Xylooligosaccharides ameliorate intestinal barrier injury by inhibiting apoptosis, RhoA/ROCK/MLCK signaling and NKG2DLs in intestinal epithelial cells.

Intestinal barrier (IB) dysfunction contributes to metabolic endotoxemia and associated diseases. Specifically, intestinal inflammation upregulates natural killer cell receptor 2D ligands (NKG2DLs) in intestinal epithelial cells (IECs), which triggers excessive immune responses and exacerbates IB injury. The prebiotic xylooligosaccharides (XOS) hold promise for ameliorating such injury by regulating NKG2DLs, yet the underlying mechanisms remain elusive. This study aimed to investigate the efficacy of XOS in improving IB injury and explore its role and molecular mechanisms in mitigating IB damage via NKG2DLs modulation. IB injury models were established in differentiated Caco-2 and MODE-K cells by treatment with 20 ng/mL tumor necrosis factor-α (TNF-α), followed by XOS intervention at concentrations of 50, 100, 150, and 200 μg/mL. Tight junction (TJ) proteins (ZO-1, Occludin) were detected using Western blotting (WB) and immunofluorescence, while barrier permeability was evaluated via transepithelial electrical resistance (TEER), phenol red permeability assay, and transmission electron microscopy (TEM). Additionally, the RhoA/ROCK/MLCK signaling pathway, apoptosis-related factors (Caspase-3, Bcl-2), and the expression of human NKG2DLs (MICA, MICB, ULBP-1) and murine NKG2DLs (Rae-1ε, H60, MULT-1) were analyzed by quantitative real-time PCR (qRT-PCR) or WB, and apoptotic cells were labeled using the TUNEL assay. To further investigate the involvement of NKG2DLs in the protective effect of XOS, RNA interference (RNAi) was performed to knock down NKG2DLs in Caco-2 and MODE-K cells. This conserved mechanism relies on Argonaute (Ago) family proteins that associate with small RNAs to mediate sequence-specific degradation of complementary target RNAs. Subsequently, both cell lines were treated with TNF-α and XOS, and the expression levels of NKG2DLs, ZO-1, and Occludin were determined by qRT-PCR or WB. Compared with TNF-α treatment alone, XOS at the concentrations used in this study significantly attenuated the downregulation and mislocalization of ZO-1 and Occludin in both Caco-2 and MODE-K cells (p < 0.05), increased TEER in Caco-2 cell (p < 0.05), reduced phenol red permeability (p < 0.05), and improved the ultrastructure of TJs and the integrity of microvilli. XOS at the tested concentrations also inhibited the activation of the RhoA/ROCK/MLCK signaling pathway (p < 0.05), suppressed Caspase-3 (p < 0.05) and the number of TUNEL-positive apoptotic cells, and alleviated TNF-α-induced overexpression of both human and murine NKG2DLs (p < 0.05). Notably, RNAi-mediated knockdown of Rae-1ε or H60 in MODE-K cells abrogated the XOS-induced restoration of ZO-1 and Occludin expression under TNF-α challenge. Collectively, under the experimental conditions of this study, XOS mitigate IB injury by inhibiting apoptosis, RhoA/ROCK/MLCK signaling and NKG2DLs in IECs.