Couplet Medicines of Aurantii Fructus Immaturus and Atractylodis Macrocephalae Rhizoma Ameliorates Colonic Motility Dysfunction in Slow Transit Constipation Mice via AMPK/Drp1 Pathway

OBJECTIVE To investigate the effects of couplet medicines of Aurantii Fructus Immaturus and Atractylodis Macrocephalae Rhizoma on colonic motility dysfunction and mitochondrial dynamics-related proteins in slow transit constipation(STC) mice, and to explore their potential therapeutic mechanism.

METHODS Fifty-six male C57BL/6J mice were randomly divided into seven groups(n=8 per group): normal group, model group, natural recovery group, Aurantii Fructus Immaturus group, Atractylodis Macrocephalae Rhizoma group, Aurantii Fructus Immaturus-Atractylodis Macrocephalae Rhizoma group and mosapride group. The STC model was established by intragastric loperamide administration for 14 d. Drug treatments were administered for 14 d, while the normal group, model group, and natural recovery group received saline. Fecal pellet count, water content, and intestinal propulsion rate were measured. Colon morphology was assessed via hematoxylin-eosin and Alcian blue-periodic acid-Schiff staining. Ultrastructural changes were observed using transmission electron microscopy. Real-time PCR quantified mRNA levels of SUMO1, SUMO2, SUMO3, OPA1, MFN1, MFN2, Drp1, FIS1, and MFF. Western blotting analyzed protein expression of OPA1, MFN1, MFN2, Drp1, p-Drp1, FIS1, MFF, PGC-1α, AMPK, and p-AMPK. Moreover, co-immunoprecipitation(Co-IP) was used to detect the binding of SUMO1 and SUMO2/3 to Drp1.

RESULTS Compared with the normal group, the model and natural recovery groups exhibited significantly reduced fecal output, water content, and intestinal propulsion rate(P<0.05 or P<0.01). Pathological staining revealed thinning of the muscle layer and acidic mucin layer, decreased goblet cells, disorganized villi, and inflammatory infiltration. TEM showed mitochondrial swelling, cristae disruption, matrix degradation, and increased autophagosomes and autolysosomes. The model and natural recovery groups had downregulated SUMO1, SUMO2, SUMO3, OPA1, MFN1, and MFN2 mRNA(P<0.01) but upregulated Drp1, FIS1, and MFF(P<0.01). Protein analysis confirmed elevated FIS1, MFF, AMPK, p-AMPK, Drp1, and p-Drp1(P<0.01) and reduced MFN1, MFN2, OPA1, and PGC-1α(P<0.01). Compared with the model group, the Aurantii Fructus Immaturus-Atractylodis Macrocephalae Rhizoma group reversed the trend of the above indicators. Co-immunoprecipitation showed that the compatibility of Aurantii Fructus Immaturus-Atractylodis Macrocephalae Rhizoma group may exert its therapeutic effect by reducing the SUMO1 modification of Drp1 and increasing its SUMO2/3 modification.

CONCLUSION The couplet medicines of Aurantii Fructus Immaturus and Atractylodis Macrocephalae Rhizoma alleviate STC by improving fecal hydration, intestinal motility, and colonic pathology, and ultrastructure, potentially via inhibiting overactivation of the AMPK/Drp1 pathway to restore mitochondrial dynamics homeostasis.