Isorhoifolin regulates S1PR3-CK2-GSK3β axis and promotes neurite regrowth and functional recovery after traumatic brain injury

BACKGROUND: Traumatic brain injury (TBI) disrupts anatomical structure and cellular signaling, yet the molecular mechanisms governing endogenous repair remain incompletely defined. Accumulating evidence implicate an increased risk of developing to neurodegenerative diseases for TBI patients, in part through chronic neuroinflammation, protein aggregation, and progressive synaptic dysfunction. However, a critical unmet need is that no approved medicine directly promotes neurite regrowth and functional recovery after TBI. PURPOSE: To identify candidate compounds that can promote neurite regrowth of injured brain neurons and improve functional outcome of TBI mice. The mechanism of action of the lead compound will be determined. STUDY DESIGN: Through an extensive screening of plant extracts, we have identified a nature compound, isorhoifolin, that promotes neurite regrowth of injured cortical and hippocampal neurons. Functional assays were conducted to assess behavioral efficacy and the direct protein targets of isorhoifolin were identified. RESULTS: Using complementary in vitro, ex vivo, and in vivo models of TBI, we demonstrated that isorhoifolin attenuated both cytosolic and mitochondrial reactive oxygen species, highlighting its role in redox homeostasis. Comparative structure-activity analyses revealed that the closely related flavonoids exhibited divergent biological efficacy, indicating that specific chemical features determine functional outcomes. In vivo, isorhoifolin crossed the blood-brain barrier and significantly improved motor coordination following experimental TBI. Transcriptomic profiling and cellular thermal shift assay (CETSA) further revealed that isorhoifolin bound directly to sphingosine-1-phosphate receptor-3 (S1PR3) and exerted temporally structured effects on injury-responsive networks. In human transcriptomic data, we found activation of S1P receptor-related pathways in TBI patients and the expression of S1PR3 was increased approximately 40%. Importantly, the current work delineates a neuron-centric role for S1PR3 in regulating structural repair that is mechanistically distinct from the known functions of S1PRs in immune cells. Biochemical assays supported a model in which isorhoifolin facilitates neurite repair through inhibiting neuronal S1PR3-CK2-GSK3β signaling axis. In parallel, isorhoifolin interacted directly with N-ribosyldihydronicotinamide:quinone reductase 2 (NQO2) based on proteomic CESTA, and genetic knockdown as well as inhibition of NQO2 in astrocytes promote neurite regrowth of injured cortical neurons. CONCLUSION: Together, these findings define mechanistically distinct yet coordinated neuronal and astrocytic pathways that are responsible for isorhoifolin-enhanced structural and functional recovery after TBI, and identify S1PR3 and NQO2 as direct and druggable targets.