Comparative evaluation of two herbal formulas for gastrointestinal function and gut microbiota modulation in rats with loperamide-induced dyspepsia

Abstract

Dyspepsia is a prevalent refractory condition that arises from various causes and lacks definitive treatment. There is an urgent need for evidence to support the use of herbal medicines in the treatment of gastroenterological disorders. This study aimed to compare the therapeutic effects of two common herbal formulas-namely, Shihosogan-tang (SST) and Yijung-tang (YJT)-on loperamide (LOP)-induced dyspepsia and to explore their potential mechanisms. A dyspepsia model was established using Sprague-Dawley rats by intraperitoneal LOP injection at a dose of 3 mg/kg/day for one week. During this period, 30% ethanol extract of SST (1.4 g/kg/day) or YJT (2 g/kg/day) was intragastrically administered in rats. Mosapride (3 mg/kg/day) was used as the positive control. Unlike YJT, SST significantly mitigated LOP-induced reductions in intestinal length, gastrointestinal transit ratio, and serum ghrelin levels. Conversely, YJT significantly enhanced defecation, ileum villus length, and muscular thickness, outperforming SST. Expression of genes related to intestinal motility (ZO-1), inflammation (IL-6), and water absorption (SERT, AQP-3) indicated that both treatments ameliorated LOP-induced changes in the duodenum. Additionally, neuropeptides and hormones (TRH, bombesin, motilin, glucagon, neurotensin, PYY), Toll-like receptors (TLR-2 and TLR-4), and growth factors (GDNF and BMP2) were noticeably altered by SST and/or YJT treatment. While neither LOP nor the herbal formulas affected gut microbiota α-diversity, SST and YJT altered β-diversity compared to LOP alone, unlike mosapride. SST improves dyspepsia more effectively than YJT, possibly through mechanisms involving intestinal hormone regulation, inflammation inhibition, and gut microbiota modulation.

Keywords: Dyspepsia; Gastrointestinal motility; Ghrelin; Gut microbiota; Herbal formula.

Fig. 1

Effects of SST and YJT on phenotypic characteristics in dyspeptic rats. (a) Experimental schedule. (b) Body mass. (c) Food intake (calculated as average food consumption, two rats per cage). (d) Percentage of water in feces. (e) Serum ghrelin levels. Data are presented as means ± SEM (n = 6–8 per group). Statistical analysis was performed using two-way analysis of variance (ANOVA), followed by LSD tests when applicable. Differences were considered statistically significant at p < 0.05. Different letters indicate statistically significant differences. Abbreviations: LOP, loperamide; MOS, mosapride; YJT, Yijung-tang; SST, Shihosogan-tang; SD, Sprague–Dawley; SEM, standard error of the mean; LSD, least significant difference.

Fig. 2

Charcoal test for determining intestinal motility. (a) Gross appearance of rat intestine. (b) Total intestinal length was measured from the pyloric sphincter to the rectum. (c) Gastrointestinal (GI) transit. (d) Number of fecal pellets remaining in the colon after sacrifice. Data are presented as means ± SEM (n = 6–8 per group). Statistical analysis was performed using two-way analysis of variance (ANOVA), followed by LSD tests when applicable. Differences were considered statistically significant at p < 0.05. *p < 0.05. Abbreviations: LOP, loperamide; MOS, mosapride; YJT, Yijung-tang; SST, Shihosogan-tang; SEM, standard error of the mean; LSD, least significant difference. GIT ratio, gastrointestinal transfer ratio.

Fig. 3

Histopathological analysis of the gut barrier. (a) Histological assessment of ileal tissue using H&E staining (n = 5). (b) Villus length. (c) Crypt length. (d) Muscularis thickness. Data are presented as means ± SEM (n = 6–8 per group). Statistical analysis was performed using two-way analysis of variance (ANOVA), followed by LSD tests when applicable. Differences were considered statistically significant at p < 0.05. *p < 0.05. Abbreviations: LOP, loperamide; MOS, mosapride; YJT, Yijung-tang; SST, Shihosogan-tang; SEM, standard error of the mean; LSD, least significant difference; H&E, hematoxylin and eosin.

Fig. 4

Improvement in intestinal dysfunction in duodenal tissue. (a) Changes in the relative mRNA expression of genes related to intestinal tight junction markers: Claudin-2 and ZO-1; inflammatory markers: TNF-α and IL-6; PCNA; and regulators of water absorption: SERT and AQP3. (b) H&E staining of the rat duodenal mucosa (400×magnification; n = 5), with goblet cells and crypts of Lieberkühn indicated by red arrows. Data are presented as means ± SEM (n = 6–8 per group). Statistical analysis was performed using two-way analysis of variance (ANOVA), followed by LSD tests when applicable. Differences were considered statistically significant at p < 0.05. ^*p < 0.05, ^**p < 0.01. Abbreviations: LOP, loperamide; MOS, mosapride; YJT, Yijung-tang; SST, Shihosogan-tang; SEM, standard error of the mean; LSD, least significant difference. ZO-1, zonula occludens-1; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; PCNA, proliferating cell nuclear antigen; SERT, serotonin transporter; AQP3, aquaporin-3; H&E, hematoxylin and eosin.

Fig. 5

Changes in intestinal markers related to GI motility. (a) Expression of genes encoding neurohumoral mediators involved in intestinal contraction, including stimulators such as TRH, bombesin, motilin, and opioids and inhibitors such as glucagon, neurotensin, and PYY. (b) mRNA expression of markers related to neurotrophic signaling and improvement in GI motility, including TLR2, TLR4, GDNF, and BMP2. Data are presented as means ± SEM (n = 6–8 per group). Statistical analysis was performed using two-way analysis of variance (ANOVA), followed by LSD tests when applicable. Differences were considered statistically significant at p < 0.05. ^*p < 0.05, ^**p < 0.01. Abbreviations: LOP, loperamide; MOS, mosapride; YJT, Yijung-tang; SST, Shihosogan-tang; SEM, standard error of the mean; LSD, least significant difference; GI, gastrointestinal; TRH, thyrotropin-releasing hormone; PYY, peptide YY; TLR2, Toll-like receptor 2; TLR4, Toll-like receptor 4; GDNF, glial cell line-derived neurotrophic factor; BMP2, bone morphogenetic protein 2.

Fig. 6

Comparative metagenomic analysis on gut microbiota. (a) Chao1 and Shannon indices. (b) Principal coordinate analysis (PCoA) plot based on Bray–Curtis dissimilarity of fecal microbiota. (c) Relative abundance of different bacterial phyla. (d) Proportion of specific bacterial genera. Data are presented as means ± SEM (n = 6–8 per group). Statistical analysis was performed using two-way analysis of variance (ANOVA), followed by LSD tests when applicable. Differences were considered statistically significant at p < 0.05. Different letters in columns indicate significant differences. Abbreviations: LOP, loperamide; MOS, mosapride; YJT, Yijung-tang; SST, Shihosogan-tang; SD, Sprague–Dawley; SEM, standard error of the mean; LSD, least significant difference.

Fig. 7

Heatmap of correlation between specific genera and mRNA expression of different intestinal markers and receptors. Pearson’s two-tailed correlation was performed using data from all groups, with |r|> 0.3 indicating a meaningful correlation. Abbreviations: TRH, thyrotropin-releasing hormone; PYY, peptide YY; TLR2, Toll-like receptor 2; TLR4, Toll-like receptor 4; GDNF, glial cell line-derived neurotrophic factor; BMP2, bone morphogenetic protein 2; ZO-1, zonula occludens-1; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; PCNA, proliferating cell nuclear antigen; SERT, serotonin transporter; AQP3, aquaporin-3.