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Background <p>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.</p> Methods <p>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.</p> Results <p>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.</p> Conclusion <p>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.</p> Graphical Abstract <p></p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月11日

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  • Maomei Song,
  • Angelica Barone,
  • Suiyi Xu,
  • Changxin Li,
  • Giuseppe Gigli,
  • Giacomo Frati,
  • Cherubino Di Lorenzo,
  • Gianluca Coppola,
  • Barbara Cortese

摘要

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.

Graphical Abstract