Identification and validation of key target genes of penning formula for treating rat chronic endometritis model

Abstract

This study analyzed transcriptome sequencing data from rat samples, categorized into control (normal), model (chronic endometritis, CE), and test (treated with Penning Formula, PNF) groups, to identify key target genes of PNF in CE treatment and elucidate the specific mechanism of PNF treatment for CE. Differentially expressed genes (DEGs) between the model-control (DEGs1) and model-test (DEGs2) groups were compared, revealing 47 PNF-related genes (PRGs), primarily involved in stress response and prolactin signaling. These PRGs were cross-referenced with 909 target genes (TGs) of 25 active ingredients of PNF obtained from public databases, resulting in the identification of three key target genes: Bhmt, Scn10a, and Esr2. Further analysis, including functional pathway enrichment, chromosomal mapping, expression profiling, gene interactions, regulatory networks, and molecular docking, highlighted their involvement in stress response and prolactin signaling, with distinct chromosomal distributions and differential expression across groups. Additionally, miRNA predictions, such as rno-miR-29a-3p, were made, and significant binding interactions between key genes and active ingredients were observed. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) validation confirmed a marked down-regulation of Bhmt, Scn10a, and Esr2 in the treatment group, corroborating the therapeutic effect of PNF on CE. This study unveils novel targets and offers valuable insights into the mechanisms underlying PNF's therapeutic action for CE.

Keywords: Active ingredient; Chronic endometritis; Key target gene; Penning formula; Regulatory network.

Fig. 1

Identification of 370 DEGs1, 464 DEGs2, and 47 PRGs. (a) Volcano plot of differentially expressed genes (DEGs1). Green dots represent down-regulated genes, red dots represent up-regulated genes, and gray dots represent genes with no significant differential expression. (b) Volcano plot of differentially expressed genes (DEGs2). Green dots represent down-regulated genes, red dots represent up-regulated genes, and gray dots represent genes with no significant differential expression. (c) Heatmap of differential expression analysis for DEGs1. Yellow indicates high expression, and purple indicates low expression. (d) Heatmap of differential expression analysis for DEGs2. Yellow indicates high expression, and purple indicates low expression. (e) Venn diagram of the intersection of DEG1 up-regulated genes and DEG2 down-regulated genes. (f) Venn diagram of the intersection of DEG1 down-regulated genes and DEG2 up-regulated genes.

Fig. 2

Related functional pathways of DEGs1, DEGs2, and PRGs. (a) Gene Ontology (GO) enrichment analysis of DEGs1. (b) GO enrichment analysis of DEGs2. (c) GO enrichment analysis of PRGs. (d) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs1. (e) KEGG enrichment analysis of DEGs2. (f) KEGG enrichment analysis of PRGs.

Fig. 3

Chromosome localization and expression of three key target genes. (a) Screening of key target genes. (b) Distribution map of key genes on chromosomes. (c) Analysis of expression levels of key target genes.

Fig. 4

Crucial functional pathways of key target genes. (a) GO enrichment analysis of key target genes. (b) KEGG enrichment analysis of key target genes.

Fig. 5

Pathway network diagram of herbs, ingredients, and key target genes. Yellow represents the Chinese medicine names, orange represents the active ingredients of the medicine, cyan represents the key target genes, and blue represents the KEGG pathways.

Fig. 6

Complex interaction network of key target genes. (a) Network diagram of key target genes and their co-expressed genes. The size of the circles (nodes) represents the “degree,” which is the number of interactions the node has with other nodes. The higher the degree, the larger the circle, indicating the importance of the node in the network or the richness of its interactions. The colors in the circles represent the functions of the related target proteins, and different line colors represent different network relationships. (b) Functional similarity analysis of key target genes. Different colors represent different genes, and the x-axis represents gene expression levels.

Fig. 7

Specific miRNA-key target genes regulatory network. (a) Venn plot of miRNA intersection predicted by EIMMo, mirDB, and Microcosm databases. (b) miRNA-key target gene regulatory network diagram. Orange represents key target genes, and blue represents miRNAs.

Fig. 8

Expression patterns of key target genes in CE, control, and treatment groups (a: Bhmt, b: Scn10a, c: Esr2).