Phosphoproteomics Uncovers a Neuroimmune Perspective on Trigeminal Neuralgia: Sexually Dimorphic Regulatory Networks Linking Calcium Channels to the Complement Cascade

Xiaojie Zhai¹Xianghong Lin¹Linlin Zhang¹Yuxuan Ren²He Miao²Mengmeng Miao²Yijun Chen¹Yiwei Zhang¹Changshun Huang^1*

• ¹Anesthesiology Department, The First Affiliated Hospital of Ningbo University, Ningbo, China
• ²Ningbo University, Ningbo, China

Background: Trigeminal neuralgia (TN) is a neuropathic pain disorder with a marked female predominance. While transcriptional changes in TN are documented, the translational and post-translational landscapes—specifically protein abundance and phosphorylation states—within the trigeminal ganglion (TG) remain largely unexplored. Understanding these layers is essential to deciphering the mechanisms behind the disease's sexual dimorphism. Methods: we utilized the chronic infraorbital nerve ligation (CION) method via an intraoral approach model in male and female Sprague-Dawley rats. Mechanical allodynia was confirmed via behavioral testing. On postoperative day 7, trigeminal ganglia were harvested to capture the somatic molecular response. We performed an integrated analysis using TMT-based quantitative proteomics and phosphoproteomics. Bioinformatics tools were employed to map differentially expressed proteins (DEPs), kinase-substrate relationships, and protein-protein interaction (PPI) networks. Results: The study quantified 5,820 proteins and 8,830 phosphopeptides. (1) A striking divergence was observed in pathological pathways. Females exhibited a robust neuroimmune signature characterized by the specific upregulation of complement components (C1QA, C1QC) and Kininogen (KNG1). In contrast, males showed alterations primarily in lipid metabolism and synaptic vesicle cycles. (2) Phosphoproteomics identified the MAPK signaling pathway as a shared mechanism. Specifically, phosphorylation of HSPB1 at Ser86 (pS86-HSPB1)—a target of the MAPKAPK2/3 cascade—was significantly elevated, linking stress signaling to cytoskeletal reorganization. (3) PPI analysis highlighted voltage-gated calcium channel subunits as central hubs connecting these sex-specific modules, validating the relevance of calcium channel modulation in TN management. Conclusions: This study presents the first dual-omics atlas of the injured trigeminal ganglion. We identify a female-specific "Complement-Kininogen" axis and a conserved "MAPK-HSPB1" phosphorylation pathway as key drivers of TN. These findings provide a molecular explanation for the clinical gender bias and suggest that therapeutic strategies may need to be sex-stratified, with complement inhibition holding particular potential for female patients.