Runchangningshen paste activates NLRP6 inflammasome-mediated autophagy to stimulate colonic mucin-2 secretion and modulates mucosal microbiota in functional constipation.

Background: Runchangningshen paste (RCNSP) is a paste made of four medicinal and edible homologous Chinese medicine mixed with honey. It is known for its ability to nourish yin and blood as well as to loosen the bowel to relieve constipation. The pathophysiology of functional constipation (FC) is associated with a reduction in mucin-2 (MUC2) secretion and microbial dysbiosis.

Aim: To investigate the underlying mechanism of RCNSP against FC through MUC2 and the gut mucosal microbiota.

Methods: Ultra-performance liquid chromatography tandem mass spectrometry characterized RCNSP composition to elucidate the material basis of action. FC model was induced loperamide gavage (16 mg/kg) twice daily for 7 days. Applying defecation function and gastrointestinal motility to assess constipation severity. Hematoxylin and eosin and Alcian blue-periodic acid-schiff staining analyzed colonic mucosal morphology. Transmission electron microscope was used to observe the ultrastructure of goblet cells (GCs). Immunofluorescence colocalization, quantitative PCR, and western blot assessed the impact of RCNSP on gene and protein expression within the NLRP6/autophagy pathway. 16S rDNA was employed to sequence the gut mucosal microbiota.

Results: RCNSP contained 12 components with potential laxative effects. It enhanced defecation function, accelerated gastrointestinal motility, and maintained colonic mucosal integrity. RCNSP treatment significantly increased GC abundance and MUC2 production while preserving GC ultrastructure. At the molecular level, RCNSP enhanced the colocalized expression of key regulatory proteins and modulated mRNA and protein expressions in the NLRP6/autophagy pathway. Through 16S rDNA sequencing analysis, RCNSP significantly altered the mucosal microbiota composition. Specifically, it increased beneficial bacterial strains while reducing harmful ones. Simultaneously, RCNSP reduced butyrate-producing bacteria like , , , and and decreased hydrogen sulfide-producing species, such as . It also reduced bile acid-inhibiting species, such as and while increasing bile acid-producing species, such as .

Conclusion: Our findings suggested that RCNSP ameliorated constipation through a dual mechanism: It stimulated colonic MUC2 secretion by activating NLRP6 inflammasome-mediated autophagy and modulated the composition of the mucosal microbiota.