To identify targets that are downstream of Andro, the authors performed a meta-analysis of multiple databases like TCMSP, SwissTargetPrediction, BATMAN-TCM, SuperPred, Comparative Toxicogenomics Database (CTD), the Drug Gene Interaction Database (DGldb), PharmMapper, DrugBank, PharmGKB, GeneCards, OMIM, and Therapeutic Target Database (TTD). The search terms they used were “doxorubicin-induced cardiotoxicity/heart failure/cardiomyopathy/myocardial injury.” They found a total of 512 Andro targets and 1719 DIC targets, of which 108 genes were common. Then, they used various bioinformatics tools such as gene ontology (GO), KEGG pathway analysis, protein-protein interaction (PPI), and STRING pathway analysis to identify the relationship between these 108 genes as well as the processes they are involved in. Using three plug-ins in the Cytoscape 3.7.2 software (CytoNCA, CytoHubba, and MCODE), they developed three core networks and identified 19 core genes. These core genes were involved in processes like apoptosis (caspase 3, caspase 9) and inflammation (IL-6, PI3K, AKT, etc.). Liu et al. [6] hypothesized that Andro offers cardioprotective effects against DIC through these pathways. To find a more direct target for Andro, they performed RNA sequencing on the hearts of mice treated with Dox and Dox in combination with Andro, and compared them to a control group, which resulted in a shortlist of 276 genes that were significantly up- or downregulated. Liu et al. [6] then overlapped the 108 genes identified by network pharmacology and 276 genes from RNA sequencing to determine that only two genes (Tap1 and Timp1) were common in them.

Liu et al. [6] elegantly used several tools to identify downstream targets and mechanisms through which Andro can offer protection against DIC. However, this study is only the first step towards developing Andro as a potential therapeutic agent. Finding and validating the mechanisms through which Andro acts will be the biggest challenge. Unfortunately, even after using various analyses and RNA sequencing, Liu et al. [6] were not able to narrow down the mechanism of Andro-mediated cardioprotection. After integrating the DEGs from target discovery databases with DEGs from the RNA sequencing (RNA-seq), they found that Tap1 and Timp1 were the only overlapping genes. This is interesting because both Tap1 and Timp1 are associated with multiple cardiac conditions. However, Liu et al. [6] only validated their expression at the mRNA level; their protein levels were not assessed in the present study. It would be interesting to see if the protein levels of Tap1 and Timp1 also follow the mRNA levels. Furthermore, a future study using Tap1 and/or Timp1 knockout mice would be useful to elucidate their potential role in Dox-induced cardiotoxicity. Furthermore, the 108 target genes identified through the database mining as well as the 276 genes identified through RNA-seq may also be analyzed to uncover the underlying mechanisms. The results of the RNA-seq experiment are actual data that is not mined through a database search. Future studies on the role of these genes and the molecular pathways they are involved in could unlock new mechanistic pathways.