食品与发酵工业 >

2024 , Vol. 50 >Issue 24: 191 - 200

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.038094

研究报告

鼠李糖乳酪杆菌KF7发酵乳上清对脂多糖诱导的Caco-2细胞单层屏障损伤的保护作用及其机制研究

  • 董奇奇 ,
  • 张怡琳 ,
  • 游春苹
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  • 1(上海海洋大学 食品学院,上海,201306)
    2(乳业生物技术国家重点实验室,上海乳业生物工程技术研究中心,光明乳业股份有限公司乳业研究院,上海,200436)
第一作者:硕士研究生(游春苹正高级工程师为通信作者,E-mail:youchunping@brightdairy.com)

收稿日期: 2023-11-28

  修回日期: 2024-02-03

  网络出版日期: 2024-12-30

基金资助

上海市优秀技术带头人计划 (20XD1430100);市国资委能力提升项目(2022013)

收起

Study on protective effects and mechanism of Lacticaseibacillus rhamnosus KF7 fermented milk supernatant on lipopolysaccharide-induced epithelial barrier damage in Caco-2 cell monolayer model

  • DONG Qiqi ,
  • ZHANG Yilin ,
  • YOU Chunping
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  • 1(School of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China)
    2(State Key Laboratory of Dairy Biotechnology, Shanghai Engineering Research Center of Dairy Biotechnology, Dairy Research Institute, Bright Dairy & Food Co., Ltd., Shanghai 200436, China)

Received date: 2023-11-28

  Revised date: 2024-02-03

  Online published: 2024-12-30

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摘要

该研究利用脂多糖诱导Caco-2细胞单层屏障模型损伤,在体外探究鼠李糖乳酪杆菌KF7发酵乳上清液对肠道屏障的保护作用。通过Cell Counting Kit-8(CCK-8)法检测KF7发酵乳上清液对细胞活力的影响;使用电阻仪和异硫氰酸荧光素-右旋糖苷检测肠上皮屏障结构的完整性和通透性;利用荧光探针以及比色法检测细胞内活性氧和丙二醛(malondialdehyde, MDA)的含量;采用qRT-PCR分析紧密连接蛋白、促炎细胞因子、抗炎细胞因子以及氧化应激标志物和炎症反应相关通路的基因表达情况。结果发现,1%~5%的鼠李糖乳酪杆菌KF7发酵乳上清液对Caco-2细胞活力无损伤,且能够保护Caco-2细胞单层屏障,降低脂多糖诱导的炎症反应(下调TNF-α、IL-1β、IL-6的基因表达,上调IL-10的基因表达)和氧化应激(降低MDA含量),上调细胞间紧密连接蛋白相关基因(Claudin-1和Occludin)的表达,改善细胞单层跨上皮电阻并降低细胞单层的通透性,从而恢复Caco-2细胞单层屏障完整性。在机制方面,鼠李糖乳酪杆菌KF7发酵乳上清液可能通过促进Nrf2/NQO1信号通路拮抗氧化应激,通过抑制TLR/MyD88/NF-κB信号通路及下游肌球蛋白轻链激酶的基因表达来降低炎症反应和肠上皮细胞间通透性。该研究结果可为鼠李糖乳酪杆菌KF7发酵乳制品作为一种有前途的保护肠道健康的功能性益生菌膳食补充剂提供一定的理论基础。

关键词： 鼠李糖乳酪杆菌KF7; Caco-2细胞; 跨上皮电阻; 肠上皮屏障; 氧化应激; 炎症反应

本文引用格式

董奇奇 , 张怡琳 , 游春苹 . 鼠李糖乳酪杆菌KF7发酵乳上清对脂多糖诱导的Caco-2细胞单层屏障损伤的保护作用及其机制研究[J]. 食品与发酵工业, 2024 , 50(24) : 191 -200 . DOI: 10.13995/j.cnki.11-1802/ts.038094

Abstract

Damage to the intestinal barrier is associated with many diseases.Preventing and improving impaired intestinal barrier function through dietary interventions, especially probiotics, may be a promising therapeutic strategy.This study utilized lipopolysaccharide to induce damage in the Caco-2 cell monolayer barrier model to investigate the protective effect of Lacticaseibacillus rhamnosus KF7 fermented milk supernatant on the intestinal barrier in vitro. Cell Counting Kit-8(CCK-8) assay was used to detect the effects of KF7 fermented milk supernatant on cell viability.A resistivity meter and fluorescein isothiocyanate-dextrose were used to examine the structural integrity and permeability of the intestinal epithelial barrier.Fluorescent probe and colorimetric assay were used to detect the intracellular levels of reactive oxygen species and malondialdehyde (MDA), and qRT-PCR was used to analyze the gene expression levels of tight junction proteins, pro-inflammatory cytokines, anti-inflammatory cytokines, and markers of oxidative stress and inflammation-related pathways.Results revealed that 1%-5% Lacticaseibacillus rhamnosus KF7 fermented milk supernatant did not impair Caco-2 cell viability, but protected the Caco-2 cell monolayer barrier, decreasing lipopolysaccharides-induced inflammatory response (down-regulation of gene expression of TNF-α, IL-1β, and IL-6, and up-regulation of IL-10) and oxidative stress (reduction of MDA content), increasing the expression of intercellular tight junction protein-related genes (Claudin-1 and Occludin), improving cell monolayer trans-epithelial electrical resistance, and reducing the permeability, thus recovered the integrity of Caco-2 cell monolayer barrier.Mechanistically, Lacticaseibacillus rhamnosus KF7 fermented milk supernatant may antagonize oxidative stress by promoting the Nrf2/NQO1 signaling pathway, and decrease the inflammatory response and intestinal epithelial inter-cellular permeability by inhibiting the TLR/MyD88/NF-κB signaling pathway and downstream myosin lightchain kinase(MLCK) gene expression.The results of this study may provide some theoretical basis for Lacticaseibacillus rhamnosus KF7 fermented dairy products as a promising functional probiotic dietary supplement for protecting intestinal health.

Key words： Lacticaseibacillus rhamnosus KF7; Caco-2 cells; transepithelial electrical resistance; intestinal epithelial barrier; oxidative stress; inflammatory response

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