Didang Tang inhibits intracerebral hemorrhage-induced neuronal injury via ASK1/MKK7/JNK signaling pathway, network pharmacology-based analyses combined with experimental validation

Abstract

BackgroundIntracerebral hemorrhage (ICH) is an acute cerebrovascular disease, which is also a principal consideration for disability. Didang tang (DDT) is a classic traditional Chinese medicine formula for treating ICH. However, its pharmacological mechanism of action has not been elucidated. Materials and methodsThe TCMSP and BATMAN-TCM databases were used to collect chemical compounds and predict targets of DDT. Protein targets in ICH were identified by GeneCards, OMIM, and DrugBank databases. DDT compounds-ICH targets and protein-protein interaction (PPI) networks were constructed for topological analysis and hub-targets screening. Further, Key biological processes and signaling pathways were identified by GO and KEGG enrichment analyses. Then, an ICH rat model and a Cobaltous Chloride (CoCl2)-induced PC12 cells model were established. Cell viability and lactate dehydrogenase (LDH) release were detected using cck8 and LDH kits. Apoptosis levels were detected by TUNEL assessment and flow cytometry. IL-1β levels were detected by ELISA, while key protein expressions were determined by Western blot. ResultsA total of 126 active compounds related to DDT and 3,263 therapeutic targets for ICH were predicted. The functional enrichment of the GO and KEGG pathways combined with literature studies suggested that DDT is most likely to influence MAPK and apoptotic signaling pathways for ICH treatment. In vitro and in vivo experiments have shown that DDT remarkably inhibited apoptosis and increased the expression of Bcl-2, while inhibiting Bax and cleaved-Caspase 3. For other enriched core proteins, DDT suppressed the phosphorylation of Src and the expression of c-Myc and IL-1β, and up-regulated the level of MMP-9. The further results showed that, DDT decreased the phosphorylation of ASK1, MKK7, JNK and c-JUN. ConclusionBased on network pharmacology and experimental validation results, our in vivo and in vitro study indicated that ASK1/MKK7/JNK pathway might be the critical target for DDT against ICH.