Key Regulatory Networks of Mitochondrial Dysfunction in Human Hippocampal Neurons under Cobaltous Chloride-Induced Hypoxia Revealed by Combined Transcriptome Analysis with Protein-Protein Interaction

Hypoxic stress in hippocampal neurons is a critical factor in the pathogenesis of neurodegenerative diseases and brain injury, yet the gene regulatory networks and cellular adaptations underlying their response remain poorly understood. We investigated the effects of CoCl₂-induced hypoxic stress on hippocampal neuronal cells (HPPNCs) using transmission electron microscopy (TEM), confocal microscopy, JC-1 staining, transcriptome sequencing, bioinformatic analysis and molecular docking. Morphological analysis revealed that hypoxia led to pronounced mitochondrial damage, as evidenced by distribution disorder, swelling, vacuolization and loss of subcellular compartmentalization. Functional assays with JC-1 staining demonstrated significant depolarization of mitochondrial membrane potential, indicating impaired mitochondrial function under hypoxic conditions. Transcriptomic profiling identified 471 differentially expressed genes (DEGs), with enrichment analyses highlighting biological processes related to oxygen sensing, metabolic reprogramming, angiogenesis and synaptic function. Pathway analysis further linked these changes to key metabolic and disease-associated networks. Protein-protein interaction (PPI) network construction revealed HSPD1, HSPA9, and HSP90AA1 as central hub proteins connecting hypoxia-responsive gene expression to mitochondrial quality control. Notably, molecular docking simulations validated high binding affinities between these hub proteins and mitochondrial function-related proteins (DRP1, OPA1), confirming robust potential protein-protein interactions. Western blotting validated the expression changes of these hub proteins, while ceRNA network analysis suggested miR-27 as a potential post-transcriptional regulator of HSPD1, HSPA9, and HSP90AA1. Collectively, our findings provide integrated morphological, functional and molecular evidence for the mechanisms of hippocampal neuronal adaptation to hypoxic stress, and identify novel regulatory nodes and pathways that may serve as future therapeutic targets for hypoxia-related neurological disorders.