A network-based method for mechanistic investigation and neuroprotective effect on treatment of tanshinone I against ischemic stroke in mouse

Ethnopharmacological relevance: Tanshinone-I (TSNI), a member of the mainly active components of Salvia miltiorrhiza Bunge (Dan Shen), which is widely used for the treatment for modern clinical diseases including cardiovascular and cerebrovascular diseases, has been reported to show the properties of anti-oxidation, anti inflammation, neuroprotection and other pharmacological actions. However, whether TSNI can improve neuron survival and neurological function against transient focal cerebral ischemia (tMCAO) in mice is still a blank field. Aim of the study: This study aims to investigate the neuroprotective effects of TSNI on ischemic stroke (IS) induced by tMCAO in mice and explore the potential mechanism of TSNI against IS by combining network pharmacology approach and experimental verification. Materials and methods: In this study, the pivotal candidate targets of TSNI against IS were screened by network pharmacology firstly. Enrichment analysis and molecular docking of those targets were performed to identify the possible mechanism of TSNI against IS. Afterwards, experiments were carried out to further verify the mechanism of TSNI against IS. The infarct volume and neurological deficit were evaluated by 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining and Longa respectively. Immunohistochemistry was used to observe neuronal death in the hippocampus and cortical regions by detecting the change of NeuN. The predicting pathways of signaling related proteins were assessed by Western blot in vitro and in vivo experiments. Results: In vivo, TSNI was found to dose-dependently decrease mice?s cerebral infarct volume induced by tMCAO. In vitro, pretreatment with TSNI could increase cell viability of HT-22 cell following oxygen-glucose deprivation (OGD/R). Moreover, the results showed that 125 candidate targets were identified, Protein kinase B (AKT) signaling pathway was significantly enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and mitogen-activated protein kinases 1 (MAPK1) and AKT1 could be bound to TSNI more firmly by molecular docking analysis, which implies that TSNI may play a role in neuroprotection through activating AKT and MAPK signaling pathways. Meanwhile, TSNI was confirmed to significantly protect neurons from injury induced by IS through activating AKT and MAPK signaling pathways. Conclusion: In conclusion, our study clarifies that the mechanism of TSNI against IS might be related to AKT and MAPK signaling pathways, which may provide the basic evidence for further development and utilization of TSNI.