Nitrate maintains mitochondrial membrane integrity by increasing Sialin-FKBP8 granules via liquid-liquid-phase separation

Mitochondria, the metabolic centers of cells, play a pivotal role in maintaining cellular homeostasis and regulating physiological processes. Mitochondrial membrane disruption and consequent dysfunction are key pathogenic mechanisms underlying various diseases, including irradiation (IR)-induced salivary gland damage. Our previous studies have demonstrated that nitrate exerts preventive effects against mitochondrial dysfunction and IR-induced salivary gland injury. However, the mechanism remains unclear. In the present study, supplementation with nitrate significantly reduced mitochondrial damage to salivary epithelial cells in the context of IR-induced salivary gland damage in vivo and cellular injury to human salivary gland cells (HSGs) in vitro. Nitrate alleviated mitochondrial membrane damage in salivary epithelial cells mainly by maintaining the integrity of the mitochondrial membrane. Mechanistically, nitrate upregulates the expression of the nitrate transporter Sialin in salivary epithelial cells, along with H⁺ importation and subsequent cytoplasmic acidification. The high expression of Sialin, coupled with a low cytoplasmic pH, synergistically promotes the interaction between Sialin and FK506-binding protein 8 (FKBP8), leading to the formation of Sialin-FKBP8 granules through liquid-liquid phase separation. These granules anchor to the outer mitochondrial membrane, thereby maintaining mitochondrial membrane integrity. Notably, the Sialin ^R494A mutation inhibited the Sialin-FKBP8 interaction and abolished the protection of nitrate. Collectively, this study is the first to elucidate the mechanism by which nitrate protects against mitochondrial membrane damage, providing a potential strategy for preventing IR-induced injury and other mitochondrial damage-related diseases.