-associated male infertility and suggest that histone lactylation may represent a potential therapeutic target.

Resumen

Antecedentes: La lactilación de histonas es una modificación epigenética que se deriva del metabolismo del lactato. Este estudio tuvo como objetivo determinar si la lactilación de histonas inducida por lactato contribuye a la regulación de la apoptosis en células espermatogoniales de ratón Germ Cell-1 (GC-1) bajo estrés hipóxico. Métodos: Se estableció un modelo celular inducido por hipoxia para examinar la acumulación de lactato, la lactilación de histonas y la apoptosis en células GC-1. Los grupos experimentales incluyeron: control en normoxia (grupo Con), hipoxia (grupo Hy), hipoxia + oxamato sódico (grupo Hy + Oxa) y tratamiento con L-lactato (grupo Nala). Los niveles de lactato, la lactilación de histonas, la expresión de genes y proteínas relacionados con la apoptosis y las tasas de apoptosis se evaluaron mediante ensayos de lactato, Western blot, inmunofluorescencia, reacción en cadena de la polimerasa cuantitativa a tiempo real (qRT-PCR) y citometría de flujo con Annexina V/yoduro de propidio (PI). Resultados: Después de 36 h de hipoxia, la viabilidad celular disminuyó a (58.55% ± 6.20%) (p < 0.001). La exposición a la hipoxia causó un aumento en la concentración intracelular de lactato e indujo lactilación de histonas (p < 0.01). En comparación con el grupo Con, las células del grupo Hy exhibieron un aumento en la expresión de los marcadores proapoptóticos Bax y caspasa-3, una expresión reducida de Bcl-2 y mayores niveles de apoptosis (p < 0.05). El grupo Hy + Oxa mostró una lactilación de histonas reducida y una disminución de la apoptosis en comparación con el grupo Hy (p < 0.05). El grupo Nala exhibió patrones similares al grupo Hy, incluido el aumento de la lactilación de histonas y la apoptosis (p < 0.05), lo que confirma que el lactato regula la apoptosis a través de la lactilación de histonas. Además, tanto la hipoxia como el lactato exógeno redujeron significativamente las razones de fosfatidilinositol 3-quinasa fosforilada (p-PI3K)/PI3K y proteína quinasa B fosforilada (p-AKT)/AKT, mientras que el oxamato restauró parcialmente su actividad (p < 0.05). Conclusiones: La acumulación de lactato inducida por hipoxia promueve la apoptosis de las células espermatogoniales GC-1 al potenciar la lactilación de histonas, particularmente en el sitio H3K18, parcialmente mediante la supresión de la vía de señalización PI3K/AKT. Estos hallazgos proporcionan nuevas perspectivas mecanísticas sobre la infertilidad masculina asociada a la hipoxia y sugieren que la lactilación de histonas puede representar una diana terapéutica potencial.

Histone lactylation; Hypoxia; Spermatogonia; Apoptosis; Lactate; GC-1 cells

Palabras Clave

Lactilación de histonas; Hipoxia; Espermatogonias; Apoptosis; Lactato; Células GC-1

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