Lv Haimiao, Yan Chenbo, Deng Lixin, Peng Zhan, Yang Dexin, Hu Wenjv, Ding Xuefen, Tong Chao, Wang Xinzhuang
College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, China.
College of Agricultural Medicine, Henan Radio and Television University, Zhengzhou, China.
Front Vet Sci. 2021 Feb 24;8:642913. doi: 10.3389/fvets.2021.642913. eCollection 2021.
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 (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 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.
分娩后上皮细胞破裂，牛子宫内膜基质细胞（bESCs）暴露于细菌和病毒的复杂环境中。宿主模式识别受体通过病原体相关分子如细胞膜上的脂多糖（LPS）激活，引发炎症反应。连翘酯苷A（FTA）是连翘（Thunb.）Vahl.的主要活性成分。连翘是一种广泛用作传统中药的开花植物，用于治疗各种炎症性疾病，如肾炎、眼肿、疥疮、溃疡和乳腺炎；然而，其对牛子宫内膜炎治疗作用的分子机制仍不清楚。本研究的目的是探讨miRNA的作用以及FTA对LPS诱导的牛子宫内膜基质细胞炎症的保护活性机制。基于先前的研究，我们分离并培养了bESCs，并将其分为LPS组和LPS+FTA组，每组三个重复。当细胞汇合度达到80%时，用0.5μg/mL的LPS或0.5μg/mL的LPS+100μg/mL的FTA处理bESCs。然后，我们进行了高通量测序（RNA-Seq），以研究FTA对LPS刺激的原代bESCs的影响及其潜在机制。我们在LPS组中鉴定出167个差异表达的miRNA；72个miRNA上调，95个下调。基因本体富集分析显示，差异表达的microRNA（DEGs）在细胞代谢过程中富集程度最高；它们大多位于细胞内，参与蛋白质、酶和离子结合。京都基因与基因组百科全书通路分析显示，DEGs在丝裂原活化蛋白激酶、肿瘤坏死因子和白细胞介素-17信号通路中富集程度最高。这些结果揭示了FTA涉及的复杂分子机制，并为未来用中药单体治疗牛子宫内膜炎的研究提供了基础。
