Institute of Food Sciences and Technologies, Ajinomoto Co., Inc, Kanagawa, 210-8681, Japan.
Amino Acids. 2021 Jul;53(7):1021-1032. doi: 10.1007/s00726-021-03001-y. Epub 2021 May 15.
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 HO 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 HO-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.
肠道氧化应激会产生促炎细胞因子，增加紧密连接（TJ）的通透性，导致肠道和全身炎症。胱氨酸（Cys2）是谷胱甘肽（GSH）的底物，能抑制炎症，但尚不清楚 Cys2 是否能局部改善肠道屏障功能障碍。因此，我们研究了 Cys2 对氧化应激诱导的 TJ 通透性和肠道炎症反应的局部作用。将 Caco-2 细胞在补充 Cys2 的培养基中培养 24 小时，然后用 HO 处理 2 小时。我们通过测量跨上皮电阻和荧光素异硫氰酸酯-4 kDa 葡聚糖的旁流来评估 TJ 通透性。我们在 Cys2 预处理后测量 Cys2 和 GSH 的浓度。评估促炎细胞因子的 mRNA 表达。此外，还通过测量整个细胞中 TJ 蛋白的表达和去污剂不溶性部分与可溶性部分的 TJ 蛋白比（IS/S 比）来评估 TJ 蛋白的水平。Cys2 处理降低了 HO 诱导的 TJ 通透性。Cys2 不改变整个细胞中 TJ 蛋白的表达，但抑制了 claudin-4 的 IS/S 比。用 Cys2 处理的细胞中细胞间 Cys2 和 GSH 的水平显著增加。Cys2 处理抑制了促炎细胞因子的 mRNA 表达，mRNA 水平与 TJ 通透性显著相关。总之，Cys2 处理局部减轻了氧化应激诱导的肠道屏障功能障碍，可能是由于 claudin-4 定位不当得到缓解。此外，Cys2 改善肠道屏障功能的作用与局部抑制氧化应激诱导的促炎反应有关。
