Mechanistic investigation of HMGB1 in an in vitro model of the trigeminovascular system under migraine-like conditions

Background: Migraine is a complex neurovascular disorder driven by abnormal activation of the trigeminovascular system (TVS), neurogenic inflammation and excessive release of calcitonin gene-related peptide (CGRP). While CGRP-targeted therapies have demonstrated clinical efficacy, the upstream molecular mechanisms sustaining CGRP dysregulation remain incompletely understood. High-mobility group box 1 (HMGB1), a pro-inflammatory damage-associated molecule implicated in neuroinflammation, has emerged as a potential contributor to migraine pathophysiology. However, its role in neuron-endothelial interactions within the TVS under migraine -relevant conditions remains unclear. Methods: To model inflammatory and hypoxic stress associated with migraine, SH-SY5Y neuron-like cells were stimulated with lipopolysaccharide (LPS) and cobalt chloride (CoCl₂). HMGB1 expression was silenced using siRNA to evaluate its role in regulating CGRP and NF-κB signaling. Conditioned medium from stimulated neuronal cells was applied to human umbilical vein endothelial cells (HUVECs) to investigate neuron–endothelial interactions, and CGRP signaling was inhibited using a receptor antagonist. Protein expression of HMGB1, CGRP, and NF-κB pathway components was assessed by Western Blot. In addition, neuronal migration and intracellular reactive oxygen species (ROS) were assessed to evaluate cellular activation under inflammatory stress conditions. Results: Inflammatory and hypoxic stimulation increased HMGB1 expression and activated NF-κB signaling in SH-SY5Y cells, accompanied by elevated CGRP production. HMGB1 knockdown reduced NF-κB activation and attenuated CGRP upregulation, suggesting that HMGB1 contributes to CGRP regulation under these conditions. Conditioned medium enriched in CGRP promoted NF-κB activation and HMGB1 expression in endothelial cells, effects that were attenuated by CGRP receptor blockade. In addition, HMGB1 enhanced neuronal migration and oxidative stress responses in stimulated SH-SY5Y cells. Conclusion: These findings support a model in which HMGB1 and CGRP participate in a positive neuron–endothelial feedback loop under inflammatory and hypoxic stress, potentially amplifying neurovascular signaling relevant to migraine. Although derived from an in vitro system, this study identifies HMGB1 as a potential upstream modulator of CGRP-associated pathways and highlights its possible contribution to peripheral mechanisms involved in trigeminovascular activation.