Zhizhu decoction alleviates slow transit constipation by regulating aryl hydrocarbon receptor through gut microbiota

Abstract

Context: Slow transit constipation (STC), the most common type of constipation, seriously affects the life of patients. Zhizhu decoction (ZZD), a traditional Chinese medicine compound, has is effective against functional constipation, but the mechanism is still unclear.

Objective: This research explores the mechanism of ZZD on STC from the perspective of metabolomics and gut microbiota.

Materials and methods: Fifty-four C57BL/6 mice were randomly divided into six groups (n = 9): control (control); STC (model); positive control (positive); low-dose (5 g/kg; L-ZZD), medium-dose (10 g/kg; M-ZZD), and high-dose (20 g/kg; H-ZZD) ZZD treatment. Following treatment of mice with ZZD for two weeks, the changes in intestinal motility, colon histology, intestinal neurotransmitters, and aryl hydrocarbon receptor (AHR) pathway determined the effects of ZZD on the pathophysiology of STC. LC-MS targeting serum metabolomics was used to analyze the regulation of ZZD on neurotransmitters, and 16S rRNA high-throughput sequencing was used to detect the regulation of the gut microbiome.

Results: ZZD had the highest content of naringin (6348.1 mg/L), and could significantly increase the 24 h defecations (1.10- to 1.42-fold), fecal moisture (1.14-fold) and intestinal transport rate (1.28-fold) of STC mice, increased the thickness of the mucosal and muscular tissue (1.18- to 2.16-fold) and regulated the neurotransmitters in the colon of STC mice. Moreover, ZZD significantly activated the AHR signaling pathway, and also affected the composition of gut microbiota in STC mice.

Discussion and conclusions: The beneficial effect and the possible mechanism of ZZD on STC could provide a theoretical basis for the broader clinical application of ZZD.

Keywords: 16S rRNA sequencing; Intestinal neurotransmitters; functional constipation; intestinal motility; metabolomics.

Figure 1.

Effects of ZZD on intestinal motility of STC mice. (A) 24 h defecation weight of STC mice at the various indicated time points. (B) The fecal water content of mice. (C) The intestinal transport rate of mice. Data were shown as mean ± SD, n = 9. **p < 0.01, ***p < 0.001, compared with the control group; ^##p < 0.01, ^###p < 0.001, compared with the STC model group. ZZD: Zhizhu Decoction; L-ZZD: low-dose ZZD treatment group; M-ZZD: medium-dose ZZD treatment group; H-ZZD: high-dose ZZD treatment group.

Figure 2.

Effects of ZZD on histological changes in STC model mice. (A) Representative images showing hematoxylin and eosin of colon tissues in mice (magnification, ×200 and/or ×400). (B) Tissue thickness of mucosal and muscular layers. Data were shown as mean ± SD, n = 9. ***p < 0.001, compared with the control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, compared with the STC model group. ZZD: Zhizhu Decoction; L-ZZD: low-dose ZZD treatment group; M-ZZD: medium-dose ZZD treatment group; H-ZZD: high-dose ZZD treatment group.

Figure 3.

Effects of ZZD on neurotransmitters in the colon of STC mice. (A) The relative content of Ach in the colon of STC mice. (B) The relative content of SP. (C) The relative content of 5-HT. (D) The relative content of VIP. Data were shown as mean ± SD, n = 9. **p < 0.01, ***p < 0.001, compared with the control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, compared with the model group. ZZD: Zhizhu Decoction; L-ZZD: low-dose ZZD treatment group; M-ZZD: medium-dose ZZD treatment group; H-ZZD: high-dose ZZD treatment group.

Figure 4.

Overall tree diagram and multivariate statistical analysis of differences in neurotransmitters among the group. (A) The overall tree diagram representing the similarity between samples is formed by calculating the distance matrix and clustering all samples by hierarchical clustering. (B) Principal Component Analysis (PCA) score plot of model group and control group, ZZD group and model group. (C) Partial Least Squares-Discriminant Analysis (PLS-DA) score plot of model group and control group, ZZD group and model group in the multivariate statistical analysis. (D) Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) score plot of model group and control group, ZZD group and model group. n = 6. M-ZZD: medium-dose ZZD treatment group.

Figure 5.

Effects of ZZD on AHR signaling pathway in the colon of STC mice. (A) The protein expression of AHR and CYP1A1 in the colon of STC mice. (B) Densitometry analysis of the intensity of the proteins. (C) The mRNA level of Ahr and Cyp1a1 in the colon of STC mice was detected by qPCR, and the relative expression was normalized to Actin expression. (D) Representative images showing immunofluorescence staining of AHR (green) and PGP9.5 (red). Magnification, ×400. (E) Mean fluorescence intensity of AHR in the colon. (F) Representative images showing immunofluorescence staining of CYP1A1 (green) and PGP9.5 (red). Magnification, 400×. Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue) in (D,F). (G) Mean fluorescence intensity of CYP1A1 in the colon. Data were shown as mean ± SD, n = 9. ***p < 0.001, compared with the control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, compared with the model group. M-ZZD: medium-dose ZZD treatment group.

Figure 6.

Diversity and composition of fecal microbiota among groups. (A) Two-dimensional ordering diagram of unweighted UniFrac based PCoA. (B) Unweighted UniFrac based UPGMA clustering tree. (C) OTU Venn diagram. n = 6. M-ZZD: medium-dose ZZD treatment group.

Figure 7.

The relative abundance of microflora in each group at the family or genus level. (A) The relative abundance at the family level. (B) The relative abundance at the genus level. Data were shown as mean ± SD, n = 6. M-ZZD: medium-dose ZZD treatment group.

Figure 8.

Functional analysis of gut microbiota. (A) Heatmap of correlation analysis between differentially expressed gut microbiota and serum metabolomics. (B)Two-dimensional ordering diagram of Bray-Curtis based PCoA for KEGG orthology (KO). (C) Differences in KEGG metabolic pathways between control and model group. (D) Differences in KEGG metabolic pathways between ZZD and control group. (E) Differences in KEGG metabolic pathways between ZZD and model group. KEGG: Kyoto Encyclopedia of Genes and Genomes. In C-E, the positive value of logFC [log2 (fold change)] on the horizontal axis represents up-regulation, the complex value represents down-regulation, and the ordinate represents different KEGG metabolic pathway labels. M-ZZD: medium-dose ZZD treatment group.