Modified Zhizhu Pill improves the loperamide-induced slow transit constipation via gut microbiota and neurotransmitters in microbiota-gut-brain axis.

BACKGROUND: Slow-transmission constipation is a type of intractable constipation with unknown etiology and unclear pathogenesis. OBJECTIVE: The intention of this study was to evaluate the therapeutic effect and possible mechanism of Modified Zhizhu Pills on loperamide-induced slow transit constipation. METHODS: The effects of the Modified Zhizhu Pill were evaluated in a rat model of constipation induced by subcutaneous administration of loperamide. Fecal parameters (fecal count, fecal water content, and fecal hardness) were measured in constipated rats. The substance, target, and pathway basis of the Modified Zhizhu Pill on constipation was investigated using network pharmacology. The microflora in rats was determined. Serum neurotransmitters (acetylcholine and 5-hydroxytryptamine) were measured in rats and their relationship with the gut microbiota was assessed. RESULTS: Modified Zhizhu Pill increased the number of bowel movements and fecal water content, and decreased fecal hardness and transit time. Network pharmacological analysis showed that Modified Zhizhu Pill can target multiple constipation-related targets and pathways through multiple potential active ingredients. Modified Zhizhu Pill alleviated loperamide-induced microbiota dysbiosis. Modified Zhizhu Pill increased serum 5-hydroxytryptamine and acetylcholine. The increase in serum 5-hydroxytryptamine and acetylcholine was associated with rat gut microbiota. CONCLUSION: These results suggest that Modified Zhizhu Pill may increase intestinal motility and ultimately relieve constipation by improving microecological dysbiosis and neurotransmission.

MiR-26b-3p Promotes Intestinal Motility Disorder by Targeting FZD10 to Inhibit GSK3ß/ß-Catenin Signaling and Induce Enteric Glial Cell Apoptosis.

Enteric glial cells (EGCs) are the major component of the enteric nervous system and affect the pathophysiological process of intestinal motility dysfunction. MicroRNAs (miRNAs) play an important role in regulating gastrointestinal homeostasis. However, the mechanism of miRNA-mediated regulation of EGCs in intestinal dysmotility remains unclear. In this study, we investigated the effect of EGC apoptosis on intestinal dysmotility, and the effect of miR-26b-3p on EGC proliferation and apoptosis in vivo and in vitro. A loperamide hydrochloride (Lop)-induced constipated mouse model and an in vitro culture system of rat EGCs were established. The transcriptome was used to predict the differentially expressed gene miR-26b-3p and the target gene Frizzled 10 (FZD10), and their targeting binding relationship was verified by luciferase. EGCs were transfected with miR-26b-3p mimic or antagomir, and the FZD10 expression was down-regulated by siRNA. Immunofluorescence and flow cytometry were used to detect EGC apoptosis. MiR-26b-3p and FZD10 expressions were examined using quantitative real-time PCR (qRT-PCR). The CCK-8 assay was used to detect EGC proliferation. The protein levels were detected by Western blotting and enzyme-linked immunosorbent assay (ELISA). The results showed that miR-26b-3p was up-regulated in the Lop group, whereas FZD10 was down-regulated, and EGC apoptosis was increased in the colon of intestinal dysmotility mice. FZD10 down-regulation and miR-26b-3p mimic significantly increased glycogen synthase kinase-3ß phosphorylation (p-GSK3ß) levels, decreased ß-catenin expression, and promoted EGC apoptosis. MiR-26b-3p antagomir alleviated intestinal dysmotility, promoted EGC increased activity of EGCs, and reduced EGC apoptosis in vivo. In conclusion, this study indicated that miR-26b-3p promotes intestinal motility disorders by targeting FZD10 to block GSK3ß/ß-catenin signaling and induces apoptosis in EGCs. Our results provide a new research target for the treatment and intervention of intestinal dysmotility.

Paeoniflorin Improved Constipation in the Loperamide-Induced Rat Model via TGR5/TRPA1 Signaling-Mediated 5-Hydroxytryptamine Secretion.

Slow transit constipation (STC) is a common type of constipation with a high incidence rate and a large number of patients. We aimed to investigate the therapeutic effects and potential mechanism of paeoniflorin (PAE) on loperamide-induced Sprague Dawley (SD) rat constipation models. Rats with loperamide-induced constipation were orally administered different concentrations of PAE (10, 20, or 40 mg/kg). In vitro, enterochromaffin (EC)-like RIN-14B cells were treated with 20, 40, or 80 µg/ml PAE. We found that PAE treatment significantly improved the symptoms of constipation and increased the intestinal transit rate. Hematoxylin and eosin (H&E) staining showed that PAE alleviated colonic tissue pathological damage. Besides, our results implied that PAE concentration-dependently promoted the content of 5-hydroxytryptamine (5-HT) catalyzed by tryptophan hydroxylase (Tph)-1 in the serum of loperamide-induced rats and in RIN-14B cells. Western blot and immunofluorescence (IF) stain indicated that PAE also promoted the expression of G protein-coupled BA receptor 1 (TGR5), transient receptor potential ankyrin 1 (TRPA1), phospholipase C (PLC)-γ1, and phosphatidylinositol 4,5-bisphosphate (PIP2) in vivo and in vitro. RIN-14B cells were cotreated with a TGR5 inhibitor (SBI-115) to explore the mechanism of PAE in regulating the 5-HT secretion. We observed inhibition of TGR5 reversed the increase of 5-HT secretion induced by PAE in RIN-14B cells. We provided evidence that PAE could promote 5-HT release from EC cells and improve constipation by activating the TRPA1 channel and PLC-γ1/PIP2 signaling. Thus, PAE may provide therapeutic effects for patients with STC.