Table_5_Role of MicroRNAs in Protective Effects of Forsythoside A Against Lipopolysaccharide-Induced Inflammation in Bovine Endometrial Stromal Cells.XLSX

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.

牛子宫内膜基质细胞（bovine endometrial stromal cells，bESCs）在分娩后因上皮细胞破裂，暴露于细菌与病毒构成的复杂环境中。宿主细胞膜上的病原体相关分子（如脂多糖（lipopolysaccharides，LPS））可激活宿主模式识别受体，进而诱发炎症应答。连翘苷A（Forsythoside A，FTA）是连翘（Forsythia suspensa (Thunb.) Vahl.）的主要活性成分，连翘是一种广泛用作传统中草药的开花植物，可用于治疗肾炎、眼肿、疥疮、溃疡及乳腺炎等多种炎症性疾病；但其针对牛子宫内膜炎的治疗作用的分子机制仍未明确。本研究旨在探讨microRNA（miRNA）的作用，以及连翘苷A对脂多糖诱导的牛子宫内膜基质细胞炎症的保护活性的分子机制。基于前期研究，我们体外分离并培养bESCs，将其分为LPS组与LPS+FTA组，每组设置3个生物学重复。当细胞汇合度达80%时，分别用0.5 μg/mL LPS，或0.5 μg/mL LPS + 100 μg/mL FTA处理细胞。随后我们通过高通量测序（RNA-Seq），探究连翘苷A对LPS刺激的原代bESCs的影响及其潜在分子机制。我们在LPS组中鉴定出167个差异表达miRNA，其中72个表达上调，95个表达下调。基因本体（Gene Ontology，GO）富集分析显示，差异表达microRNA（DEGs）在细胞代谢过程中富集最为显著；它们主要定位于细胞内，并参与蛋白质结合、酶结合及离子结合过程。京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）通路分析显示，差异表达microRNA（DEGs）显著富集于丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）、肿瘤坏死因子（tumor necrosis factor，TNF）及白细胞介素-17（Interleukin-17，IL-17）信号通路。本研究结果揭示了连翘苷A发挥保护作用的复杂分子机制，为未来利用中草药单体治疗牛子宫内膜炎的相关研究提供了理论依据。