Modulation of Macrophages TLR4-mediated Transcriptional Response by Lacticaseibacillus rhamnosus CRL1505 and Lactiplantibacillus plantarum CRL1506. Modulation of Macrophages TLR4-mediated Transcriptional Response by Lacticaseibacillus rhamnosus CRL1505 and Lactiplantibacillus plantarum CRL1506

The aim of this work was to gain insight into the mechanisms involved in the immunomodulatory properties of L. rhamnosus CRL1505 and L. plantarum CRL1506 in the context of TLR4-mediated inflammation, particularly focused on macrophages. Therefore, in vitro studies in murine macrophages were conducted to evaluate the ability of the lactobacilli strains to modulate the transcriptomic response after LPS stimulation. Overall design: Raw 264.7 mouse macrophage cells (5x10^5 cells/well) were inoculated in 12-well plates and incubated overnight. The culture medium was changed, and macrophages were stimulated with lactobacilli strains CRL1505 or CRL1506 (5x10^7 cells/mL) for 24 h. Macrophages were washed to eliminate lactobacilli and stimulated with 50 ng/mL LPS (InvivoGen, San Diego, USA) for 12 h. Total RNA was extracted using the RNeasy Mini kit (Qiagen, Tokyo, Japan). The RNA labelling and hybridization was performed using the SurePrint G3 Mouse GE 8x60K ver2.0 Microarray from Hokkaido System Science Co., Ltd., Sapporo, Japan. Microarray scanning was performed with the Microarray Scanner from Agilent Technologies, while digitalization was done using Agilent Feature Extraction 10.7.3.1.

本研究旨在深入阐明鼠李糖乳杆菌（L. rhamnosus）CRL1505与植物乳杆菌（L. plantarum）CRL1506在Toll样受体4（TLR4）介导的炎症反应中发挥免疫调节特性的相关机制，研究重点聚焦于巨噬细胞。为此，本研究开展了鼠源巨噬细胞的体外实验，以评估这两株乳杆菌菌株在脂多糖（LPS）刺激后对细胞转录组应答的调节能力。
实验总体设计：将RAW 264.7小鼠巨噬细胞（5×10^5 个/孔）接种于12孔细胞培养板中，静置过夜培养。随后更换细胞培养液，分别以浓度为5×10^7 个/mL的CRL1505或CRL1506乳杆菌菌株刺激巨噬细胞24小时。洗涤细胞以去除残留乳杆菌后，使用50 ng/mL的脂多糖（LPS，InvivoGen，美国圣地亚哥）刺激细胞12小时。采用RNeasy Mini试剂盒（Qiagen，日本东京）提取总RNA。RNA标记与杂交实验采用日本札幌北海道系统科学株式会社生产的SurePrint G3 Mouse GE 8x60K ver2.0微阵列芯片完成。微阵列扫描使用安捷伦科技（Agilent Technologies）的微阵列扫描仪完成，数据数字化则通过安捷伦Feature Extraction 10.7.3.1软件实现。