Systems pharmacology, proteomics and in vivo studies identification of mechanisms of cerebral ischemia injury amelioration by Huanglian Jiedu Decoction

Ethnopharmacological relevance: Huanglian Jiedu Decoction (HLJDD) has the effect of clearing heat and detoxifying, and has been considered as an effective prescription for cerebral ischemia (CI) for thousands of years in traditional Chinese medicine (TCM). It can improve the quality of life of patients with ischemic stroke, but its pharmacological mechanism remains unclear. Aim of the study: The study aimed to explore the pharmacological action and potential mechanism of HLJDD against CI by systems pharmacology, proteomics and in vivo experiments. Materials and methods: In this study, databases such as TCMIP V2.0 and Genecards were used to predict compounds, targets and CI related targets, and network topology criteria of protein-protein interaction (PPI) network was used to screen core targets. The Database for Annotation, Visualization and Integrated Discovery database (DAVID) was used to discover biological processes and pathways. In addition, molecular docking was performed between the screened core biological active compounds and targets to verify the binding activity. Finally, proteomics and Western blot were performed on cerebral cortex tissues of middle cerebral artery occlusion (MCAO) model rats with HLJDD intervention to further verify the predicted results. Results: 77 compounds and 308 targets of HLJDD were identified, 54 of which were predicted to be associated with cerebral ischemia. PPI network and enrichment results showed that 8 targets, including AKT1, PTGS2 and TLR4, were core targets of HLJDD in CI. And 19 signaling pathways, including Rap1 signaling pathway, cAMP signaling pathway and arachidonic acid metabolism, were identified as key pathways to the therapeutic activity of HLJDD in CI. Combined with proteomics studies, we identified that Rap1 signaling pathway and upstream and downstream targets were the key mechanisms. Molecular biology experiments showed that RAP1A and AKT expression levels were significantly up-regulated in middle cerebral artery occlusion (MCAO) rats treated with HLJDD (P < 0.0001), GRIN1 expression level was significantly down-regulated (P < 0.0001). However, ACTB expression level was slightly down-regulated (P > 0.05), which may be related to the biological function. Conclusion: This study confirms the pharmacological effect of HLJDD on cerebral ischemia. These results indicate that HLJDD mediates various pathways such as inhibition of apoptosis, regulation of oxygen balance, inhibition of excitatory toxicity and maintenance of basic cell functions to improve CI by regulating Rap1 signaling pathway.