Abstract
Intestinal oxidative stress produces pro-inflammatory cytokines, which increase tight junction (TJ) permeability, leading to intestinal and systemic inflammation. Cystine (Cys2) is a substrate of glutathione (GSH) and inhibits inflammation, however, it is unclear whether Cys2 locally improves intestinal barrier dysfunction. Thus, we investigated the local effects of Cys2 on oxidative stress-induced TJ permeability and intestinal inflammatory responses. Caco-2 cells were cultured in a Cys2-supplemented medium for 24 h and then treated with H2O2 for 2 h. We assessed TJ permeability by measuring transepithelial electrical resistance and the paracellular flux of fluorescein isothiocyanate–dextran 4 kDa. We measured the concentration of Cys2 and GSH after Cys2 pretreatment. The mRNA expression of pro-inflammatory cytokines was assessed. In addition, the levels of TJ proteins were assessed by measuring the expression of TJ proteins in the whole cells and the ratio of TJ proteins in the detergent-insoluble fractions to soluble fractions (IS/S ratio). Cys2 treatment reduced H2O2-induced TJ permeability. Cys2 did not change the expression of TJ proteins in the whole cells, however, suppressed the IS/S ratio of claudin-4. Intercellular levels of Cys2 and GSH significantly increased in cells treated with Cys2. Cys2 treatment suppressed the mRNA expression of pro-inflammatory cytokines, and the mRNA levels were significantly correlated with TJ permeability. In conclusion, Cys2 treatment locally reduced oxidative stress-induced intestinal barrier dysfunction possively due to the mitigation of claudin-4 dislocalization. Furthermore, the effect of Cys2 on the improvement of intestinal barrier function is related to the local suppression of oxidative stress-induced pro-inflammatory responses.
Highlights
The intestinal epithelium, composed of a single layer of cells, is a mucosal barrier that maintains the luminal environment of the intestine (Turner 2009) and regulates the permeation of luminal noxious substances, such as bacteria, toxins, and dietary antigens, into the intestinal cells (Claude and Goodenough 1973; Farquhar and Palade 1963; Powell 1981)
To investigate the effect of Cys2 on the expression of tight junction (TJ) proteins, we measured the expression of zonula occludens-1 (ZO-1), occludin, and claudin-1 and -4 in Caco-2 cells incubated with H2O2 for 2 h by western blotting (Fig. 3a)
There were no significant differences between the control and H 2O2-treated groups in the expression of TJ proteins in the whole cells (Fig. 3b–e)
Summary
The intestinal epithelium, composed of a single layer of cells, is a mucosal barrier that maintains the luminal environment of the intestine (Turner 2009) and regulates the permeation of luminal noxious substances, such as bacteria, toxins, and dietary antigens, into the intestinal cells (Claude and Goodenough 1973; Farquhar and Palade 1963; Powell 1981) Stressful stimuli, such as pregnancy, administration of drugs, and endurance exercise, induce intestinal barrier dysfunction, causing intestinal inflammation (Camilleri 2019). The increase in TJ permeability leads to the translocation of lipopolysaccharide (LPS) into the blood circulation, triggering the release of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), IL-1β, or IL-6 (Raetz and Whitfield 2002) These cytokines activate NF-κB signaling pathways in epithelial cells, decreasing the expression of TJ proteins, such as ZO-1 and occludin, and increasing TJ permeability (Al-Sadi and Ma 2007; Ma et al 2004). The suppression of oxidative stress or pro-inflammatory cytokines protects the intestinal barrier function
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