铁皮石斛多糖对小肠上皮细胞肠道屏障的保护作用

Protective Effect of Dendrobium officinale Polysaccharide on Small Intestinal Epithelial Barrier

摘要:

该研究用铁皮石斛多糖（Dendrobium officinale Polysaccharide, DOP）诱导IEC-6肠上皮细胞分泌的外泌体探讨对TNF-α诱导的小肠上皮细胞肠道屏障保护作用。超速离心提取外泌体，透射电观察外泌体形态，Western blot检测标志蛋白HSP70、TSG101和CD63，纳米颗粒跟踪分析（Nanoparticle Tracking Analysis, NTA）检测外泌体粒径；Western blot检测铁皮石斛多糖对肠上皮组织紧密连接蛋白表达的影响，测谷胱甘肽过氧化物酶（Glutathione Peroxidase, GSH-PX）、超氧化物歧化酶（Superoxide Dismutase, SOD）和过氧化氢酶（Catalase, CAT）的活性。结果表明：超速离心法获得的颗粒形状呈圆盘状，粒径为30~150 nm，含标志性蛋白TSG101、HSP70和CD63，鉴定为外泌体；DOP孵育产生的外泌体DE可被IEC-6细胞摄取，DOP组小鼠IEC-6上皮细胞ZO-1、Occludin和MUC1蛋白的表达量分别增加了104.63%、70.09%和52.34%，抗氧化酶SOD、CAT和GSH-PX活性分别提高了52.16%、43.40%、122.89%。综上所述，铁皮石斛多糖通过外泌体提高小肠上皮细胞抗氧化酶活性，增强紧密连接蛋白表达来修护肠道屏障功能损伤，为炎症性肠病的治疗提供参考。

Abstract:

Exosome secretion by IEC-6 intestinal epithelial cells was induced by Dendrobium officinale polysaccharide (DOP) to investigate the TNF-α-induced protective effect of the small intestinal epithelial barrier. The secreted exosomes were extracted by ultracentrifugation. The morphology of exosomes was observed by transmission electron microscopy, the marker proteins HSP70, TSG101, and CD63 were detected by western blot, and the particle sizes of exosomes were determined by nanoparticle tracking analysis. The effects of DOP on the expression of tight junction proteins in intestinal epithelial tissues were clarified by western blot. The activities of glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), and catalase (CAT) were measured. The effects of DOP on the expression of tight junction proteins in the intestinal epithelium were detected by Western blot, and the activities of oxidative stress kinase GSH-PX, SOD, and CAT were determined. The results showed that the particles were disc-shaped (diameter: 30~150 nm) and contained the signature proteins TSG101, HSP70, and CD63. The particles were identified as exosomes. The exosome DE produced by DOP incubation can be taken up by IEC-6 cells. The expression of ZO-1, Occludin, and MUC1 proteins in IEC-6 cells of mice in the DOP group increased by 104.63%, 70.09%, and 52.34%, respectively. The activities of antioxidant enzymes SOD, CAT, and GSH-PX were increased by 52.16%, 43.40%, and 122.89%, respectively. In summary, DOP can improve the antioxidant enzyme activity and enhance the expression of tight junction proteins in intestinal epithelial cells through exosomes to repair intestinal barrier damage.