PurposeThis study investigated the mechanism by which Rosmarinic acid (RA) may alleviate doxorubicin (DOX)- induced cardiomyocyte apoptosis.MethodsThe 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.ResultsA 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.ConclusionsThe 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.
Hanming Yu,Miao Li,Xin Kang,Yi Dai,Fei Li,Yuan Zhang,Qiuhong Li
Background Nitro-aromatic compounds (NACs) are pervasive environmental pollutants with recognized pulmonary toxicity, yet their mechanistic involvement in pulmonary fibrosis remains incompletely understood. Methods We applied an integrative framework combining network toxicology, transcriptomic analysis, prognostic modeling, immune infiltration profiling, molecular docking, and molecular dynamics (MD) simulations to investigate the fibrogenic effects of two representative NACs, 1-nitropyrene (1-NP) and nitrobenzene (NB). Identification and evaluation of NACs-related targets and their clinical relevance using the idiopathic pulmonary fibrosis (IPF) GEO dataset. Results Network analysis using eight topological algorithms identified ten key genes occupying central positions in the NACs-associated fibrosis-related candidate network. Among these, BCL2L1 and MYC exhibit significant prognostic significance in IPF.Risk models based on hub genes effectively stratify patient survival rates and are closely associated with immune dysregulation, characterized by macrophage aggregation and CD8⁺ T cell exhaustion. Molecular docking analysis indicates that 1-NP and NB exhibit strong binding affinity with the anti-apoptotic protein BCL2L1 . Notably, 100-ns MD simulations revealed that NACs binding was associated with significant conformational destabilization and partial unfolding of BCL2L1 , as evidenced by sustained increases in Root mean square deviation (RMSD), radius of gyration (Rg), and solvent-accessible surface area (SASA). These findings support a non-classical mechanism of structural toxicity, wherein NACs exert their effects by disrupting protein stability rather than acting as classical enzyme inhibitors. Conclusions This study suggests a potential mechanism by which environmental NACs may contribute to fibrotic progression by disrupting the structural stability of BCL2L1 and reshaping the immune microenvironment. Our findings elucidate a potential molecular association between environmental exposure and fibrosis progression, suggesting that BCL2L1 -mediated structural toxicity may serve as a potential biomarker and therapeutic target for pollution-related pulmonary fibrosis.
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