Salidroside Alleviates Mitochondrial Ca2+ Overload on Hypoxic Ht22 Cells by Balancing Mcu-Mediated Mitochondrial Fusion/Fission

Strong evidence indicated that the antioxidant, anti-inflammatory, and anti-apoptotic pharmacological activities of salidroside (Sal) contributed to the maintenance of mitochondrial function in hypoxic neurons. However, its underlying molecular mechanism that regulates Ca2+ homeostasis and neuron function has been puzzling people. This study aims to reveal the molecular mechanism of Sal in alleviating mitochondrial Ca2+ overload and maintaining mitochondrial function and homeostasis. Cell viability was measured by CCK-8 and Calcein AM assays. The levels of MDA, SOD and GSH-PX were determined by ELISA. Ca2+ levels were determined with Fluo-3 AM probe. MPTP assay kit was used to evaluate mitochondrial membrane permeability. Apoptosis was detected by Annexin V-FITC kit, YO-PRO-1/PI and TUNEL. Mito-Tracker Red CMXRos and TEM were employed to detect mitochondrial morphology. Mitochondrial membrane potential and function were evaluated by JC-1 and Seahorse analysis. Immunofluorescence and western blot analysis were performed to test MCU-mediated mitochondrial fusion/fission related protein expression. In HT22 cells, hypoxia leads to the increasing of MCU and p-Drp1 ser616, the decreasing of OPA1, which causes the imbalance of mitochondrial homeostasis and apoptosis. This study demonstrated that Sal exerts anti-hypoxic brain protection from inhibiting oxidative stress, reducing intracellular Ca2+ concentration, and maintaining the normal morphology and function of mitochondria. Mechanistically, Sal inhibits MCU-mediated activation of Drp1 and enhances the expression of OPA1 to maintain mitochondrial homeostasis, as well as suppress the activity of Caspase-3. Altogether, this research reveals the mechanism by which Sal promotes sequestration mitochondrial and Ca2+ of HT22 cells and inhibits apoptosis via MCU-mediated mitochondrial fusion/fission.