Abstract：
Recent advancements in canine intestinal organoid research have paved the way for the development of enhanced in vitro models, crucial for exploring intestinal physiology and diseases. Despite these strides, there is a notable gap in creating specific in vitro models that focus on intestinal inflammation. Our study aims to bridge this gap by investigating the impact of proinflammatory cytokines on canine intestinal epithelial cells (IECs) within the context of organoid models. Canine intestinal organoids were treated with proinflammatory cytokines TNF-α, IFN-γ, and IL-1β. The expression of stem cell markers Lgr5, Sox9, Hopx, and Olfm4 was evaluated through RT-qPCR, while membrane integrity was assessed using immunofluorescence staining for tight junction proteins and transport assays for permeability. IFN-γ significantly decreased Lgr5 expression, a key intestinal stem cell marker, at both 24 and 48 h post-treatment (p=0.030 and p=0.002, respectively). Conversely, TNF-α increased Olfm4 expression during the same intervals (p=0.018 and p=0.011, respectively). A reduction in EdU-positive cells, indicative of decreased cell proliferation, was observed following IFN-γ treatment. Additionally, a decrease in tight junction proteins E-cadherin and ZO-1 (p<0.001 and p=0.003, respectively) and increased permeability in IECs (p=0.012) were noted, particularly following treatment with IFN-γ. The study highlights the profound impact of proinflammatory cytokines on canine IECs, influencing both stem cell dynamics and membrane integrity. These insights shed light on the intricate cellular processes underlying inflammation in the gut and open avenues for more in-depth research into the long-term effects of inflammation on intestinal health.
摘要：
犬肠道类器官研究的最新进展为增强的体外模型的开发铺平了道路。对于探索肠道生理和疾病至关重要。尽管取得了这些进展,在创建专注于肠道炎症的特定体外模型方面存在显著差距.我们的研究旨在通过在类器官模型的背景下研究促炎细胞因子对犬肠上皮细胞（IECs）的影响来弥合这一差距。犬肠道类器官用促炎细胞因子TNF-α治疗，IFN-γ，和IL-1β。干细胞标志物Lgr5、Sox9、Hopx的表达,并通过RT-qPCR评估Olfm4，而使用免疫荧光染色的紧密连接蛋白和转运测定的通透性来评估膜的完整性。IFN-γ显著降低Lgr5表达，一个关键的肠道干细胞标记，在治疗后24和48小时（分别为p=0.030和p=0.002）。相反，TNF-α在相同的时间间隔内增加Olfm4的表达（分别为p=0.018和p=0.011）。EdU阳性细胞减少,指示细胞增殖减少，在IFN-γ处理后观察到。此外,注意到IEC中紧密连接蛋白E-cadherin和ZO-1的减少（分别为p<0.001和p=0.003）和通透性增加（p=0.012），特别是在用IFN-γ治疗之后。该研究强调了促炎细胞因子对犬IEC的深远影响，影响干细胞动力学和膜完整性。这些见解揭示了肠道炎症背后复杂的细胞过程，并为更深入地研究炎症对肠道健康的长期影响开辟了途径。
