Acteoside alleviates slow transit constipation with attenuation of NLRP3 inflammasome activation and preservation of enteric neurons.

BACKGROUND: Slow 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. METHODS: The 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. RESULTS: ACT 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. CONCLUSION: ACT 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.