Aurantii fructus immaturus (Rutaceae) flavonoid ameliorated constipation by regulating colonic microbiota and miRNA/mRNA network

<i>Aurantii fructus immaturus</i> flavonoid (AFIF) is the main constituent of <i>Aurantii fructus immaturus</i> (Rutaceae) (AFI), and is effective against constipation. This study explores the mechanism of AFIF against antibiotics-induced constipation (AC) in mice. Forty six-week-old female C57BL/6 mice were randomly divided into 4 groups (<i>n</i> = 10): control, control + AFIF, model, and model + AFIF groups. The AC model was established by antibiotics mixture for 8 days. Mice were gavaged daily with AFIF (0.1 mL/10 g, 3 g/mL) for 2 weeks. Hematoxylin and eosin (H&E) staining and periodic acid-Schiff (PAS) staining were used for histological analysis. The colonic microbiota was analyzed by 16sRNA sequencing. Transcriptome sequencing was used to detect miRNA and mRNA expression profiles. The results showed that AFIF treatment improved constipation in AC mice: increased fecal number, fecal wet weight, fecal water content, and intestinal propulsion rate; decreased average weight of individual feces. AFIF improved the colonic pathological injury and increased acetylcholine (ACH), gastrin (GAS), motilin (MTL), substance P (SP), and vasoactive intestinal peptide (VIP) levels. Moreover, AFIF might improve AC by regulating colonic microbiota and a "miRNA-mRNA" regulatory network related to cell junction and neuroactive function. This study also found the colonic microbiota at the genus level was connected to the expressions and target mRNA expressions (including Ccdc85b, Dlgap2, Elavl4, and Shisa6) of mmu-miR-5100 and mmu-miR-18b-5p. In conclusions, AFIF could improve AC via regulating colonic microbiota and a "miRNA-mRNA" regulatory network, which provide a theoretical basis for expanding its clinical application.