Abstract：
Nonsteroidal anti-inflammatory drugs (NSAIDs) possess anti-inflammatory, antipyretic, and analgesic properties and are among the most commonly used drugs. Although the cause of NSAID-induced gastric ulcers is well understood, the mechanism behind small intestinal ulcers remains elusive. In this study, we examined the mechanism through which indomethacin (IM), a prominent NSAID, induces small intestinal ulcers, both in vitro and in vivo. In IEC6 cells, a small intestinal epithelial cell line, IM treatment elevated levels of LC3-II and p62. These expression levels remained unaltered after treatment with chloroquine or bafilomycin, which are vacuolar ATPase (V-ATPase) inhibitors. IM treatment reduced the activity of cathepsin B, a lysosomal protein hydrolytic enzyme, and increased the lysosomal pH. There was a notable increase in subcellular colocalization of LC3 with Lamp2, a lysosome marker, post IM treatment. The increased lysosomal pH and decreased cathepsin B activity were reversed by pretreatment with rapamycin (Rapa) or glucose starvation, both of which stabilize V-ATPase assembly. To validate the in vitro findings in vivo, we established an IM-induced small intestine ulcer mouse model. In this model, we observed multiple ulcerations and heightened inflammation following IM administration. However, pretreatment with Rapa or fasting, which stabilize V-ATPase assembly, mitigated the IM-induced small intestinal ulcers in mice. Coimmunoprecipitation studies demonstrated that IM binds to V-ATPase in vitro and in vivo. These findings suggest that IM induces small intestinal injury through lysosomal dysfunction, likely due to the disassembly of lysosomal V-ATPase caused by direct binding. Moreover, Rapa or starvation can prevent this injury by stabilizing the assembly. SIGNIFICANCE STATEMENT: This study elucidates the largely unknown mechanisms behind small intestinal ulceration induced by indomethacin and reveals the involvement of lysosomal dysfunction via vacuolar ATPase disassembly. The significance lies in identifying potential preventative interventions, such as rapamycin treatment or glucose starvation, offering pivotal insights that extend beyond nonsteroidal anti-inflammatory drugs-induced ulcers to broader gastrointestinal pathologies and treatments, thereby providing a foundation for novel therapeutic strategies aimed at a wide array of gastrointestinal disorders.
摘要：
非甾体抗炎药（NSAIDs）具有抗炎作用，退烧药,和镇痛特性，是最常用的药物之一。尽管NSAID引起的胃溃疡的原因已广为人知，小肠溃疡背后的机制仍然难以捉摸。在这项研究中,我们研究了消炎痛(IM)，一种突出的NSAID,引起小肠溃疡，体外和体内IEC6细胞，小肠上皮细胞系，IM治疗升高了LC3-Ⅱ和p62的水平。用氯喹或巴菲霉素治疗后，这些表达水平保持不变，它们是液泡ATP酶(V-ATP酶)抑制剂。IM处理降低了组织蛋白酶B的活性，溶酶体蛋白水解酶，并增加溶酶体的pH值。LC3与溶酶体标记物Lamp2的亚细胞共定位显着增加，IM后处理。通过用雷帕霉素（Rapa）或葡萄糖饥饿预处理，可以逆转溶酶体pH升高和组织蛋白酶B活性降低。两者都稳定V-ATP酶组装。为了验证体内的体外发现，我们建立了IM诱导的小肠溃疡小鼠模型。在这个模型中,我们观察到IM给药后多处溃疡和炎症加剧.然而,用Rapa或禁食预处理，稳定V-ATP酶组装，减轻IM诱导的小鼠小肠溃疡。免疫共沉淀研究表明IM在体外和体内与V-ATPase结合。这些发现表明IM通过溶酶体功能障碍诱导小肠损伤。可能是由于直接结合引起的溶酶体V-ATPase的拆解。此外，Rapa或饥饿可以通过稳定组件来防止这种伤害。意义陈述这项研究阐明了吲哚美辛诱导的小肠溃疡背后的未知机制，并揭示了通过V-ATPase分解参与的溶酶体功能障碍。意义在于确定潜在的预防性干预措施，如雷帕霉素治疗或葡萄糖饥饿，提供超越NSAID引起的溃疡，更广泛的胃肠道病理和治疗的关键见解，从而为针对广泛的胃肠道疾病的新型治疗策略提供了基础。
