Combined Bioinformatic Analysis and RNA Sequencing Uncover Key Tinnitus-Related Molecules in the Cingulate Gyrus

Tinnitus arises from maladaptive plasticity and abnormal synaptic function, not only in auditory pathways but also in limbic regions. Among these, the cingulate gyrus plays a pivotal part in shaping the emotional burden and sensory filtering disturbances often seen in tinnitus patients. Yet, the specific molecular players operating within this region remain poorly understood, and most existing bioinformatic predictions have not been tested in living systems. Here, we combined multi-database mining with in vivo transcriptomic profiling to identify and validate key proteins and pathways linked to tinnitus in the cingulate gyrus. We built and compared protein-protein interaction networks representing auditory perception, acoustic challenge, and tinnitus states, thereby pinning down candidate hub molecules. To verify these candidates, we used a noise-induced mouse model of tinnitus, confirmed the phenotype via gap prepulse inhibition of acoustic startle (GPIAS), and then performed RNA-seq and qPCR on cingulate gyrus tissue. The bioinformatic screens pointed to AKT1 and TNF as central hubs, suggestive of an inflammatory-survival signaling axis. In our animal experiments, RNA-seq showed a clear rise in Akt1 transcript levels in the tinnitus group, and functional enrichment analysis pointed to marked changes in extracellular matrix organization, cell adhesion, and cytokine receptor pathways. qPCR further corroborated the Akt1 upregulation. Taken together, our integrative strategy establishes AKT1 and TNF as topologically central hub proteins in the cingulate gyrus tinnitus network, and validates Akt1 upregulation in vivo, offering potential biomarker candidates and therapeutic leads for future studies.