Naringenin from Citrus aurantium L. alleviates loperamide-induced constipation by inhibiting excessive autophagy-mediated apoptosis in interstitial cells of Cajal via the AKT1 signaling pathway.

Abstract

Constipation is a prevalent gastrointestinal disorder frequently associated with the dysfunction and loss of Interstitial Cells of Cajal (ICCs). Zhishi, the dried young fruit of Citrus aurantium L. (CAL), is a traditional medicine used for constipation; however, its specific bioactive constituents and mechanisms of action remain unclear. This study aimed to investigate the therapeutic effects of CAL flavonoid extract and its primary components on constipation. CAL flavonoid extract significantly alleviated symptoms in a loperamide-induced mouse model of constipation by improving fecal parameters, accelerating gastrointestinal transit, ameliorating colonic pathological damage and increasing intestinal barrier integrity. These protective effects were linked to the inhibition of ICCs apoptosis. Combined LC-MS/MS analysis and network pharmacology identified naringenin as a key bioactive flavonoid and AKT1 as its primary molecular target. This interaction was supported by molecular docking, which showed a strong binding affinity between naringenin and AKT1. In vivo experiments confirmed that naringenin administration recapitulated the effects of the CAL flavonoid extract, protecting ICCs from excessive autophagy, apoptosis, and oxidative stress. Further in vitro studies using primary ICCs isolated from constipated mice demonstrated that naringenin enhanced cell viability, inhibited apoptosis, and mitigated mitochondrial dysfunction and oxidative stress. Crucially, these protective effects of naringenin were abolished when AKT1 expression was silenced using siRNA, confirming that naringenin functions through the AKT1 pathway. In conclusion, this study demonstrates that naringenin alleviates constipation by targeting the AKT1 signaling pathway to inhibit excessive autophagy-induced apoptosis and oxidative stress in ICCs, providing a scientific basis for the traditional use of CAL.