副干酪乳酪杆菌BD5115代谢产物对肠上皮屏障完整性的增强作用及其效应物质的初步研究  

Enhancing effect of Lacticaseibacillus paracasei BD5115 metabolites on intestinal epithelial barrier integrity and the effectors: a preliminary study

作　　者：王超越 韩瑨[2] 郭诚笑 唐月方 吴正钧[2] WANG Chao-yue;HAN Jin;GUO Cheng-xiao;TANG Yue-fang;WU Zheng-jun(College of Food Sciences&Technology,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]上海海洋大学食品学院,上海201306 [2]光明乳业股份有限公司乳业研究院,上海乳业生物工程技术研究中心,乳业生物技术国家重点实验室

出　　处：《中国微生态学杂志》2022年第11期1254-1261,1268,共9页Chinese Journal of Microecology

基　　金：上海乳业生物工程技术研究中心(19DZ2281400)。

摘　　要：目的 筛选具有改善肠上皮屏障功能潜力的益生菌,并进一步探究其作用机制及其生物效应物质。方法 以Caco-2细胞单层跨上皮电阻(transepithelial electrical resistance,TEER)和异硫氰酸荧光素―葡聚糖(FITC-Dextran)的透过率为评价指标,比较不同乳酪杆菌发酵脱脂乳上清对细胞单层完整性的影响及发酵时间对副干酪乳酪杆菌BD5115发酵乳上清活性的影响;采用实时荧光定量PCR(real-time quantitative PCR,RT-qPCR)检测乳酪杆菌发酵乳上清及其代谢产物对Caco-2细胞间紧密连接相关基因表达的影响;采用超高效液相色谱和四极杆―静电场轨道阱高分辨质谱仪联用对发酵24 h和6 h的BD5115发酵乳上清中的差异代谢物进行筛选;采用细胞免疫荧光对Caco-2细胞中紧密连接蛋白Occludin进行定位。结果 干酪乳酪杆菌ATCC334和副干酪乳酪杆菌BD5115发酵乳上清能显著增加细胞单层TEER及降低FITC-Dextran的透过率,上调Occludin基因表达,且BD5115发酵乳上清对细胞单层完整性的增强作用优于ATCC334。另外,BD5115发酵乳上清的活性与其发酵时间有关,其代谢产物吲哚-3-甲醛能显著上调Occludin基因的表达。结论 益生菌对肠上皮屏障功能的改善作用具有菌株特异性,BD5115发酵脱脂乳产生的吲哚-3-甲醛可能是其效应物质之一。Objective To screen probiotics strains with the potential to improve intestinal epithelial barrier function, and unravel the mechanisms of action and effectors of the probiotics. Methods Using transepithelial electrical resistance(TEER) and paracelular permeability of Dextran-fluorescein isothiocyanate(FITC-Dextran) of Caco-2 cell layer as the evaluation indexes, the effect of the supernatant of fermented skim milk by different Lacticaseibacillus strains on the integrity of Caco-2 cell layer was observed. Besides, the influence of cultivation period on the activity of L. paracasei BD5115fermented milk supernatant were investigated. Real-time quantitative PCR(RT-qPCR) was used to detect the impact of the supernatant of Lacticaseibacillus fermented milk as well as some differential metabolites on the expression of genes encoding proteins in charge of the tight junction between Caco-2 cells in the monolayer. The differential metabolites in the fermented milk by L. paracasei BD5115 cultivated for 6 hours vs 24 hours were screened using high performance liquid chromatography tandem mass spectrometry(UHPLC-HRMS). The location of the tight junction protein Occludin on the surface of Caco-2 cells was visualized using immunofluorescence staining. Results Among the tested Lacticaseibacillus strains, the supernatant of fermented skim milk by L. casei ATCC334 and that by L. paracasei BD5115 significantly increased TEER of Caco-2 monolayer and up-regulated the expression of Occludin encoding gene in the mammal cells, while decreased the transmittance of FITC-Dextran of the monolayer. Compared with ATCC334, the supernatant of BD5115 fermented skim milk demonstrated a stronger capability in enhancing the Caco-2 cell monolayer integrity, and the activity of the supernatant was cultivation period-dependant. Indole-3-formaldehyde, one of the differential metabolites, significantly up-regulated the expression of Occludin encoding gene in Caco-2 cells. Conclusion The effect of probiotics in improving the intestinal barrier integrity

关 键 词：乳酪杆菌 肠上皮屏障 代谢产物 紧密连接蛋白 效应物质 

分 类 号：R446.5[医药卫生—诊断学]