LC-HRMS of cultured retinal pigment epithelial cells treated with zinc and interleukin

Zinc deficiency and chronic low‑grade inflammation are prevalent conditions that compromise epithelial integrity and metabolic homeostasis, particularly in ageing populations. Here, we investigated the concentration-dependent effects of Zn treatment on a well-differentiated human epithelial cell model exposed to cytokine-induced inflammatory stress, combining metallomics, transcriptomics, and metabolomics. Exposure to isotopically enriched Zn (125-, 200-, 250-µM 68ZnSO4) increased intracellular Zn availability and promoted MT metal-loading, revealing a functional Zn window with 200 µM achieving optimal Zn-MT stoichiometry (Zn7.4Cu0.4MT). Zinc pre‑exposure also attenuated cytokine‑responsive inflammatory gene networks while maintaining epithelial differentiation markers, indicating a protective transcriptional program that counteracts inflammation‑induced de‑differentiation. Metabolomic profiling showed that Zn partially restored lipid species suppressed by inflammatory stress, including anti‑inflammatory fatty acid derivatives and selected sphingolipids (docosenamide and sphinganine), while modulating broader lipid classes in a dose‑dependent manner. Together, these multi-layered responses demonstrate that Zn modulates epithelial integrity through coordinated remodeling of metal homeostasis, inflammatory signaling, and lipid metabolism. These findings support the concept that Zn status influences epithelial resistance to inflammatory stress through MT‑mediated buffering and lipid pathway regulation, providing mechanistic insight relevant for nutritional strategies in populations at risk of Zn deficiency.锌缺乏与慢性低度炎症是两类高发病症，会破坏上皮完整性与代谢稳态，在老年人群中尤为显著。本研究结合金属组学（metallomics）、转录组学（transcriptomics）与代谢组学（metabolomics）技术，探究了锌（Zn）处理的浓度依赖性效应，实验对象为经细胞因子诱导炎性应激的高分化人上皮细胞模型。使用同位素富集锌（125、200、250μM 68ZnSO4）处理细胞后，细胞内锌的生物可利用度得以提升，同时促进了金属硫蛋白（metallothionein，MT）的金属负载；结果显示存在一个功能性锌浓度窗口，其中200μM剂量可实现最优的锌-金属硫蛋白化学计量比（Zn7.4Cu0.4MT）。锌预暴露还可削弱细胞因子介导的炎性基因网络表达，同时维持上皮细胞分化标志物水平，这表明存在一种保护性转录程序，可对抗炎性应激诱导的去分化过程。代谢组学分析显示，锌可部分逆转炎性应激所抑制的脂质组分，包括抗炎性脂肪酸衍生物以及特定鞘脂类（二十二碳烯酰胺与鞘氨醇），同时以浓度依赖性方式调控更广泛的脂质家族。综上，这些多层次的应答反应表明，锌可通过协同重塑金属稳态、炎性信号通路与脂质代谢，调控上皮细胞完整性。本研究结果支持“锌营养状态可通过金属硫蛋白介导的缓冲作用与脂质通路调控，影响上皮细胞对抗炎性应激的能力”这一观点，为锌缺乏高风险人群的营养干预策略提供了机制层面的理论依据。