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Bovine endometrial stromal cells (bESCs) are exposed to a complex environment of bacteria and viruses due to the rupture of epithelial cells after delivery. Inflammatory responses are elicited by the activation of host pattern recognition receptors through pathogen-related molecules such as lipopolysaccharides (LPS) on the cell membrane. Forsythoside A (FTA) is a major active constituent of Forsythia suspensa (Thunb.) Vahl. is a flowering plant widely employed as a traditional Chinese herbal medicine to treat various inflammatory diseases such as nephritis, eye swelling, scabies, ulcers, and mastitis; however, the molecular mechanisms underlying its therapeutic effects on bovine endometritis are still unclear. The aim of this study was to explore the role of miRNA and the mechanisms underlying the protective activity of FTA on the inflammation of bovine endometrial stromal cells induced by LPS. Based on previous research, we isolated and cultured bESCs in vitro and categorized them into LPS and LPS+FTA groups with three replicates. Upon reaching 80% confluence, the bESCs were treated with 0.5 μg/mL of LPS or 0.5 μg/mL of LPS + 100 μg/mL of FTA. We, then, performed high-throughput sequencing (RNA-Seq) to investigate the effects of FTA on LPS-stimulated primary bESCs and their underlying mechanisms. We identified 167 miRNAs differentially expressed in the LPS groups; 72 miRNAs were up-regulated, and 95 were down-regulated. Gene ontology enrichment analysis revealed that differentially expressed microRNA (DEGs) were most enriched during the cellular metabolic process; they were mostly located intracellularly and participated in protein, enzyme, and ion binding. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the DEGs were most enriched in the mitogen-activated protein kinase, tumor necrosis factor, and Interleukin-17 signaling pathways. These results reveal the complex molecular mechanism involved in the FTA and provide a basis for future studies of bovine endometritis treatment with traditional Chinese medicine monomer.

Abstract Background: Endometritis adversely affects the ability of cattle to reproduce, and significantly reduces milk production. Consequently, it has great influence on the economic value of dairy cows. The endometrium is mainly composed of epithelial and stromal cells and they produce the first immune response to invading pathogens. Epithelial cells are the first cellular barrier through which bacteria enter the uterine endometrium. However, most of the epithelial cells are disrupted and stromal cells are exposed to an inflammatory environment when endometritis occurs, especially postpartum. A loss of the protective epithelium allows bacteria or toxins to access the underlying stromal cells. The activation of Toll-like receptor（TLRs）on stromal cells induces increased production of cytokines and chemokines. Understanding the genome-wide characterization of the bovine endometritis will be beneficial for prevention and treatment of endometritis. In this study, whole-transcriptomic gene changes in bovine stromal cells treated with LPS were compared with those treated with PBS (control group) and were analyzed by RNA sequencing (RNA-seq). Results: Compared with the control group, a total of 366 differentially expressed genes (DEGs) were identified in LPS-induced group (234 upregulated and 132 downregulated genes), with an adjusted P-value＜0.05 by DESeq. Gene ontology (GO) enrichment analysis revealed DEGs were most enriched in lymphocyte activation, interleukin-1 receptor binding, regulation of cell activation, and lymphocyte-activated interleukin-12 production. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed DEGs were most enriched in TNF signaling pathway, Toll-like receptor signaling pathway, cytokine-cytokine receptor interaction, nucleotide-binding oligomerization domain-like (NOD-like) receptor signaling pathway, NF-κB signaling pathway, and chemokine signaling pathway.Conclusion: The results of this study unraveled endometrial stromal cells transcriptome profile alterations in bovine affected by LPS which may have a significant effect on the eliminating or reducing inflammation by comprehending molecular mechanisms and authenticating unique genes related to endometritis.

Endometritis adversely affects the ability of cattle to reproduce and significantly reduces milk production. The is mainly composed of epithelial and stromal cells, and they produce the first immune response to invading pathogens. However, most of the epithelial cells are disrupted, and stromal cells are exposed to an inflammatory environment when endometritis occurs, especially postpartum. Many bacteria and toxins start attacking stromal cell due to loss of epithelium, which stimulates Toll-like receptor (TLRs) on stromal cells and causes upregulated expression of cytokines. Understanding the genome-wide characterization of bovine endometritis will be beneficial for prevention and treatment of endometritis. In this study, whole-transcriptomic gene changes in bovine endometrial stromal cells (BESCs) treated with LPS were compared with those treated with PBS (control group) and were analyzed by RNA sequencing. Compared with the control group, a total of 366 differentially expressed genes (DEGs) were identified in the LPS-induced group (234 upregulated and 132 downregulated genes), with an adjusted P < 0.05 by DESeq. Gene Ontology (GO) enrichment analysis revealed that DEGs were most enriched in interleukin-1 receptor binding, regulation of cell activation, and lymphocyte-activated interleukin-12 production. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed DEGs were most enriched in the TNF signaling pathway, Toll-like receptor signaling pathway, cytokine–cytokine receptor interaction, NF-κB signaling pathway, and chemokine signaling pathway. The results of this study unraveled BESCs affected with LPS transcriptome profile alterations, which may have a significant effect on treatment inflammation by comprehending molecular mechanisms and authenticating unique genes related to endometritis.

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