Potential Mechanisms of Tetramethylpyrazine in the Treatment of Traumatic Brain Injury Based on Network Pharmacology, Molecular Docking, Molecular Dynamics Simulations, and in vivo Experiments.

Background: Traumatic brain injury (TBI) is a leading cause of global disability and mortality. Tetramethylpyrazine (TMP), an active compound from Chuanxiong, holds promise for treating cerebrovascular diseases, but its precise mechanism of action against TBI remains incompletely understood. This study aimed to elucidate the therapeutic effects and underlying mechanisms of TMP in TBI. Methods: Potential targets of TMP against TBI were identified using Swiss Target Prediction, PharmMapper, and GeneCards databases. Core targets and mechanisms were predicted through network pharmacology, molecular docking, and molecular dynamics (MD) simulations. These computational predictions were then experimentally validated in a rat TBI model, employing behavioral tests, ELISA, RT-qPCR, and Western blot analysis. Results: Through network pharmacology analysis, 39 potential targets associated with TMP were identified. Molecular docking and MD simulations manifested that key genes like MMP3, MMP2, MMP13, and GSK3B, showed a strong binding affinity to TMP. GO analysis and KEGG analysis corroborated that such targets strongly related to the IL-17 signaling pathway and the relaxin signaling pathway. In vivo tests proved that TMP could improve the modified Neurological Severity Score (mNSS) and foot defect test scores among rats. ELISA confirmed that TMP could decrease the expression of inflammatory factors, encompassing interleukin 1 beta (IL-1β), interleukin 6 (IL-6), interleukin 17A (IL-17A), and tumor necrosis factor-alpha (TNF-α). Furthermore, RT-qPCR analysis exhibited that the levels of MMP3, MMP2, MMP13, and GSK3B were increased within the rat cortex after TBI. Significantly, TMP treatment alleviated such upregulation. Western blot analysis validated that TMP down-regulated the expression of p-GSK3β (Ser9), active MMP13, active MMP3, and P65 NF-κB proteins after TBI, while TMP increased the expression of occludin protein. Conclusion: This study demonstrates that TMP exerts therapeutic effects on TBI by targeting the IL-17 and relaxin signalling pathways, providing evidence for its potential as a clinical therapy.