Background Intestinal stem cells (ISCs) play a pivotal role in maintaining intestinal homeostasis and facilitating the restoration of intestinal mucosal barrier integrity. Glutamine (Gln) is a crucial energy substrate in the intestine, promoting the proliferation of ISCs and mitigating damage to the intestinal mucosal barrier after burn injury. However, the underlying mechanism has not yet been fully elucidated. The objective of this study was to explore the mechanism by which Gln facilitates the proliferation of ISCs. Methods A mouse burn model was established to investigate the impact of Gln on intestinal function. Subsequently, crypts were isolated, and changes in TP53-induced glycolysis and apoptosis regulator (TIGAR) expression were assessed using real-time quantitative polymerase chain reaction (RT-qPCR), western blotting, immunohistochemistry, and immunofluorescence. The effects of TIGAR on cell proliferation were validated through CCK-8, EdU, and clonogenicity assays. Furthermore, the effect of TIGAR on Yes-associated protein (YAP) nuclear translocation and ferroptosis was examined by western blotting and immunofluorescence staining. Finally, dot blot analysis and methylation-specific PCR were performed to evaluate the effect of Gln on TIGAR promoter methylation. Results The mRNA and protein levels of TIGAR decreased after burn injury, and supplementation with Gln increased the expression of TIGAR. TIGAR accelerates the nuclear translocation of YAP, thereby increasing the proliferation of ISCs. Concurrently, TIGAR promotes the synthesis of nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione to suppress ferroptosis in ISCs. Subsequent investigations demonstrated that Gln inhibits TIGAR promoter methylation by increasing the expression of the demethylase ten-eleven translocation. This change increased TIGAR transcription, increased NADPH synthesis, and reduced oxidative stress, thereby facilitating the restoration of intestinal mucosal barrier integrity post-burn injury. Conclusions Our data confirmed the inhibitory effect of Gln on TIGAR promoter methylation, which facilitates YAP translocation into the nucleus and suppresses ferroptosis, ultimately promoting the proliferation of ISCs.

中文翻译：

---

谷氨酰胺通过抑制烧伤小鼠中TP53诱导的糖酵解和凋亡调节启动子甲基化促进肠道干细胞增殖


背景肠干细胞（ISC）在维持肠道稳态和促进肠粘膜屏障完整性恢复方面发挥着关键作用。谷氨酰胺 (Gln) 是肠道中重要的能量底物，可促进 ISC 增殖并减轻烧伤后肠粘膜屏障的损伤。然而，其根本机制尚未完全阐明。本研究的目的是探讨 Gln 促进 ISC 增殖的机制。方法建立小鼠烧伤模型，探讨Gln对肠道功能的影响。随后，分离隐窝，并使用实时定量聚合酶链反应 (RT-qPCR)、蛋白质印迹、免疫组织化学和免疫荧光评估 TP53 诱导的糖酵解和凋亡调节因子 (TIGAR) 表达的变化。 TIGAR 对细胞增殖的影响通过 CCK-8、EdU 和克隆形成分析进行了验证。此外，通过蛋白质印迹和免疫荧光染色检查了 TIGAR 对 Yes 相关蛋白 (YAP) 核易位和铁死亡的影响。最后，进行斑点印迹分析和甲基化特异性PCR来评估Gln对TIGAR启动子甲基化的影响。结果烧伤后TIGAR mRNA和蛋白水平下降，补充Gln可增加TIGAR表达。 TIGAR 加速 YAP 的核转位，从而增加 ISC 的增殖。同时，TIGAR 促进烟酰胺腺嘌呤二核苷酸磷酸 (NADPH) 和谷胱甘肽的合成，以抑制 ISC 中的铁死亡。 随后的研究表明，Gln 通过增加去甲基化酶 10-11 易位的表达来抑制 TIGAR 启动子甲基化。这种变化增加了 TIGAR 转录，增加了 NADPH 合成，并减少了氧化应激，从而促进烧伤后肠粘膜屏障完整性的恢复。结论 我们的数据证实了 Gln 对 TIGAR 启动子甲基化的抑制作用，促进 YAP 转位到细胞核并抑制铁死亡，最终促进 ISC 的增殖。