Ethanol Extract of Mao Jian Green Tea Attenuates Gastrointestinal Symptoms in a Rat Model of Irritable Bowel Syndrome with Constipation via the 5-hydroxytryptamine Signaling Pathway

Abstract

In a previous study, we demonstrated that the hydro extract of Mao Jian Green Tea (MJGT) promotes gastrointestinal motility. In this study, the effect of MJGT ethanol extract (MJGT_EE) in treating irritable bowel syndrome with constipation (IBS-C) in a rat model constructed via maternal separation combined with an ice water stimulation was investigated. First, a successful model construction was confirmed through the determination of the fecal water content (FWC) and the smallest colorectal distension (CRD) volume. Then, the overall regulatory effects of MJGT_EE on the gastrointestinal tract were preliminarily evaluated through gastric emptying and small intestinal propulsion tests. Our findings indicated that MJGT_EE significantly increased FWC (p < 0.01) and the smallest CRD volume (p < 0.05) and promoted gastric emptying and small intestinal propulsion (p < 0.01). Furthermore, mechanistically, MJGT_EE reduced intestinal sensitivity by regulating the expression of proteins related to the serotonin (5-hydroxytryptamine; 5-HT) pathway. More specifically, it decreased tryptophan hydroxylase (TPH) expression (p < 0.05) and increased serotonin transporter (SERT) expression (p < 0.05), thereby decreasing 5-HT secretion (p < 0.01), activating the calmodulin (CaM)/myosin light chain kinase (MLCK) pathway, and increasing 5-HT₄ receptor (5-HT₄R) expression (p < 0.05). Moreover, MJGT_EE enhanced the diversity of gut microbiota, increased the proportion of beneficial bacteria, and regulated the number of 5-HT-related bacteria. Flavonoids may play the role of being active ingredients in MJGT_EE. These findings suggest that MJGT_EE could serve as a potential therapeutic pathway for IBS-C.

Keywords: 5-hydroxytryptamine; Mao Jian Green Tea; ethanol extract; flavonoids; gastrointestinal motility; gut microbiota.

Figure 1

The timeline from rat model establishment, administration to sampling (A). Comparison of fecal water content (B) and pressure threshold (C) in rats of different groups. Capital and lowercase letters above the bar indicate the difference significance at the 0.01 or 0.05 levels, respectively. (n = 8 for each group). NC: negative control group; MG: model group, MSP: mosapride group, MJGT_EE: Mao Jian Green Tea ethanol extract group.

Figure 2

Effect of MJGT_EE on gastric emptying rate (A) and intestinal propulsive rate (B) in vivo. Capital letters above the bar indicate the difference significance at the 0.01 level. (n = 8 for each group). NC: negative control group; MG: model group, MSP: mosapride group, MJGT_EE: Mao Jian Green Tea ethanol extract group.

Figure 3

H&E staining to observe the effect of MJGT_EE on the colonic tissues of IBS-C rats (200×). (A) The colonic tissues from normal rats; (B) the colonic tissues from rats with IBS-C; (C) the colonic tissues of IBS-C rats after 30 days of MJGT_EE treatment; (D) the colonic tissues of IBS-C rats after 30 days of MSP treatment. (n = 8 for each group). NC: negative control group; MG: model group, MSP: mosapride group, MJGT_EE: Mao Jian Green Tea ethanol extract group.

Figure 4

Immunohistochemical staining analysis to observe the effect of MJGT_EE on the colonic tissues of IBS-C rats. (A) The colonic tissues from normal rats; (B) the colonic tissues from rats with IBS-C; (C) the colonic tissues of IBS-C rats after 30 days of MJGT_EE treatment; (D) the colonic tissues of IBS-C rats after 30 days of MSP treatment; (E) the relative expression of 5-HT. Capital letters above the bar indicate the difference significance at the 0.01 level. (n = 8 for each group). NC: negative control group; MG: model group, MSP: mosapride group, MJGT_EE: Mao Jian Green Tea ethanol extract group; 5-HT: 5-hydroxytryptamine.

Figure 5

Western blotting analysis of TPH1 (A), TPH2 (B), SERT (C), 5-HT₃ receptors (D), 5-HT₄ receptors (E), CaM (F), and MLCK (G) in colonic tissues of each group. Capital and lowercase letters above the bar indicate the difference significance at the 0.01 or 0.05 levels, respectively. (n = 8 for each group). NC: negative control group; MG: model group, MSP: mosapride group, MJGT_EE: Mao Jian Green Tea ethanol extract group; 5-HT: 5-hydroxytryptamine; TPH1: tryptophan hydroxylase 1; TPH2: tryptophan hydroxylase 2; SERT: serotonin transporter; CAM: calmodulin; MLCK: myosin light chain kinase; 5-HT₃R: 5-HT₃ receptor; 5-HT₄R: 5-HT₄ receptor.

Figure 6

PCoA analysis of the OTU level of intestinal flora in different groups rats (A); venn diagram of the distribution of OTUs in the four groups (B). (n = 4 for each group). NC: negative control group; MG: model group, MSP: mosapride group, MJGT_EE: Mao Jian Green Tea ethanol extract group.

Figure 7

The variations in intestinal flora composition are displayed the phylum (A) and family (B) and genus level (C), respectively. (n = 4 for each group). NC: negative control group; MG: model group, MSP: mosapride group, MJGT_EE: Mao Jian Green Tea ethanol extract group.