Herb Pair of <scp> <i>Aurantii Fructus Immaturus–Atractylodis Macrocephalae Rhizoma</i> </scp> Alleviates Slow‐Transit Constipation in Rats by Regulating Mitophagy and Modulating Gut Microbiota

BACKGROUND: Slow transit constipation (STC) is a prevalent functional gastrointestinal disorder that significantly affects people's quality of life. Aurantii Fructus Immaturus (Zhishi, ZS) and Atractylodis Macrocephalae Rhizoma (Baizhu, BZ) are a classic herbal pair in traditional Chinese medicine (TCM) for regulating spleen-stomach function, respectively recognized for their properties of "replenishing qi and fortifying the spleen" and "dispersing stagnation and promoting digestion". This study aimed to elucidate the mechanisms by which ZSBZ ameliorates STC through regulating mitophagy in ICCs and restructuring gut microbiota composition. METHODS: concentration and mitochondrial membrane potential (MMP) in cells isolated from colonic tissues. Mitophagy-related protein and mRNA expression were analyzed by Western blotting and reverse transcription quantitative polymerase chain reaction (RT-qPCR). Apoptosis in rat colonic tissues was detected using TUNEL assay. Gut microbiota composition was analyzed by 16S rRNA gene sequencing, and its metabolic functional potential was predicted. RESULTS: concentration in cells isolated from colonic tissues. Mechanistically, ZSBZ attenuated excessive mitophagy in colonic ICCs by downregulating autophagy-related genes and proteins, restoring p62 expression, and suppressing the elevated mRNA and protein expression of the mitophagy-related markers PINK1 and Parkin. In addition, 16S rRNA gene sequencing demonstrated that ZSBZ reshaped the gut microbiota in STC rats, characterized by a decreased abundance of Proteobacteria and an increased abundance of Bacteroidota. KEGG functional prediction further indicated that ZSBZ regulated mitochondrial metabolism-related pathways, possibly contributing to the amelioration of colonic dysmotility. CONCLUSION: ZSBZ significantly alleviated constipation symptoms in STC rats. The mechanism may involve inhibition of excessive mitophagy in ICCs and restructuring of gut microbiota composition. This study provides compelling theoretical support for ZSBZ as a therapeutic strategy against STC and offers novel insights into intestinal motility restoration in humans.