A newly discovered bioactive equivalence of combinatorial components of Angong Niuhuang pill improves ischemic stroke via the PI3K/AKT axis.

Ethnopharmacological Relevance: Angong Niuhuang pill (ANP) is effective in preventing and treating ischemic stroke, however, the pharmacodynamic substances and mechanism of ANP have not been scientifically clarified.

Aim Of The Study: This study aims to identify the bioactive equivalence of combinatorial components (BECCs) of ANP for treating ischemic stroke and discuss the underlying mechanisms.

Materials And Methods: Network pharmacology was performed to screen key compounds and predict potential pathways. The effect of BECCs on ischemic stroke was screened and verified in ponatinib-induced zebrafish model and mice middle cerebral artery occlusion (MCAO) model. Finally, the mechanism of BECCs was preliminarily investigated.

Results: Through network pharmacology, the degree values of each component in ANP were determined, and five candidate BECCs were obtained by combining the content of the components in the original prescription. The BECCs V has the same efficacy as the original formula in reducing the movement disorder and neuronal injury of zebrafish cerebral ischemia models and lowering the neurologic deficits and cerebral infarction volume of mouse MCAO models. Mechanistically, BECCs V and ANP blocked neuronal autophagy through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) axis, inhibited microglial inflammatory activation through the PI3K/AKT/hypoxia inducible factor-1α (HIF-1α) axis, protected microvascular endothelial function through the PI3K/AKT/forkhead box O3 (FoxO3a) axis, thereby improving ischemic cerebral injury.

Conclusions: The newly discovered BECCs V is equivalent to ANP in regulating the motor function recovery rate and neuroprotective rate of zebrafish and the neurological deficit scores and the average infarct volume of MCAO mice. This study suggests that the PI3K/AKT signaling axis plays a key role in neuronal autophagy, microglial inflammatory activation and microvascular endothelial dysfunction induced by cerebral ischemic injury, suggesting that the regulation of PI3K/AKT may be a potential therapeutic strategy for neuroprotection and ischemic stroke injury.