Role of MicroRNAs in Protective Effects of Forsythoside A Against Lipopolysaccharide-Induced Inflammation in Bovine Endometrial Stromal Cells

Abstract

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.

Keywords: bovine endometrial stromal cells; endometritis; forsythoside A; lipopolysaccharide; microRNA.

Figure 1

Chemical structure of forsythoside A (FTA).

Figure 2

Effect of FTA on bESC viability. Cells were cultured with different concentrations of FTA (25, 50, 100, 200, 400, 600, 800, and 1,000 μg/ml) for 12 h. The cell viability was determined by CCK8 assay. The values presented are the means ± SEM.

Figure 3

Comparison of miRNA expression patterns between lipopolysaccharide (LPS) and forsythoside A (FTA) + LPS groups. (A) Heat map of differentially expressed miRNAs. High and low expression levels of miRNA are shown in red and green, respectively. Each group included three biological repeats. (B) A volcano plot of differentially expressed miRNAs. Differences in miRNA up-regulation, down-regulation, and unchanged levels are represented by red, blue, and gray points, respectively.

Figure 4

Functional annotation and enrichment analysis of differentially expressed miRNAs. (A) The results of Gene Ontology (GO) enrichment analysis of differentially expressed GO classified miRNA target genes according to molecular functional (MF), biological process (BP), and cellular component (CC) analysis, and the top 10 GO term items with the smallest P-value or the most significant enrichment were selected for display in each GO classification. The X-axis represents gene function, and the Y-axis represents gene percentage. (B) Scatter diagram of GO enrichment analysis of differentially expressed target genes of miRNA. (C) Scatter diagram of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed target genes of miRNA. The ratio of the number of differential miRNA target genes enriched in the pathway to the number of annotated differential miRNA target genes.

Figure 5

Validation of features of DEGs through qRT-PCR. The relative expression level of target miRNAs was calculated using the 2^−ΔΔCt method. Data represent the mean ± SEM (n = 3).The Log₂₋fold change was the average log₂₋fold change between samples.

Figure 6

Forsythoside A (FTA) attenuates lipopolysaccharide (LPS)-induced endometritis by causing significant changes in the number of miRNAs in bESCs to regulate the target genes involved in different pathway. Through the activation of the NF-κB signaling pathway, a complex inflammatory response occurs, and pro-inflammatory cytokines and chemokines are produced and released. FTA can increase the expression of miRNA-146a, miR-29E, miR-677, and miR-2904, and negatively regulate the target genes, TLR4, TRL2, and DHX58, to reduce the production of TLR4, TRL2, and DHX58 receptor proteins. MiR-1434-5p and miR-2332 can attenuate the activation of the NF-κB signaling pathway by targeting TBK1 and TRAF6, respectively. MiR-205, miR-2904, miR-31, miR-302a, miR-302b, miR302-d, and miR-1434-5p target multiple cytokines and chemokines to reduce the production of inflammatory factors and reduce the inflammatory response. For example, bta-miR-205 targeted several inflammatory cytokines, including IL1A, IL27, IL2RA, and IL21. Bta-miR-2904 targeted IL1A. IL6ST is a signal transducer of IL-6 and a target gene of bta-miR-302a, bta-miR-302b, and bta-miR302-d. Bta-miR-1434-5p targeted chemokines CXCL5, CXCL8, and CXCL9. IL1B is a target gene of bta-miR-31. The black arrow indicates the mechanism underlying the inflammatory response of bESCs stimulated by LPS, and the green arrow indicates that miRNA expression partly increases under the action of FTA. The red T-shaped line indicates that miRNA targets inflammatory cytokines and reduces the expression of inflammatory cytokines, and red arrows indicate that inflammatory factors or proteins can promote the expression of downstream inflammatory factors or proteins.