Naringenin attenuates slow-transit constipation by regulating the AMPK/mTOR/ULK1 signaling pathway: In vivo and in vitro studies.

Slow-transit constipation (STC) is a widespread functional gastrointestinal condition distinguished by decreased colonic motility as an essential clinical characteristic. The excessive autophagy of interstitial cells of Cajal (ICCs) causes phenotypic changes and functional abnormalities, which are important in colonic dysmotility. Naringenin (NAR) has been shown to regulate gastrointestinal motility disorders. The present study aimed to elucidate the regulatory role of NAR in autophagy in STC and its underlying mechanism. Loperamide (Lop)-induced ICCs from STC mice and L-Glutamic acid (GA)-induced ICCs from rats were utilized as in vivo and in vitro models. The experiments involved various techniques, such as flow cytometry, Western blotting, CCK-8 assay, siRNA transfection, real-time PCR (RT-qPCR), co-immunoprecipitation, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), histological analysis, transmission electron microscopy (TEM), and immunohistochemistry (IHC). Our results indicated that NAR improved the survival and apoptosis of ICCs after GA by inhibiting autophagy through the partial suppression of the AMPK/mTOR/ULK1 signaling pathway Western blot analysis revealed that NAR treatment significantly reduced phosphorylation ratios of key autophagy regulators: pT172-AMPK/AMPK, pS2448-mTOR/mTOR, and pS757-ULK1/ULK1 in ICCs after GA. Critically, the level of pS757-ULK1 demonstrated a strong mechanistic association with autophagic initiation and modulation. NAR inhibits the autophagic degradation of pS757-ULK1 by weakening the interactions between pS757-ULK1 and the selective autophagy receptor genes NDP52 or OPTN. Further research revealed that NAR significantly increased fecal moisture content and small intestinal propulsion rate in constipated mice, while elevating serum excitatory neurotransmitters (GAS, 5-HT, MTL, SP) and reducing inhibitory neurotransmitters (VIP, SS). Additionally, it ameliorated pathological damage in Lop-induced STC mice and upregulated ANO1 and c-Kit expression in colonic tissues. Its molecular mechanism is consistent with the in vitro results. In summary, NAR inhibits excessive autophagy in ICC through the AMPK/mTOR/ULK1 pathway, thereby improving STC colonic dysmotility and underscoring its promise as a therapeutic option for STC.