Network pharmacology and experimental verification: Rosmarinic acid alleviates doxorubicin-induced cardiomyocyte apoptosis by regulating BCL2L1

Purpose This study investigated the mechanism by which Rosmarinic acid (RA) may alleviate doxorubicin (DOX)- induced cardiomyocyte apoptosis. Methods The target genes of RA, DOX-related differentially expressed genes, and GEO database related genes were retrieved by bioinformatics analyses. The results of these analyses were further intersected to identify candidate genes. The protein-protein interaction network was constructed to develop the pharmacophore model. The molecular docking was simulated to determine the core target B-cell lymphoma 2-like 1 (BCL2L1) for subsequent molecular mechanism investigation in vitro . The effects of DOX and RA on the apoptosis of H9c2 cells were assessed using the CCK8 assay. The present study investigated the effect of RA on DOX-induced oxidative stress in cardiomyocytes. This investigation was conducted using an ELISA test and a DCFH-DA probe. The JC-1 probe was utilized to assess the effect of RA on DOX-induced cardiomyocyte mitochondrial membrane permeability. A Western blot assay was conducted to ascertain the activation of multiple signaling molecules, including those belonging to the BCL-2 and caspase-3 families, within the apoptosis pathway. Results A total of 17 differentially expressed genes (DEGs) were screened, and five genes were selected as hub DEGs. A subsequent KEGG enrichment analysis revealed that these DEGs were significantly enriched in various biological processes and pathways, including the MAPK signaling pathway, autophagy, apoptosis, and the TNF signaling pathway. The pharmacophore model and molecular docking of five candidate targets with RA were successfully established. It is noteworthy that DOX treatment led to a suppression of SOD and GSH levels, an exacerbation of oxidative stress, and a promotion of cardiomyocyte apoptosis. Furthermore, it has been demonstrated to suppress mitochondrial membrane permeability. Subsequent RT-qPCR analysis of the hub genes revealed that only BCL2L1 exhibited significant alterations. Treatment with DOX altered the expression levels of apoptosis-associated proteins, BCL-2 family members, and caspase-3 family members. However, the administration of RA mitigated the deleterious effects of DOX on cardiomyocytes. Conclusions The protective effects of RA may against myocardial cell apoptosis are likely mediated through its activation of BCL2L1 and inhibition of caspase cascade protein expression in myocardial cells.