Objective: The traditional Chinese medicine (TCM) formulation Xiaoyao San (XYS) has a good clinical effect in treating ischemic stroke (IS). We explored the mechanism and material basis of XYS in IS treatment. Methods: Network pharmacology was used to construct a network of XYS components and IS targets. R software was used to analyze the biological process and pathway analysis of the targets of XYS in IS treatment. In vitro, a model of apoptosis of PC12 cells induced by oxygen-glucose deprivation/reperfusion (OGD/R) was established to evaluate the neuroprotective effect of XYS and its influence on the expression of apoptotic protein-related genes. The affinity between the potentially active compounds in XYS and apoptotic proteins was evaluated by molecular docking. Results: XYS was shown to have 136 chemical components that exert potential anti-IS activity by acting on 175 proteins. Bioinformatics analysis showed that apoptosis and the phosphoinositide 3-kinase/protein kinase B (PI3K-Akt) signaling pathway were the main signaling pathways of XYS. In vitro experiments showed that XYS could improve the effect of OGD/R on PC12-cell activity (EC₅₀ = 0.43 mg/mL) and inhibit apoptosis. The main mechanisms were related to the improvement of oxidative stress and regulation of apoptosis-related gene expression. Molecular docking showed that C22, C102 and other components in XYS had a strong affinity with apoptosis-related proteins. Conclusion: Network pharmacology, in vitro experiments, and molecular docking were used, for the first time, to study the material basis and molecular mechanism of XYS in IS treatment from the perspective of multiple targets and multiple pathways. We provided a new approach for the future study of TCM formulations in the treatment of complex diseases.

Stroke is a fatal neurological disease, which seriously threatens human health and life. Ischemic stroke (IS) is the most common type of stroke in clinic. Its pathogenesis is very complex, mainly caused by nerve damage caused by brain blood supply disorder. Previous studies have confirmed that natural products play important roles in improving neurological disorders. Furthermore, our previous results also suggested that Shenxiong Tongmai granule, a clinically used herbal medicines’ prescription, has a good ameliorating effect on IS. In the present study, we found that Monomethyl lithospermate (MOL), a constituent of Shenxiong Tongmai granule, significantly improved the neurological damage in middle cerebral artery occlusion (MCAO) rats. MOL can significantly improve the neurological deficit score of MCAO rats, and improve the damage of hippocampal neurons caused by ischemia-reperfusion (IR). At the same time, we also found that MOL could reduce the level of oxidative stress in the brain tissues of MCAO rats. Furthermore, the oxygen and glucose deprivation/Reoxygenation (OGD/R)-induced SHSY-5Y cell model was established in vitro to investigate the pharmacological activity and molecular mechanisms of MOL in improving the nerve injury of IS rats. The results showed that MOL could increase the cell viability of SHSY-5Y cells, inhibit the mitochondrial membrane potential (MMOP) collapse and suppress apoptosis. In addition, MOL also ameliorated the elevated oxidative stress level caused by OGR/R treatment in SHSY-5Y cells. Further mechanistic studies showed that MOL could activate the PI3K/AKT pathway via promoting the phosphorylation of PI3K and AKT in MCAO rats and OGR/R-induced SHSY-5Y cells, which could be partially blocked by addition of PI3K/AKT pathway inhibitor of LY294002. Taken together, our current study suggested that MOL exerts a protective effect against neural damage caused by IS in vivo and in vitro by activating the PI3K/AKT pathway.