关键词: 甘草次酸, 猪肠道类器官, 氧化应激

Abstract:

【Background】Oxidative stress is a key factor compromising intestinal health and impeding the growth and development of pigs. Consequently, the identification and development of natural and effective antioxidant compounds are of great significance for enhancing animal health and production efficiency. 18β-Glycyrrhetinic acid (GA), a pentacyclic triterpenoid extracted from Glycyrrhiza spp., has been recognized for its antioxidant activity. 【Objective】This study aimed to establish an oxidative stress injury model based on porcine intestinal organoids to assess the antioxidant efficacy of GA and to explore its potential underlying mechanisms. The findings were intended to provide a theoretical foundation for the application of GA as a functional antioxidant additive in livestock and poultry nutrition. 【Method】Intestinal crypts were isolated and collected from the jejunal tissues of piglets and used to construct porcine intestinal organoids via a three-dimensional culture system. The structural and functional integrity of the organoids was confirmed by immunofluorescence staining for intestinal epithelial stem cell and differentiation markers, including LGR5, β-catenin, Ki67, PCNA, c-Myc, CDX2, and lysozyme, indicating the presence of typical intestinal epithelial lineages. After 48 hours of passaging, organoids were treated with hydrogen peroxide (H₂O₂) at concentrations of 0, 100, 250, 500, 750, 1 000, 1 500, and 2 000 μmol·L^-1 for three days to induce oxidative stress, respectively. The optimal H₂O₂ concentration for model establishment was determined through comprehensive evaluation of morphological parameters (budding rate, branching coefficient, and bud condition), cellular proliferation activity (EdU incorporation and Ki67 expression), intracellular reactive oxygen species (ROS) levels (detected by fluorescent probes), and expression of key genes in the Wnt/β-catenin signaling pathway (Lgr5, C-myc, Cyclin D1, β-catenin, PCNA, and AXIN2). Subsequently, GA at concentrations of 10, 50, and 100 μmol·L^-1 was applied to the oxidative stress model to assess its protective effect. The antioxidant capacity and underlying mechanism of GA were investigated by analyzing organoid morphology, proliferation activity, intracellular ROS accumulation, and expression of representative proteins associated with the Wnt/β-catenin signaling pathway (β-catenin, PCNA, LGR5, and CDX2). 【Result】After 48 hours of passaging, treatment of porcine intestinal organoids with 1 mmol·L^-1 H₂O₂ for three days successfully established a stable oxidative stress injury model. This was demonstrated by a significant decrease in the organoid budding rate and branching coefficient, accompanied by a marked reduction in the proportion of EdU-positive cells and Ki67 expression. Moreover, oxidative stress induced activation of the Wnt/β-catenin signaling pathway, as evidenced by significant upregulation of C-myc expression. Under normal culture conditions, GA at concentrations of 10, 50, and 100 μmol·L^-1 exerted no adverse effects on organoid growth. However, under oxidative stress conditions, 100 μmol·L^-1 GA exhibited the most pronounced protective effect, significantly enhancing the budding rate and branching coefficient, and promoting cell proliferation as indicated by increased EdU-positive cell proportion and elevated Ki67 expression. Additionally, GA treatment effectively reduced ROS accumulation and inhibited the activation of the Wnt/β-catenin pathway, demonstrated by the downregulation of β-catenin, PCNA, and LGR5 protein expression levels. 【Conclusion】In this study, a porcine intestinal organoid model of oxidative stress was successfully established. Based on this model, the protective role of GA against oxidative damage in organoids was confirmed, and its underlying mechanism was preliminarily elucidated. GA exerted notable antioxidant activity by improving organoid morphological development, promoting cellular proliferation, reducing intracellular ROS levels, and downregulating the Wnt/β-catenin signaling pathway. These findings laid a theoretical foundation for the functional development and practical application of GA in the nutritional regulation and health management of livestock and poultry.

Key words: 18β-glycyrrhetinic acid, porcine intestinal organoid, oxidative stress