The Role of Probiotics in Lipopolysaccharide-Induced Autophagy in Intestinal Epithelial Cells
The Role of Probiotics in Lipopolysaccharide-Induced Autophagy in Intestinal Epithelial Cells
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益生菌在脂多糖诱导的肠上皮细胞自噬中的作用
DOI：
10.1159/000445597
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发表时间：
2016-01-01
影响因子：
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通讯作者：
Lin, Rong
中科院分区：
文献类型：
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作者：
Han, Chaoqun;Ding, Zhen;Lin, Rong
背景/目的：自噬功能障碍与肠道内环境平衡的丧失有关。革兰氏阴性菌产生的脂多糖(LPS)是肠上皮细胞(IEC)自噬的主要启动因子。虽然益生菌已经被认为参与了许多治疗特性和参与宿主防御反应，但益生菌发挥这些积极作用的分子机制仍不清楚。这项研究评估了益生菌对内毒素诱导的物理屏障功能障碍的影响，以及益生菌作用于IECS的潜在机制，重点是自噬。方法：在大鼠IEC18细胞中建立内毒素诱导的自噬模型，在一定时间内给予双歧杆菌培养上清处理。将GFP-mCherry-LC3串联导入IEC18细胞后，通过共聚焦显微镜和透射电子显微镜观察到自噬小体的存在。用免疫印迹法检测自噬相关蛋白水平，用上皮伏特计测定跨皮细胞电阻(TEER)。结果：益生菌处理可有效抑制脂多糖诱导的自噬，表现为微管相关轻链3(Lc3)-II/Lc3-I比值降低，自噬空泡减少，GFP-mCherry-Lc3点状分布减少。此外，益生菌阻止了氯喹(CQ)对自噬通量和自噬溶酶体融合的抑制，这表明未能将LAMP1和组织蛋白酶D招募到溶酶体中。有趣的是，ATG16L1基因敲除并不能抑制益生菌对内毒素诱导的自噬的影响。此外，益生菌可以预防屏障功能的减弱。结论：我们提供的证据表明，益生菌介导的自噬可能参与了对内毒素诱导的肠上皮毒性的肠道保护作用，并可能成为益生菌促进和维持肠道动态平衡的一种新机制。
Background/Aims: Dysfunction of autophagy has been associated with loss of intestinal homeostasis. Lipopolysaccharide (LPS) from Gram-negative bacteria is known to be a major initiator of intestinal epithelial cell (IEC) autophagy. Although probiotics have been recognized to be involved in many therapeutic properties and participate in host defense responses, the molecular mechanisms by which probiotics exert these positive effects remain unknown. This study assessed the effect of probiotics on LPS-induced physical barrier dysfunction and the underlying mechanism of probiotic action in IECs with a focus on autophagy. Methods: A LPS-induced autophagic model was established in rat IEC18 cells wherein cells were treated with culture medium supernatants of Bifidobacteria following LPS intervention at indicated times. Autophagosomes in IEC18 cells were visualized by confocal microscopy after transfection with a tandem GFP-mCherry-LC3 construct and also by transmission electron microscopy. Autophagy-associated protein levels were analyzed by western blot and transepithelial electrical resistance (TEER) was measured using an epithelial voltohmmeter. Results: Probiotic treatment could effectively inhibit LPS-induced autophagy, as evidenced by the decreased ratio of microtubule-associated light chain 3 (LC3)-II/LC3-I, fewer autophagic vacuoles, and reduced punctate distribution of GFP-mCherry-LC3. In addition, probiotics prevented chloroquine (CQ) inhibition of autophagic flux and autophagolysosomal fusion as indicated by a failure to recruit LAMP1 and cathepsin D to lysosomes. Interestingly, ATG16L1 knockdown did not inhibit the effect of probiotics on LPS-induced autophagy. Furthermore, the diminished barrier function could be prevented by probiotics. Conclusions: We provide evidence that autophagy mediation by probiotics may be involved in enteroprotection against LPS-induced intestinal epithelial toxicity, and could serve as a novel mechanism through which probiotics promote and maintain gut homeostasis.