This study aims to investigate the impact of total flavonoids extracted from Forsythia suspensa leaf on zebrafish ulcerative colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS).The FSLF was purified using D101 macroporous resin and its components were analyzed via UPLC-MS.Thirty-six wild zebrafish were divided into a control group,model group,low-dose FSLF group (10 μg/mL),medium-dose FSLF group (20 μg/mL),high-dose FSLF group (40 μg/mL),and dexamethasone group (50 μg/mL).ulcerative colitis was induced in all groups except for the blank group by anal injection of 160 mmol/L TNBS.The colonic histopathology and goblet cell number of zebrafish were observed using hematoxylin-eosin and alcian blue staining.The levels of reactive oxygen species (ROS) and superoxide dismutase (SOD) in zebrafish colon tissue were assessed using a detection kit.Fluorescence quantitative PCR detected the presence of MyD88 and TNF receptor associated factor 6 (TRAF6) in the colon tissue of zebrafish.Additionally,mRNA expression of nuclear factor-κB (NF-κB) p65,tumor necrosis factor-α (TNF-α),interleukin-1β (IL-1β),and interleukin-10 (IL-10) was also observed.The highest concentration of total flavonoids was found in the 30% ethanol-eluting fraction of FSLF,UPLC-MS analysis identified nine flavonoids,including rutin,isoquercetin,kaempferol-3-glucorhamnoside,hypericin,kaempferin-3-O-rutinoside,quercetin,Hesperidin,kaempferol and porphyrin.Compared to the model group,FSLF demonstrates an improvement in the colonic structure of zebrafish,as evidenced by an increase in goblet cell number (P<0.001),a decrease in ROS content (P<0.05),downregulation of MyD88/TRAF6/NF-κBp65,TNF-α,IL-1β mRNA expression levels,and an increase in SOD activity (P<0.01) and IL-10 mRNA expression levels (P<0.05).This study indicates that FSLF exerts a significant therapeutic impact on zebrafish ulcerative colitis,potentially through the modulation of inflammatory factors TNF-α and IL-1β,as well as the enhancement of IL-10 mRNA expression via regulation of the NF-κB signaling pathway.