Ju Huang Jing Li Zhijun Geng Lixia Yin Minzhu Niu Qingqing Li Xinyue Liu Xinke Cheng Xiaofeng Zhang Xue Song Yueyue Wang Lian Wang Lugen Zuo Jianguo Hu ^*

• The first affiliated hospital of Bengbu medical university, Bengbu, China

Background: Crohn's disease (CD) patients have the pathological phenomenon of excessive apoptosis of intestinal epithelial cells (IECs), which leads to damage to the intestinal barrier structure and function and may participate in the progression of colitis. Resisting IEC apoptosis and protecting the intestinal barrier is the key to alleviating colitis. Natural plant monomers have been reported to have multiple pharmacological effects, especially with the potential to treat CD; in this study, we focused on Cynaroside (Cyn) to explore its effect on IEC apoptosis and observe its pharmacological effects on the intestinal barrier and colitis. Methods: We used the 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced CD-like colitis mice model as the research object. By observing the colitis symptoms, levels of inflammatory factors, etc., we evaluated the therapeutic effect of Cyn on CD-like colitis. Immunofluorescence and western blotting were used to detect the expression and localization of tight junction (TJ) proteins and analyze the structure of the intestinal barrier. FITC-dextran (FD4), TEER values and bacterial translocation were used to analyze the function of the intestinal barrier. Based on network pharmacology enrichment analysis, Cyn showed the effect of inhibiting cell apoptosis. Using the TNF-α-induced mice colonic organoids model and TNBS mice, we explored the effect and mechanism of Cyn inhibiting IECs apoptosis on intestinal barrier function and colitis. Results: Our findings demonstrate that Cyn significantly alleviates TNBS-induced colitis symptoms in mice, and lower levels of inflammatory factors compared to the model group. In addition, the Cyn intervention group showed significant improvements in the intestinal barrier structure and function. By integrating network pharmacology prediction analysis with our validation results obtained from animal models and colonic organoids, we demonstrated that Cyn significantly inhibits IECs apoptosis in mice. KEGG enrichment analysis and signaling pathway agonists (740Y-P) intervention experiments confirmed that Cyn can inhibit PI3K/AKT signaling activation. Conclusions: Cyn can inhibit IEC apoptosis by blocking PI3K/AKT signaling, which may be the key reason for its role in protecting the intestinal barrier and improving CD-like colitis. This study also proved that the Chinese medicine monomer Cyn holds promise as a potential therapeutic agent for the treatment of CD.