datasheet5_Pure Total Flavonoids From Citrus Protect Against Nonsteroidal Anti-inflammatory Drug-Induced Small Intestine Injury by Promoting Autophagy in vivo and in vitro.zip

Small intestine injury is an adverse effect of non-steroidal anti-inflammatory drugs (NSAIDs) that urgently needs to be addressed for their safe application. Although pure total flavonoids from citrus (PTFC) have been marketed for the treatment of digestive diseases, their effects on small intestine injury and the underlying mechanism of action remain unknown. This study aimed to investigate the potential role of autophagy in the mechanism of NSAID (diclofenac)-induced intestinal injury in vivo and in vitro and to demonstrate the protective effects of PTFC against NSAID-induced small intestine disease. The results of qRT-PCR, western blotting, and immunohistochemistry showed that the expression levels of autophagy-related 5 (Atg5), light chain 3 (LC3)-II, and tight junction (TJ) proteins ZO-1, claudin-1, and occludin were decreased in rats with NSAID-induced small intestine injury and diclofenac-treated IEC-6 cells compared with the control groups. In the PTFC group, Atg5 and LC3-II expression, TJ protein expression, and the LC3-II/LC3-I ratio increased. Furthermore, the mechanism by which PTFC promotes autophagy in vivo and in vitro was evaluated by western blotting. Expression levels of p-PI3K and p-Akt increased in the intestine disease-induced rat model group compared with the control, but decreased in the PTFC group. Autophagy of IEC-6 cells was upregulated after treatment with a PI3K inhibitor, and the upregulation was significantly more after PTFC treatment, suggesting PTFC promoted autophagy through the PI3K/Akt signaling pathway. In conclusion, PTFC protected intestinal barrier integrity by promoting autophagy, which demonstrates its potential as a therapeutic candidate for NSAID-induced small intestine injury.

小肠损伤是非甾体抗炎药（non-steroidal anti-inflammatory drugs, NSAIDs）临床安全应用中亟待解决的不良反应。尽管柑橘纯总黄酮（pure total flavonoids from citrus, PTFC）已获批用于消化系统疾病的治疗，但其对小肠损伤的干预作用及具体分子机制仍有待阐明。本研究旨在探讨自噬（autophagy）在非甾体抗炎药（双氯芬酸，diclofenac）诱导的体内外肠损伤机制中的潜在作用，并阐明柑橘纯总黄酮对非甾体抗炎药诱导小肠损伤的保护效应。实时荧光定量聚合酶链反应（qRT-PCR）、蛋白质免疫印迹（western blotting）及免疫组织化学（immunohistochemistry）结果显示，与对照组相比，非甾体抗炎药诱导小肠损伤模型大鼠及双氯芬酸处理的IEC-6细胞中，自噬相关5（Atg5）、微管相关蛋白1轻链3II型（LC3-II）以及紧密连接（TJ）蛋白ZO-1、闭合蛋白-1（claudin-1）和咬合蛋白（occludin）的表达水平均显著降低。在柑橘纯总黄酮干预组中，Atg5与LC3-II的表达水平、紧密连接蛋白表达量以及LC3-II/LC3-I比值均显著升高。此外，本研究通过蛋白质免疫印迹法分析了柑橘纯总黄酮在体内外促进自噬的分子机制。与对照组相比，肠损伤模型大鼠体内磷酸化磷脂酰肌醇3-激酶（p-PI3K）与磷酸化蛋白激酶B（p-Akt）的表达水平显著升高，而柑橘纯总黄酮干预组中二者表达水平则显著降低。经PI3K抑制剂处理后，IEC-6细胞的自噬水平显著上调，而联合柑橘纯总黄酮干预后该上调效应更为显著，提示柑橘纯总黄酮通过调控PI3K/Akt信号通路促进细胞自噬。综上，柑橘纯总黄酮可通过促进细胞自噬维持肠屏障完整性，为非甾体抗炎药诱导小肠损伤的潜在治疗候选药物提供了实验依据。