Xuanhuang Runtong Tablets Relieve Slow Transit Constipation in Mice by Regulating TLR5/IL-17A Signaling Mediated by Gut Microbes

Abstract

This study aims to investigate the regulation effects of Xuanhuang Runtong tablets (XHRTs) on intestinal microbes and inflammatory signal toll receptor 5 (TLR5)/interleukin-17A (IL-17A) in STC mice. First, high-performance liquid chromatography (HPLC) was used to verify the composition of XHRT and quality control. Then, the defecation ability of STC mice was evaluated by measuring fecal water content and intestinal transit function. The pathological examination of colonic mucosa was observed by Alcian Blue and periodic acid Schiff (AB-PAS) staining. 16S ribosomal DNA (16S rDNA) genes were sequenced to detect the fecal microbiota. Western blotting, immunofluorescence, and real-time fluorescence quantitative PCR (qRT-PCR) were applied to detect the expression of aquaporin 3 (AQP3), connexin 43 (Cx43), TLR5, and IL-17A. The defecation function of the STC mice was significantly decreased. The amount of mucus secretion and the thickness of the colonic mucus layer were decreased, and the number of microbial species in the intestinal wall, such as Firmicutes/Bacteroidetes, anaerobic bacteria, and Alistipes, were also decreased. In addition, the expression of AQP3 and Cx43 was disordered, and the inflammatory factorsTLR5 and IL-17A were activated in the colon. The changes in the above indicators were significantly reversed by XHRT. This study demonstrates that XHRT provides a new strategy for the treatment of slow transit constipation by regulating the activation of the intestinal inflammatory signal TLR5/IL-17A mediated by gut microbes.

Figure 1

Fingerprints of XHRT and reference substances. (2. Echinacoside, 4. Naringin, 5. Neohesperidin, 9. Aloin, 15. Aloe-Emodin).

Figure 2

XHRT improved intestinal transit in mice with slow transit constipation induced by loperamide hydrochloride. (a) Comparison of fecal dry weight on day 0 and 7 days after loperamide induction. (b) Changes in body weight of mice at 0 d, 7 d, and 14 d. (c) The effect of XHRT on the time to discharge the first black stool in STC mice, (d) the number of black stools discharged within six hours, and (e) the water content of stool in 6 hours. (f–h) The effect of XHRT on the small intestine transport of STC mice was evaluated by the ink intestinal propulsion exercise. (f) The total length of the small intestine, (g) the length of ink advancing in the small intestine, and (h) the small intestine transport rate. STC: slow transit constipation; XHRT: Xuanhuang Runtong tablet; values are expressed as the mean ± SD, ^∗∗P  < 0.01 vs. control group; ^#P  < 0.05, ^##P  < 0.01 vs. STC model group.

Figure 3

XHRT improved the morphological and structural changes of the colonic mucosa of STC mice treated with loperamide hydrochloride. AB-PAS staining was used to assess the pathogenicity of mouse colon mucosal tissue (a) magnification, 100 times; after XHRT treatment, the thickness of colonic mucosa changed (b) and mucous cell content (c) in STC mice changed; STC: slow transit constipation; XHRT: Xuanhuang Runtong tablet; AB-PAS: Alcian Blue and periodic acid Schiff. Values were expressed as the mean ± SD, ^∗∗P  < 0.01 vs. control group; ^#P  < 0.05, ^##P  < 0.01 vs. STC model group.

Figure 4

XHRT regulated the structure of the intestinal microbial community in STC mice induced by loperamide hydrochloride. (a) Venn diagram representing the number of OTUs identified in the gut microbiome of STC mice treated with XHRT; mice treated with different doses of XHRT (4.056, 2.028, and 1.014 g/kg) were designated as XHRT-H, XHRT-M, and XHRT-L groups and 16S rDNA microbial gene sequencing. (b) The sparse curve was established based on the Chao1 index of 16S rDNA microbial analysis. (c) PLS-DA analysis based on the full OTUs level. XHRT-H, -M, and -L: Xuanhuang Runtong tablet high dosage, medium dosage, and low dosage. PLS-DA: partial least squares regression analysis method.

Figure 5

Identification of alpha diversity and beta diversity of the microbiome in STC mice treated with XHRT. (a–c) Microbial abundance in the group was compared based on Chao1 (a), observed species (b), and Shannon (c) indices. (d) The diversity of the microbiome between groups by UPGMA cluster analysis was evaluated. STC: slow transit constipation; XHRT: Xuanhuang Runtong tablet. UPGMA: unweighted pair group method. Values are expressed as the mean ± SD, ^∗∗P < 0.01 vs. control group; ^#P  < 0.05, ^##P  < 0.01 vs. STC model group.

Figure 6

In STC mice treated with XHRT, the relative abundance and family-level composition of the phylum (a), order (b), and genus (c) were assessed. The significance of differences between multiple groups at the phylum level through species differences was analyzed (d). Significant comparison of the difference in bacteria between the two groups and classification levels of intestinal microbes at the genus level (e–g).

Figure 7

XHRT regulated the expression of colonic aquaporin AQP3 and basal-related protein Cx43 in the colonic mucosa of STC mice induced by loperamide hydrochloride. Western blotting was used to detect and analyze the expression of AQP3 and Cx43 in mouse colon tissue (a). Quantity One software analyzed protein gray bands and counted relative protein expression (b). STC: slow transit constipation; XHRT: Xuanhuang Runtong tablet; AQP3: aquaporin 3; Cx43, gap junction protein. Values were expressed as the mean ± SD, ^∗P  < 0.05, ^∗∗P  < 0.01 vs. control group; ^#P  < 0.05, ^##P  < 0.01 vs. STC model group.

Figure 8

XHRT regulated the expression of TLR5/IL-17A related to the intestinal mucosal immune function in the STC mice induced by loperamide hydrochloride (a).Immunofluorescence method used antibodies targeting TLR5 and IL-17A proteins, observed the protein localization and fluorescence intensity under a laser confocal microscope, and used the average fluorescence intensity of the protein to reflect the relative expression level of each protein (b) (400×). (c) The relative expression of TLR5 mRNA and IL-17A mRNA in colon tissue by real-time fluorescent quantitative PCR with GAPDH as an internal reference was detected. STC: slow transit constipation; XHRT: Xuanhuang Runtong tablet; TLR5: toll receptor 5; IL-17A: interleukin-17A; GAPDH: glyceraldehyde 3-phosphate dehydrogenase. Values were expressed as the mean ± SD, ^∗∗P  < 0.01 vs. control group; ^#P  < 0.05, ^##P  < 0.01 vs. STC model group.