BackgroundSlow transit constipation (STC) is characterized by delayed intestinal transit and impaired defecation, with enteric nervous system (ENS) injury and inflammatory dysregulation contributing to its pathogenesis. Acteoside (ACT), a natural phenylethanoid glycoside, has anti-inflammatory and neuroprotective properties. However, its therapeutic effect and mechanism in STC remain unclear.MethodsThe effects of ACT were investigated in a loperamide (LOP)-induced STC-like mouse model. Intestinal motility, colonic morphology, and ENS integrity were evaluated by functional assays, hematoxylin and eosin (H&E) staining, and immunofluorescence. Transcriptomic analysis was performed to screen ACT-responsive inflammatory pathways. NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation and cytokine production were assessed by Western blotting, enzyme-linked immunosorbent assay (ELISA), and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Nlrp3-knockout (Nlrp3-KO) mice were used for genetic validation.ResultsACT shortened defecation latency, increased fecal water content and pellet number, and improved intestinal propulsion. ACT also alleviated colonic histopathological injury, restored muscular layer thickness, preserved HuC/D-positive myenteric neurons, and increased PGP9.5-positive nerve fiber density. Transcriptomic analysis identified NLRP3-related inflammatory signaling within broader high-dose ACT-responsive transcriptional changes. At the molecular level, high-dose ACT reduced the protein levels of NLRP3, cleaved-caspase-1, and cleaved-interleukin-1β (IL-1β) expression and reduced IL-1β, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) production. Nlrp3 deficiency partially reproduced ACT-mediated protection against STC-like phenotypes, inflammasome activation, and ENS injury, whereas ACT retained additional protective effects in Nlrp3-KO mice, suggesting that NLRP3-independent mechanisms may also contribute.ConclusionACT alleviates LOP-induced STC-like injury, and this protection is associated with attenuation of NLRP3 inflammasome activation and preservation of ENS integrity, while additional NLRP3-independent mechanisms may also contribute.