Utilizing network pharmacology, pharmacodynamics, molecular biology studies, and molecular docking techniques to uncover the mechanism of the Ligusticum wallichii-borneol medication combination for the treatment of ischemic stroke in rat models

Objective.To use pharmacodynamics, molecular biology studies, network pharmacology, and molecular docking to study the mechanism of action of the Ligusticum wallichii (known as Chuanxiong in China, CX) and borneol (known as Bingpian in China, BP) medication pair (CXBP) for the treatment of ischemic stroke. Methods. The TCMSP, ETCM, and SymMap databases provided the effective chemical components and targets of CXBP, while the databases OMIM, GeneCards, TTD, Pubmed, Web of Science, CNKI, Wanfang Data, and VIP Database provided targets relevant to ischemic stroke. PPI network maps of important targets were created using the String database, and GO and KEGG enrichment analyses were carried out using the Metascape database. The "Disease-Pathway-Target-Component-Drug" network was constructed in Cytoscape, and Pymol and Autodock tools were used to confirm molecular docking. Rat models of MCAO was established, and neurological scores, TTC staining and Nissl's staining were performed. Finally, the key components were verified by enzyme-linked immunosorbent assay (ELISA), real-time fluorescence quantitative PCR, and immunohistochemistry. Results.There were 33 active ingredients and 419 potential targets in CXBP, involving the key active ingredients Z-6,8',7,3'-diligustilide, Cedrene,(+)- Alpha-Funebrene, POL, Dipterocarpol, Oleanolic acid, 1-Acetyl- beta-carboline, Erythrodiol, and the key targets were transcription factor ESR1, pro-inflammatory factor PRKCA, and anti-inflammatory factor PTPN6; KEGG pathway analysis enriched 179 signaling pathways, while the results of GO enrichment comprised 2911 biological processes, 398 molecular activities, and 203 cellular components. The neurological function score and TTC staining of the brain tissue infarct region were significantly lower following CXBP intervention compared to the MCAO group, which was corroborated by experimental evidence; Nissl's staining revealed that, following CXBP intervention, the cellular morphology was more intact and there were considerably more Nissl's vesicles in the infarcted area than in the MCAO group; By using ELISA, it was possible to see substantial changes in the expression levels of PRKCA, PTPN6, ESR1, and TNF-α as well as a considerable down-regulation of the levels of IL-1β, IL-6, and TNF-α as compared to the MCAO group. The expression levels of TNF-α, IL-6, and IL-1β were markedly downregulated; The PCR results indicated that, in comparison with the MCAO group, PRKCA significantly decreased, IL-1β, IL-6, and TNF-α expression levels were significantly down-regulated, and ESR1 and PTPN6 significantly increased. Immunohistochemistry revealed that, in comparison with the MCAO group, the CXBP group and the nimodipine group, had significantly higher ESR1 and PTPN6 expression levels and significantly lower PRKCA. Conclusions.In order to improve cerebral ischemia and reperfusion injury, CXBP may act through Z-6,8',7,3'-diligustilide, Cedrene,(+)-Alpha-Funebrene, POL, Dipterocarpol, Oleanolic acid, 1-Acetyl-beta-carboline, Erythrodiol, and other important active ingredients. CXBP acts on key targets ESR1, PRKCA, and PTPN6 to regulate multiple key signaling pathways.