Integrating UHPLC-MS, Network Pharmacology, and Molecular Docking techniques to explore the neuroprotective effect of Mirificin.

Abstract

Ischemic stroke (IS) is a complex cerebrovascular disease with a high global incidence, recurrence rate, and disability. Despite the limited availability of clinical therapies for IS, further research is essential to develop traditional Chinese medicine (TCM)-based neuroprotective treatments. This study aims to investigate the neuroprotective properties of mirificin against neuronal damage and uncover the pharmacological mechanisms involved. Mirificin levels in rat cerebrospinal fluid (CSF) and plasma following oral administration of Naomaitong (NMT) were analyzed using ultra high-performance liquid chromatography-mass spectrometry (UHPLC-MS). Middle cserebral artery occlusion (MCAO) models were utilized to assess the effects of mirificin, with cerebral infarction size determined using TTC staining and neurobehavioral evaluation. Oxygen-glucose deprivation/reperfusion (OGD/R) experiments were conducted on PC12 cells to evaluate cell viability through CCK-8 and LDH assays. The predictive analysis of mirificin's potential therapeutic targets and pharmacological mechanisms was conducted employing advanced computational strategies such as Network Pharmacology (NP) and molecular docking simulations, which were further verified by corresponding antagonists. Mirificin was present in drug-containing plasma and CSF. Mirificin reduced infarct size, improved neurological function in vivo, and exerted dose-dependent neuroprotective effects towards OGD/R-injured PC12 cells. Moreover, the core targets of mirificin were predicted to be VEGFR2, CAT, MMP9, and HSP1A1. Antagonists of VEGFR2 and HSP1A1 inhibited the neuroprotective effects of mirificin. The neuroprotective effect of mirificin against OGD/R-induced neuronal injury appears to be mediated, at least in part, by targeting VEGFR2 and HSP1A1.