Pinostrobin targets the PI3K/AKT/CCL2 axis in intestinal epithelial cells to inhibit intestinal macrophage infiltration and alleviate dextran sulfate sodium-induced colitis in mice

ZHANG Keni; QIAO Tong; YIN Lin; HUANG Ju; GENG Zhijun; ZUO Lugen; HU Jianguo; LI Jing

doi:10.12122/j.issn.1673-4254.2025.10.16

Abstract：Objective To investigate the mechanism through which pinostrobin(PSB)alleviates dextran sulfate sodium(DSS)-induced colitis in mice.Methods C57BL/6 mice were randomized into control group,DSS model group,and PSB intervention(30,60,and 120 mg/kg)groups.Colitis severity of the mice was assessed by examining body weight changes,disease activity index(DAI),colon length,and histopathology.The expressions of tight junction proteins ZO-1 and claudin-1 in the colon tissues were examined using immunofluorescence staining,and macrophage infiltration and polarization were analyzed with flow cytometry.ELISA and RT-qPCR were used for detecting the expressions of inflammatory factors(TNF-α and IL-6)and chemokines(CCL2,CXCL10,and CX3CL1)in the colon tissues,and PI3K/AKT phosphorylation levels were analyzed with Western blotting.In cultured Caco-2 and RAW264.7 cells,the effect of PSB on CCL2-mediated macrophage migration was assessed using Transwell assay.Network pharmacology analysis was performed to predict the key pathways that mediate the therapeutic effect of PSB.Results In DSS-induced mouse models,PSB at 60 mg/kg optimally alleviated colitis,shown by reduced weight loss and DAI scores and increased colon length.PSB treatment significantly upregulated ZO-1 and claudin-1 expressions in the colon tissues,inhibited colonic macrophage infiltration,and promoted the shift of macrophage polarization from M1 to M2 type.In cultured intestinal epithelial cells,PSB significantly inhibited PI3K/AKT phosphorylation and suppressed chemokine CCL2 expression.PSB treatment obviously blocked CCL2-mediated macrophage migration of RAW264.7 cells,which could be reversed by exogenous CCL2.Network pharmacology analysis and rescue experiments confirmed PI3K/AKT and CCL2 signaling as the core targets of PSB.Conclusion PSB alleviates DSS-induced colitis in mice by targeting intestinal epithelial PI3K/AKT signaling,reducing CCL2 secretion,and blocking macrophage chemotaxis and migration,highlighting the potential of PSB as a novel natural compound for treatment of inflammatory bowel disease.