ObjectiveThis paper aims to explore the role and mechanism of the gut microbe metabolite trimethylamine oxide (TMAO) in regulating ferroptosis in colon cells through the reactive oxygen species (ROS)/c-Jun N-terminal kinase (JNK)/glutathione peroxidase 4 (GPX4) signaling pathway in slow transit constipation (STC).MethodsA total of 50 rats were selected and randomly assigned to the blank group, the STC group, the TMAO group, the SP600125 (JNK inhibitor) group, and the TMAO+SP600125 group, with 10 rats in each group. Except for the blank group, all other groups were orally administered 10 mg/kg/d of compound difenofibrate suspension via gavage to establish the STC model. After successful modeling, the TMAO group received daily intraperitoneal injections of TMAO (100 mg/kg); the SP600125 group received intraperitoneal injection of SP600125 (15 mg/kg) twice a week; the TMAO+SP600125 group received daily intraperitoneal injections of TMAO (100 mg/kg) along with SP600125 (15 mg/kg) twice a week. All treatments lasted for 3 weeks. Each group was given a single gavage of activated carbon suspension to observe the time required for the first black feces excretion and the small intestinal propulsion rate. H-E staining was used to observe pathological changes in colon tissues. Colorimetric assays were employed to detect the levels of superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), and ferrous ions (Fe²⁺) in colon tissues. Immunofluorescence assay was used to detect the expression of ROC in colon tissue. Immunohistochemical staining was used to detect the expression of JNK protein in colon tissues. Western blotting was used to detect the expression levels of ferroptosis-related proteins.ResultsCompared with the blank group, the STC group has a longer time required for the first black feces excretion, along with significantly increased levels of MDA, Fe²⁺, ROS, and JNK protein expression (P<0.05), while the small intestine propulsion rate, SOD and GSH levels, as well as the expression levels of GPX4 and FTH1 proteins are significantly reduced (P<0.05). After modeling, colonic mucosal tissue in rats shows bleeding, morphological structural damage, epithelial cell degeneration, and necrosis, and inflammatory cell infiltration. TMAO can cause more significant changes in the above indicators of STC rats, while SP600125 can reverse the effect of TMAO on the above indicators of STC rats.ConclusionTMAO can exacerbate the condition of STC, and its mechanism of action may be to induce ferroptosis in colon cells by regulating the ROS/JNK/GPX4 signaling pathway.