Xiao Cheng Qi (XCQ) decoction, an ancient Chinese herbal mixture, has been used in treating slow-transit constipation (STC) for years. The underlying action mechanism in relieving the clinical symptoms is unclear. Several lines of evidence point to a strong link between constipation and gut microbiota. Short-chain fatty acids (SCFAs) and microbial metabolites have been shown to affect 5-HT synthesis by activating the GPR43 receptor localized on intestinal enterochromaffin cells, since 5-HT receptors are known to influence colonic peristalsis. The objective of this study was to evaluate the efficacy of XCQ in alleviating clinical symptoms in a mouse model of STC induced by loperamide. The application of loperamide leads to a decrease in intestinal transport and fecal water, which is used to establish the animal model of STC. In addition, the relationship between constipation and gut microbiota was determined. The herbal materials, composed of Rhei Radix et Rhizoma (Rhizomes of Rheum palmatum L., Polygonaceae) 55.2 g, Magnoliae Officinalis Cortex (Barks of Magnolia officinalis Rehd. et Wils, Magnoliaceae) 27.6 g, and Aurantii Fructus Immaturus (Fruitlet of Citrus aurantium L., Rutaceae) 36.0 g, were extracted with water to prepare the XCQ decoction. The constipated mice were induced with loperamide (10 mg/kg/day), and then treated with an oral dose of XCQ herbal extract (2.0, 4.0, and 8.0 g/kg/day) two times a day. Mosapride was administered as a positive drug. In loperamide-induced STC mice, the therapeutic parameters of XCQ-treated mice were determined, i.e., (i) symptoms of constipation, composition of gut microbiota, and amount of short-chain fatty acids in feces; (ii) plasma level of 5-HT; and (iii) expressions of the GPR43 and 5-HT4 receptor in colon. XCQ ameliorated the constipation symptoms of loperamide-induced STC mice. In gut microbiota, the treatment of XCQ in STC mice increased the relative abundances of Lactobacillus, Prevotellaceae_UCG_001, Prevotellaceae_NK3B31_group, Muribaculaceae, and Roseburia in feces and decreased the relative abundances of Desulfovibrio, Tuzzerella, and Lachnospiraceae_ NK4A136_group. The levels of SCFAs in stools from the STC group were significantly lower than those the control group, and were greatly elevated via treatment with XCQ. Compared with the STC group, XCQ increased the plasma level of 5-HT and the colonic expressions of the GPR43 and 5-HT4 receptor, significantly. The underlying mechanism of XCQ in anti-constipation could be related to the modulation of gut microbiota, the increase in SCFAs, the increase in plasma 5-HT, and the colonic expressions of the GPR43 and 5-HT4 receptor. Our results indicate that XCQ is a potent natural product that could be a therapeutic strategy for constipation.
Keywords: slow transit constipation; microbe; short-chain fatty acids; Chinese medicine
Constipation is a risk factor for many gastrointestinal disorders, with a global prevalence ranging from 14% to 30% of the population [[
Several lines of evidence indicate that gut microbiota play an important role in developing chronic constipation. Disorder of gut microbiota happens during the onset and progression of chronic constipation, as characterized primarily by an increased abundance of Bacteroides [[
Xiao Cheng Qi (XCQ) decoction is a traditional Chinese herbal formula that was recorded by "Shang Han Lun" around 200 A.D., composed of Rhei Radix et Rhizoma (Rhizomes of Rheum palmatum L., Polygonaceae), Magnoliae Officinalis Cortex (Barks of Magnolia officinalis Rehd. et Wils, Magnoliaceae), and Aurantii Fructus Immaturus (Fruitlet of Citrus aurantium L., Rutaceae). This herbal decoction is frequently used to treat illnesses of the digestive system in clinical practice, including recovery from post-operative gastrointestinal function, intestinal infarction, and constipation after stroke. Each herb in the XCQ decoction plays a specific role in treating constipation. Rhei Radix et Rhizoma is a laxative herb known to promote bowel movements and relieve constipation; this herb is also known to have anti-inflammatory and anti-bacterial properties. In addition, Rhei Radix et Rhizoma can stimulate the secretion of bile and digestive enzymes, aiding in the digestion and absorption of food. Magnoliae Officinalis Cortex is a herb that can promote the movement of Qi (energy) in the body and relieve stagnation. According to Chinese medicinal theory, constipation is often attributed to the stagnation of Qi in the gut, and Magnoliae Officinalis Cortex is able to alleviate this stagnation, thereafter improving gut motility and relieving constipation. Aurantii Fructus Immaturus is a herb that can regulate the Qi in the gut and promote bowel movements, which has a mild laxative effect in relieving constipation, which also reduces abdominal bloating and discomfort, common symptoms of constipation. Overall, the three herbs in XCQ help to regulate gut motility, relieve constipation, reduce inflammation, and promote overall digestive health [[
In XCQ decoction, Rhei Radix et Rhizoma serves as a purgative in helping constipation by promoting the release of colonic mucus from mast cells and by enhancing the microenvironment in the intestine [[
The herbal extract of XCQ was subject to HPLC analysis. A fingerprint containing thirteen major peaks was generated. By comparing their retention time with the known standards, representative naringin, neohesperidin, aloe-emodin, rhein, honokiol, magnolol, emodin, chrysophanol, and physcion were identified (Figure S1 in Supplementary Materials). A quantitative analysis of XCQ extract was performed, showing the amounts of naringin (~9.84%), neohesperidin (~13.93%), anthraquinones (~0.56%, i.e., aloe-emodin, rhein, emodin, chrysophanol, and physcion), and honokiol and magnolol (~0.32%).
A mouse STC model was established via an oral gavage administration of loperamide, and thereafter different dosages of XCQ were applied; the experimental outline is shown (Figure 1). The body weights of different treatments showed no significant change (Figure S2A in Supplementary Materials); however, the fecal water content of the STC group was significantly decreased on day 7 after the treatment of loperamide, i.e., a development of the disease model. On day 14, fecal water content was markedly recovered following XCQ treatments in all doses and in the mosapride-treated group (Figure S2B in Supplementary Materials). The STC group showed significantly lower intestinal motility than that of control group (p < 0.01), indicating that STC mice were successfully established with the expected symptoms (Figure 2A). The treatments of middle and high doses of XCQ (XCQ
The refractive curve of each sample is shown, indicating the sufficient depth of RNA sequencing (Figure S3A,B in Supplementary Materials). The α-diversity analysis showed that both the Shannon and Chao indexes of STC group were less than those of the control group at the OTU level (Figure S3C,D in Supplementary Materials). Mosapride, and medium and high doses of XCQ showed an elevation in Shannon analysis (Figure S3C in Supplementary Materials). PCoA and NMDS methods were employed to determine the β-diversity of each group at the OTU level, where the features of the untreated control and STC group were well distinguished (Figure 4A). In addition, the treatment groups were different from the model group.
The community analysis was carried out at phylum and genus levels. Lachnospiraceae_NK4A136_group, a dominant genus belonging to the phylum of Firmicutes, was highly enriched in the STC group and was less abundant in other groups (Figure 4B). The increase in the abundance of the Firmicutes phylum in the STC group was due to a significant rise in the Lachnospiraceae_NK4A136_group genus from Firmicutes, as compared with that in the other groups. The abundance of Firmicutes in the XCQ treatment group was lower than that in the STC group because the amount of Lachnospiraceae_NK4A136_group downregulated in the XCQ treatment group. According to the literature, Lachnospiraceae_ NK4A136_group is an indicator of gut dysbiosis, which shows greater abundance during severe gut dysbiosis [[
5-HT is a mediator controlling several physiological processes in the gastrointestinal tract, including intestinal secretion and gut motility [[
The protein levels of the GPR43 (~55 kDa) and 5-HT4 receptor (~55 kDa) in the colon of STC mice were determined via Western blotting (Figure 8B). In STC mice, the expressions of the GPR43 and 5-HT4 receptor were reduced (Figure 8B–D). Treatment with XCQ or mosapride recovered the STC-reduced protein expressions, i.e., those of the GPR43 (p < 0.05) and 5-HT4 receptor (p < 0.05), as compared to those in the STC group (Figure 8B–D). Additionally, the immunohistochemical results showed a significant reduction in the protein expressions of the GPR43 (p < 0.05) (Figure 9A,B) and 5-HT4 receptor (p < 0.001) (Figure 10A,B) in the colon of STC mice. As compared to the STC group, XCQ administration significantly raised the protein expressions of the GPR43 (p < 0.01) (Figure 9B) and 5-HT4 receptor (p < 0.001) (Figure 10B). The effects of XCQ were robust and as sound as those of mosapride.
HPLC-grade acetonitrile, methanol, and formic acid were obtained from Merck (Darmstadt, Germany). Millipore Milli-Q water system supplied 18 MΩ cm
The ground powders of herbal materials, composed of Rhei Radix et Rhizoma (Rhizomes of R. palmatum) 55.2 g, Magnoliae Officinalis Cortex (Barks of M. officinalis) 27.6 g, and Aurantii Fructus Immaturus (Fruitlet of C. aurantium) 36.0 g, in accordance with the traditional dosage of this herbal mixture, were immersed in 8 volumes of water (1:8, w/v) for 45 min before being extracted twice by boiling, then placed under gentle heating (300 W) until the liquid reached 240 mL. The decoction was filtered through 8 layers of gauze. The combined herbal extract was evaporated under a vacuum and lyophilized. The samples were stored at 4 °C.
The HPLC analyses of XCQ were conducted by comparing the retention time of peaks with chemical standards. Methanol was added to make the mixture of standards (purity > 98%), containing naringin, neohesperidin, honokiol, magnolol, rhein, aloe-emodin, emodin, chrysophanol, and physcion, obtained from Weikeqi Biological Technology Co., Ltd. (Chengdu, China). Briefly, 20 mg of XCQ was dissolved in 1 mL of 80% methanol and analyzed via HPLC-DAD with a Waters CORTECS T3 column (2.1 mm × 150 mm, 1.6 µm). The injected volume was set as 1 µL. Solvents were as follows: solvent A, 0.1% diluted aqueous phosphoric acid, and solvent B, methanol. The elution procedure was as follows: solvent B increased from 3% to 21% at 0 to 5 min; 21% to 36% at 5 to 20 min; 36% to 50% at 20 to 32 min; 50% to 62% at 32 to 42 min; 62% to 85% at 42 to 50 min; 85% to 95% at 50 to 60 min. The column temperature was 30 °C, and the absorbance at 260 nm was collected.
Balb/c mice (5–6 weeks old with a body weight of 20 ± 2 g) were purchased from Guangdong Medical Laboratory Animal Center. Mice were kept under specific pathogen-free conditions at Laboratory Animal Center, Kangmeihuada Gene Technology (Shenzhen, China), with 44–65% humidity and a 12-h light/dark cycle at 22–26 °C, and free access to food and water. The experimental protocols were approved and followed the guidelines of Chinese legislation on the ethical use and care of laboratory animals.
After adjusting to the environment for 7 days, 84 pathogen-free mice were utilized to examine the preventative effects of XCQ on STC. The methods and procedures used in this animal experiment were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of The Hong Kong University of Science & Technology, reviewed and approved by the Animal Ethics Committee of Experimental Animals of The Hong Kong University of Science & Technology. The mice were randomly divided into six groups (n = 14): the control group, STC group, mosapride (a positive control and a gastroprokinetic agent acting as a selective 5-HT4 receptor agonist) group, XCQ
On the 21st day of experimental treatment, the parameters of constipation, e.g., the water content of feces, small intestinal transit, and time for first black stool defecation, were examined as previously described [[
Using gas chromatography (GC), the amount of SCFAs were determined in mouse stool samples. Stool samples weighing 50 mg were homogenized by adding 100 µL of ultrapure water. The supernatant was then centrifuged, and the sample solution was obtained by combining 90 µL of the supernatant with 10 µL of an internal standard (2-ethyl butyric acid). The supernatants were analyzed using an Agilent GC-FID machine, equipped with an Agilent DB-WAXERT column (30 m × 0.25 mm × 0.25 μm). Helium (flow rate: 1.8 mL/min, split ratio 20:1) was used as the carrier gas. The injection temperature was 250 °C, and the GC temperature program was as follows: an initial temperature of 50 °C was raised to 130 °C at a rate of 15 °C/min and then to 200 °C and maintained for 2 min. The concentrations of SCFAs were calculated using standard curves for acetic acid, propionic acid, butyric acid, isobutyric acid, and valeric acid.
The CTAB/SDS method was used to extract total DNA from the samples. DNA concentration and purity were measured, and diluted to 1 ng/µL using sterile water. The primers 515F (5′-GTG CCA GCM GCC GCG GTA A-3′) and 806R (5′-GGA CTA CHV GGG TWT CTA AT-3′) with the barcode were used to amplify the V4 region of bacterial the 16S ribosomal RNA gene. Sequencing libraries were generated using TruSeq
Total proteins were extracted from the tissues using a lysis buffer, and the concentration was measured. Proteins were separated using SDS-PAGE and transferred to a nitrocellulose membrane, blocking with a 5% skim milk solution. Then, the relevant primary antibodies (GPR43 and 5-HT4 receptor) were incubated overnight on the membranes. The blots were washed three times before being exposed to the secondary antibody for an hour at room temperature. With the use of the improved chemiluminescent reagent (ECL), each antigen–antibody combination was discovered. The images were analyzed using Image-Pro Plus software version 1.53k (Media Cybernetics, Rockville, MD, USA).
The expressions of the GPR43 and 5-HT4 receptor in colonic tissues were analyzed via by immunohistochemistry, as described in [[
Statistical analyses were performed via a one-way ANOVA with Tukey's post hoc test using GraphPad Prism software Version 7 (GraphPad Software, La Jolla, CA, USA). Statistical significance was determined as (*) or (#) when p < 0.05.
For thousands of years in China, numerous ailments have been prescribed for medical treatments using Chinese herbal medicine [[
An over-the-counter anti-diarrheal drug, loperamide, causes difficulties in defecation by binding to the μ-opioid receptors in the intestinal circulation and longitudinal muscles. This increases the gastrointestinal transport time, slows down intestinal peristalsis, and inhibits the contraction of intestinal smooth muscle. The application of loperamide could lead to a decrease in intestinal transport and fecal water, and is being used to establish the animal model of STC. In this study, XCQ mediation was found to improve constipation-related parameters, constipation-associated colonic lesions, defecation, fecal water content, colonic mobility, and colonic lesions related to constipation in STC mice. These results suggest that XCQ could help in improving defecation and intestinal motility.
In line with the clinical outcome of XCQ mediation, the constipation-related parameters, and constipation-associated colonic lesions, identified in STC mice, were significantly recovered in mice treated with XCQ. In addition, defecation, fecal water content, colonic mobility, and colonic lesions relating to constipation were markedly improved. This result suggested that XCQ could intervene in defecation and intestinal motility.
Targeting 5-HT signaling is a recent approach for STC's drug development. For example, mosapride, a gastroprokinetic agent acting as a selective 5-HT4 receptor agonist, has been marketed today to enhance gastrointestinal motility [[
Modulating the population of gut microbiota and the metabolism of SCFAs has emerged as a promising strategy for new drug development. The amount of B. bifidum in the gut could enhance the expression of the 5-HT4 receptor, thereby promoting colonic peristalsis and the secretion of intestinal fluid [[
This study has some limitations. Although 5-HT plays a crucial role in regulating bowel functions, the mechanistic action of 5-HT in gut function is complex. 5-HT induces smooth muscle contraction or relaxation through various subtypes of receptors. The activation of cholinergic excitatory neurons could be mediated by 5-HT3 and 5-HT4 receptors, and nitric oxide-inhibited enteric motor neurons could be mediated by 5-HT4, 5-HT1A, and 5-HT1D receptors, leading to a relaxation of the distal colon. Intestinal bacteria may lead to the upregulation of serotonin-selective reuptake transporter (SERT) expression, and as a result cause a decrease in 5-HT concentrations in colonic mucosa. In addition, some researchers have reported that increased 5-HT concentrations in the blood or colonic mucosa were closely related to constipation [[
This study focuses on the therapeutic role and mechanistic action of the XCQ herbal decoction in treating STC. XCQ exhibits an anti-constipation effect in loperamide-induced STC mice. Under the influence of XCQ in STC mice, the population of gut microbiota, the levels of SCFAs, the plasma level of 5-HT, and the expressions of the GPR43 and 5-HT4 receptor in the colon are restored back to those under a normal situation. Our results indicate that XCQ is a potent natural product that could be a therapeutic strategy for treating constipation. The pharmacological studies of natural products were conducted from 2022 to 2023. The authors are listed in order of their respective contributions to the work conducted during the study.
Graph: Figure 1 Schematic outline of STC model establishment and XCQ treatment. Mice were divided into six groups: control, normal mice treated with saline; STC, loperamide (10 mg/kg/day)-induced STC mice treated with saline; mosapride, loperamide-induced STC mice treated with mosapride (2.5 mg/kg/day); XCQLD (low dose of XCQ 2 g/kg/day), XCQMD (medium dose of XCQ 4 g/kg/day) and XCQHD (high dose of XCQ 8 g/kg/day), LOP-induced STC mice treated with XCQ herbal extracts. The drug treatments were given via gavage from day 8 to day 21.
Graph: Figure 2 XCQ ameliorates the constipation symptoms in STC mice. The experimental protocol and groups are listed in Figure 1. At 21 days, the mice and their feces were subjected to analysis. (A) The percentage of the small intestinal transit rate. (B) The content of fecal water content. (C) The first black stool defecation time. (D) The weight of feces. (E) The shape of collected feces. Values are presented as mean ± SEM, n = 3–5. Significant differences were assessed using a one-way ANOVA: ## p < 0.01 vs. the control group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the STC group.
Graph: Figure 3 Effects of XCQ on histology of colonic tissue in STC mice. The experimental protocol and groups are listed in Figure 1. At 21 days, the colon samples were subjected to analysis. Colonic tissues were stained via H&E staining. Representative images are shown, n = 5. The stars indicate colonic mucosal injury and inflammation in the colon tissues of STC mice.
Graph: Figure 4 XCQ regulates gut microbiota in STC mice. The experimental protocol and groups are listed in Figure 1. At 21 days, the feces were subjected to analysis. (A) PCoA and NMDS analysis based on Bray–Curtis distance shows the β-diversity of each group at the OTU level. (B) The community distribution at phylum and genus levels. Each group is represented by different color/symbol combinations, n = 4.
Graph: Figure 5 A cladogram of LEfSe analysis shows the differently enriched gut microbiomes based on a comparison with the model groups. LDA score > 4; n = 4.
Graph: Figure 6 XCQ regulates the composition of microbes in STC mice. The experimental protocol and groups are listed in Figure 1. At 21 days, the feces were subjected to analysis. The relative abundance of different bacterial taxa, as indicated here, was calibrated. The control group's data were used as the basal values, and the data of other groups were normalized. Values are presented as fold of change (×basal) in mean ± SEM, n = 4. Significance differences were assessed via one-way ANOVA: # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 and ##### p < 0.00001vs. the control group; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. the STC group.
Graph: Figure 7 XCQ regulating the levels of SCFAs in stools of STC mice. The experimental protocol and groups are listed in Figure 1. At 21 days, the feces were subjected to analysis. The amounts of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and total SCFAs in the collected stools were determined via GC-FID. Values are presented as mean ± SEM, n = 3. Significance differences were assessed via one-way ANOVA: # p < 0.05 and ## p < 0.01 vs. the control group; * p < 0.05 and ** p < 0.01 vs. the STC group.
Graph: Figure 8 XCQ regulating the level of 5-HT in the plasma and the expression of the GPR43 and 5-HT4 receptor in colon in STC mice. The experimental protocol and groups are listed in Figure 1. At 21 days, the plasma and colon samples were subjected to analysis. (A) The plasma level of 5-HT in different groups. (B) The relative abundances of the GPR43 (~55 kDa) and 5-HT4 receptor (~55 kDa) colon tissues determined via Western blotting, and a representative gel image. (C) The protein expression of GPR43 in the colon. (D) The protein expression of the 5-HT4 receptor in colon. Values presented as mean ± SEM, n = 3. Significant differences were assessed via one-way ANOVA: ## p < 0.01 vs. the control group; * p < 0.05 and ** p < 0.01 vs. the STC group.
Graph: Figure 9 XCQ regulates the expression of GPR43 in the colon tissues of STC mice. The experimental protocol and groups are listed in Figure 1. At 21 days, the colon samples were subjected to analysis. (A) Immunohistochemical analysis of colonic sections from different groups. Representative images are shown, n = 4. (B) Quantification of the immunostaining of the GPR43 receptor in the colonic tissues of different groups using Image-Pro Plus software (Media Cybernetics). Values are presented as a percentage of the change in mean ± SEM, n = 5. Significance differences were assessed via one-way ANOVA: # p < 0.05, vs. the control group; ** p < 0.01, and *** p < 0.001 vs. the STC group.
Graph: Figure 10 XCQ regulates the expression of the 5-HT4 receptor in the colon tissues of STC mice. The experimental protocol and groups are listed in Figure 1. At 21 days, the colon samples were subjected to analysis. (A) Immunohistochemical analysis of colonic sections from different groups. Representative images are shown, n = 4. (B) Quantification of the immunostaining of the 5-HT4 receptor in the colonic tissues of different groups using Image-Pro Plus software (Media Cybernetics). Values are presented as a percentage of the change in mean ± SEM, n = 5. Significance differences were assessed via one-way ANOVA: ### p < 0.001 vs. the control group; ** p < 0.01 and *** p < 0.001 vs. the STC group.
A.T.: conceptualization, methodology, investigation, visualization, writing—original draft. H.W.: methodology. J.W.: visualization, methodology, Writing—review and editing. Z.W.: methodology. T.D.: methodology, resources. Y.H.: methodology. D.Z.: methodology, resources. D.S.: methodology, resources. K.W.K.T.: conceptualization, supervision, writing—review and editing. All authors have read and agreed to the published version of the manuscript.
The animal study protocol was approved by the Ethics Committee of Experimental Animals of The Hong Kong University of Science & Technology. (protocol code: AEP-2023-0022 and date of approval: March 2023).
Not applicable.
Data is contained within the article and supplementary material.
The authors declare no conflicts of interest. Authors Dequan Zhu and Dongmei Sun were employed by the company Guangdong Efong Pharmaceutical Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.
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By Amanguli Tuohongerbieke; Huaiyou Wang; Jiahui Wu; Zhengqi Wang; Tingxia Dong; Yamiao Huang; Dequan Zhu; Dongmei Sun and Karl Wah Keung Tsim
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