Integration of transcriptome profiling to identify key genes involved in the interplay between oxidative stress and mitophagy in major depressive disorder, followed by multidimensional phenotypic validation.

Major depressive disorder (MDD) is recognized as a pressing global public health burden. However, its molecular mechanisms remain incompletely understood. In this study, an integrative analysis of transcriptome datasets from the GEO database was conducted. GEO2R and the R programming language were used to identify differentially expressed genes (DEGs) related to oxidative stress and mitophagy. Key hub genes, such as EEF2, CCT3, EIF3I, and RPS5, were further identified through enrichment analysis and protein-protein interaction (PPI) network construction. Following validation using an independent human dataset, we established a corticosterone-induced C8-D1A cell model. Reactive oxygen species and mitochondrial membrane potential were measured via flow cytometry. The results demonstrated that this model reliably recapitulates key pathological features of elevated oxidative stress and mitochondrial dysfunction in MDD. Finally, using an in vivo mouse model, we assessed synapse-associated proteins and mitophagy markers using Western blotting and measured the mRNA expression levels of candidate genes by qPCR to comprehensively validate the associations between the expression of the aforementioned genes and oxidative stress, mitophagy, and synaptic damage. This study combined bioinformatics screening and multidimensional phenotypic validation to construct an MDD-specific molecular regulatory network focused on carbon metabolism, thereby elucidating the interplay between four genes and oxidative stress and mitophagy. Although CCT3 and RPS5 demonstrated modest diagnostic utility in the independent validation dataset (AUC ≈ 0.6, Padj < 0.05), subsequent in vivo experiments revealed that the mRNA expression levels of these genes were significantly downregulated in MDD models (EEF2: P < 0.05; CCT3: P < 0.005; EIF3I: P < 0.05). Furthermore, the expression levels of these genes were positively correlated with those of synaptic proteins and negatively correlated with those of mitophagy markers. The downregulation of these genes may impair protein synthesis and folding, which acts in synergy with oxidative stress and mitochondrial dysfunction to perpetuate the vicious cycle of bioenergetic crisis and proteostasis collapse in MDD. Although this study did not experimentally validate the regulatory functions of the target genes or identify highly specific diagnostic biomarkers, it offers a novel molecular perspective for deciphering the complex pathology of MDD. Notably, this highlights the synergistic interaction between translational regulation and metabolic homeostasis. Further validation in larger independent cohorts is warranted to assess the viability of these genes as mechanistic therapeutic targets.