Ziziphora clinopodioides Flavonoids exerts neuroprotective effects through PI3K/Akt pathway to improve ischemic...

Ziziphora clinopodioides Lam. is a traditional Uyghur medicinal herb used for centuries in Xinjiang, China, to treat cardiovascular and cerebrovascular disorders. According to local ethnopharmacological records, it is traditionally employed to promote blood circulation, remove blood stasis, calm the heart, and relieve chest tightness and palpitations. Modern studies have shown that its total flavonoids (ZCF) possess anti-inflammatory and antioxidant activities. Ischemic stroke (IS) is a leading cause of death and disability, involving neuroinflammation, oxidative stress, and blood-brain barrier (BBB) disruption. However, the neuroprotective mechanism of ZCF against IS and its active constituents remain unclear.

This study aimed to elucidate the neuroprotective mechanism of ZCF against IS and to identify its potential active flavonoids.

Network pharmacology and transcriptomics were used to predict core pathways, followed by in vitro and in vivo experiments. Neurological deficits, infarct volume, BBB integrity, and PI3K/Akt-related inflammatory/oxidative markers were assessed. The PI3K inhibitor LY294002 was used, and active flavonoids were screened by molecular docking, molecular dynamics simulations, and ELISA.

Integrated transcriptomics and network pharmacology identified that ZCF modulates the PI3K/Akt pathway. In MCAO/R-treated rats, ZCF reduced cerebral infarction, and preserved neuronal morphology. ZCF protected BBB integrity by restoring ZO-1, Occludin, Claudin-5, reducing MMP-9 and MMP-2 levels, rebalancing the Ang-1/Ang-2 ratio, decreasing NaF permeability, and increasing TEER. ZCF activated PI3K/Akt signaling in both bEnd.3 cells and MCAO/R-treated rat brain tissues, and the PI3K inhibitor LY294002 reversed ZCF-mediated regulation of tight junction proteins, matrix metalloproteinases, and Ang-1/Ang-2, suggesting that ZCF preserves BBB integrity at least partly through a PI3K/Akt-dependent mechanism. ZCF inhibited the TLR4/NF-κB/NLRP3 axis, reduced IL-1β, IL-6, TNF-α and MDA, and up-regulated Nrf2 and SOD, and these effects were reversed by LY294002. Molecular docking and MD simulations suggested that Diosmin, Glycitin, and Tilianin may form stable binding with PI3K. Diosmin, Glycitin and Tilianin could significantly restore the activity of PI3K in PC12 cells treated with OGD/R, reduce the levels of IL-1β, IL-6, TNF-α and MDA, and enhance the activity of SOD.

Under preconditioning conditions, ZCF maintains BBB and inhibits neuroinflammation and oxidative stress through integrity and PI3K/Akt dependence, thereby exerting anti-IS protection. Diosmin, Glycitin, and Tilianin are potential active flavonoids that may contribute to PI3K/Akt activation.