Citronellal Alleviates Cognitive Impairment by Inhibiting NHE1-Mediated Mitochondrial Damage in Vascular Dementia

The present study aimed to explore the protective effect of citronellal (CT) on cognitive impairment and its underlying mechanism involving NHE1‑dependent mitochondrial damage both in vivo and in vitro. In vivo, a total of 36 rats (n = 6 per group) were randomly assigned to six groups: Sham, VaD, Sham + CT, Sham+LiCl, VaD + CT, and VaD + CT+LiCl. The VaD model was established by permanent bilateral common carotid artery occlusion. Learning and memory were evaluated using the Morris water maze test. Histopathological changes in the hippocampal CA3 region were examined by Hematoxylin and eosin, Neuronal Nuclei (NeuN), and Golgi-Cox staining. Apoptosis and proliferation were detected by Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining and 5-Ethynyl-2'-deoxyuridine (EdU) staining, respectively. Mitochondrial ultrastructure and membrane potential were assessed by Transmission electron microscope and JC-1 staining. The reactive oxygen species (ROS), nitric oxide (NO), superoxide dismutase (SOD) and malondialdehyde (MDA) levels were measured by dihydroethidium staining, water-soluble tetrazolium, thiobarbituric acid, and nitrate reductase methods, respectively. Protein expression was determined by immunohistochemistry, immunofluorescence, and Western blot. In vitro, Mouse hippocampal neuronal cells were treated with H₂O₂ or CT at 25-400 µM or 1-20 µg/L. Cell viability, mRNA levels, mitochondrial respiratory efficiency, membrane potential, and ROS levels were measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR), JC-1, and Mito-SOX staining. Protein expression was detected by immunofluorescence and Western blot. In vivo findings demonstrated that CT alleviates cognitive impairment and regulated the expression of proteins within the NHE1/PI3K/AKT signaling axis (F (5, 12) = 203.3, P < 0.01). In vitro, H₂O₂ impaired mitochondrial function and dysregulated apoptosis- and NHE1-related signaling in HT-22 cells, while CT treatment significantly reversed these alterations (F (3, 20) = 93.02, P < 0.05). CT prevented mitochondrial dysfunction, mitochondrial apoptosis and oxidative injury both in vitro and in vivo by regulating the NHE1/PI3K/AKT signaling axis.