Xiaoxuming decoction enhanced neuroprotection after cerebral ischemia/reperfusion via the JAK2/STAT3 signaling pathway based on UPLC/HRMS, network pharmacology and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiao-xu-ming decoction (XXMD), a prominent traditional Chinese medicinal formula historically revered for stroke treatment, demonstrates pronounced efficacy in ameliorating ischemic stroke injury.

AIM OF THE STUDY: This study aims to investigate the effects and mechanisms of XXMD on neuroprotection subsequent to cerebral ischemia/reperfusion in vivo and in vitro.

MATERIALS AND

METHODS

Neurobehavioral test, TTC staining, HE staining and nissl staining were used to examine the neuroprotective effect of XXMD on cerebral ischemia-reperfusion injury induced by middle cerebral artery occlusion (MCAO) in rats. Additionally, we assessed cell viability and injury with CCK8 and lactate dehydrogenase (LDH) assays. The changes in neuronal ultra-structure were observed after oxygen-glucose deprivation and reoxygenation (OGD/R) by transmission electron microscopy (TEM). Network analysis combined with ultrahighperformance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) predicted the mechanism of XXMD on ischemic stroke injury. Furthermore, the expression of neuroplasticity-related proteins neurofilament 200 (NF200), microtubule-associated protein 2 (MAP2), postsynaptic density protein 95 (PSD95), synaptophysin (SYN), phosphorylated Janus kinase2 (p-JAK2), and phosphorylated signal transduction and activator of transcription 3 (p-STAT3) was evaluated by immunofluorescence staining and Western blot analyses.

RESULTS

XXMD significantly improved Ethology, infarct area and pathological changes after MCAO and reperfusion, reducing morphological and ultrastructural alterations and decreased cell viability in HT22 cells induced by OGD/R. Network pharmacology showed that 1153 compounds of XXMD were matched. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that XXMD treated ischemia stroke mainly regulating inflammation reaction-related signaling pathways, atherosclerosish-related signaling pathways. Molecular docking results showed that TP53, AKT1, STAT3, and IL6 are closely bound to the corresponding active ingredients. XXMD treatment significantly reversed the above alternations. XXMD or AG490 up-regulated the expression of neuroplasticity-associated proteins, and reduced phosphorylation of JAK2, STAT3 expression following OGD/R.

CONCLUSION

XXMD exerts neuroprotective effects by promoting neural plasticity via regulating the JAK2/STAT3 pathway, indicating a promising alternative therapeutic strategy for ischemic stroke.