Shouhui Tongbian Capsule (SHTC) was produced by Lunan Pharmaceutical Group Co., Ltd. (Linyi, China, manufacture batch number: 26210523). SHTC is composed of 120 g Polygonum multiflorum Thunb, 160 g Aloe barbadensis Miller, 140 g Cassiae Semen (Cassia obtusifolia L.), 75 g Wolfberry (Lycium barbarum L.), 75 g Corii Colla Asini (Equus asinus L.), 50 g Panax ginseng C. A. Meyer, 50 g Atractylodes macrocephala Koidz, 120 g Citrus aurantium L. Moxapride citrate tablets were purchased from Jiangsu Haosen Pharmaceutical Group Co., Ltd. (Jiangsu, China). 5-fluorouracil (5-FU, B25419) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Activated carbon (C7261), gum Arabic (G8130), and Trizol (Lot#Q30704) were obtained from Beijing TransGen Biotechnology Co., Ltd. (Beijing, China). SYBR Green PCR Master Mix (CW2623) and High Capacity cDNA Reverse Transcription kit (CW2569) were obtained from Applied Biosystems (Waltham, MA, USA). Mouse Gastric Actin ELISA Kit (MTL, JL10463), Mouse Acetylcholinesterase ELISA Kit (AChE, JL20661), Mouse Endothelin 1 ELISA Kit (ET-1, JL12801) were purchased from Jiangsu Jianglai Biotechnology Co., Ltd. (Nanjing, China). LY294002 was purchased from MedChemExpress (New Jersey, USA) and sodium acetate was purchased from MACKLIN (Shanghai, China).

The UPLC method was referenced from previous literature with minor modifications (Liang et al., 2022; Ma et al., 2022). 0.5 g SHTC was added with 50 mL 50% methanol and sonicated in an ultrasonic water bath for 60 min. The supernatant was collected and filtered through a 0.22 μm filter membrane for further analysis. The concentrations of 2,3,5,4′-tetrahydroxyl diphenylethylene-2-o-glucoside, Naringin, and Aloin in the extracts of SHTC were analyzed using the Waters ACQUITY UPLC H-class system (Waters Corporation, Milford, MA) and C18 column (Waters ACQUITYTM UPLC BEH C18 column, 2.1 mm × 100 mm, 1.7 μm). The following settings were used for UPLC analysis: mobile phase, 0.05% phosphoric acid aqueous solution (A); acetonitrile (B) at an elution flow rate of 0.3 mL/min; column temperature 30°C. Gradient elution with A (0–5.0 min, 85–80% A; 5.0–7.0 min, 80% A; 7.0–10.0 min, 80–85% A; 10.0–12.0 min, 85% A). The detection wavelength is 220 nm.

C57BL/6 male mice (20–22 g) were purchased from Beijing Spelford Biotechnology Co. Ltd. and housed under standard conditions (20 ± 2°C, 50–60% humidity, and 12 h light/dark cycle) in the Experimental Animal Center of Tianjin University of Chinese Medicine. The mice were allowed free access to mouse chow (components: soybean meal, fish meal, vegetable oil, bran, maize, wheat middlings, vitamins, minerals, etc.; purchased from Beijing Huafukang Biotechnology Co., Ltd., batch number: 1022), and the drinking water was purified water sterilized by high pressure. All animal studies were approved by the Science and Technological Committee and the Animal Use and Care Committee of Tianjin University of Traditional Chinese Medicine (TCM-LAEC2022205).

After acclimation for 1 week, the mice were randomly divided into normal group (Nor), model group (Mod), Mosapride Citrate group (Mos), SHTC low dose group (SH-L), SHTC medium dose group (SH-M), SHTC high dose group (SH-H), with 8 mice per group. All the mice except the ones in the control group, were injected intraperitoneally with 46 mg/kg 5-FU [the administered dose of 5-FU was determined according to the report of Justino et al. (2015) and McQuade et al. (2019) and the pre-experiment] on the first day of the experiment. Then every other day for 12 days for a total of 6 injections, the mice in the normal group were injected with the same dose of saline. SHTC or mosapride citrate was given 1 h after intraperitoneal injection of 5-FU. The mice in the Mos group and SH-L/M/H groups were orally administered Mosapride Citrate (20 mg/kg) or SHTC (100, 200, 400 mg/kg). All treatments were administered once daily for 12 consecutive days. The doses of Mosapride Citrate and SHTC were determined according to the eport of Suchitra et al. (2003) and Bai et al. (2022). The mice in the Nor group and Mod group were given the same volume of saline. On the tenth and twelfth days of the experiment, all mice were fasted for 16 h and gavaged with carbon powder suspension to detect the time of the first black stool and the rate of small intestinal propulsion. The experimental design is shown in Figure 1A.

Preparation of carbon powder suspension: The preparation method was referenced from published literature with minor modifications (Lin C. Y. et al., 2015). Accurately weigh 100 g of gum arabic and add it to 800 mL of water. Boil the solution until it becomes transparent, then add 50 g of activated carbon and boil the solution three times. After the solution cools, dilute it to 1,000 mL with water, store it at 4°C, and mix well before use.

Sixty mice were randomly divided into the donor group (n = 30) and the recipient group (n = 30). Among them, the donor mice were randomly divided into Nor group (n = 10), 5-FU group (n = 10), and 5-FU + SH group (n = 10). All donor mice except those in the Nor group were intraperitoneally injected with 46 mg/kg of 5-FU on the first day of the experiment, followed by intraperitoneal injections every other day, and mice in the 5-FU + SH group were gavaged with 400 mg/kg of SHTC every day for 8 days. The recipient mice were randomly divided into FMT-Nor group (n = 10), FMT-Mod group (n = 10) and FMT-SH group (n = 10). All recipient mice were gavaged with 0.2 mL of the antibiotic mixture, referred to as ABX (ampicillin 100 mg/kg; metronidazole 100 mg/kg; neomycin 100 mg/kg; and vancomycin 50 mg/kg) daily for 7 days prior to the start of the experiment. After 2 days of intestinal rest, all recipient mice were intraperitoneally injected with 46 mg/kg of 5-FU every other day to induce constipation. Meanwhile, each group of recipient mice received freshly prepared fecal supernatant (200 μL/20 g) from the corresponding donor mice by gavage for 8 days. On the sixth day of administration, all recipient mice were fasted for 16 h. After administration of fecal supernatant for 30 min, carbon powder suspension was gavaged and the time to the first black stool, 4-h fecal excretion and fecal water content was measured. On the eighth day of the experiment, all the recipient mice were fasted for 16 h, given fecal supernatant for 30 min, and then gavaged with carbon powder suspension. Twenty minutes later, all mice were sacrificed, the rate of small intestine propulsion was calculated, and mouse serum and colon tissue were collected for subsequent experiments.

Fecal supernatant preparation: This method was referenced from published literature (Gray et al., 2024). Feces from each group of donor mice are collected daily, diluted with PBS (100 mg/mL), vigorously vortexed to form a homogeneous suspension. The suspension is centrifuged at 600 × g for 10 min at 4°C, and the supernatant is collected for subsequent use.

Mice were given 10 ml/kg activated carbon suspension at the concentration of 5%, and then the mice were immediately placed into metabolic cages and the time of the first black stool was started to be recorded, and the feces were collected for 4 h. The feces were weighed before and after they were dried at 60°C in a drying oven. The water content of the feces was calculated according to the following formula: (wet weight of feces- dry weight of feces)/wet weight of feces× 100%.

The intestinal propulsion rate was measured according to the method reported by Hao M et al. with minor modifications (Hao et al., 2023). At the end of the animal experiment, the mice were fasted overnight for 16 h to empty the intestinal contents. On the day of sampling, 10 ml/kg of 5% activated carbon suspension was given to the mice orally. After 20 min, the abdominal cavity of each mouse was opened rapidly, the intestine from the pylorus to the ileocecal valve was collected, and the whole length of the small intestine was measured and recorded. The small intestinal propulsive rates were calculated according to the following formula: Small intestine propulsive rate (%) = (black semi-solid paste front distance/whole length of the small intestine) × 100%.

The levels of excitatory gastrointestinal hormones acetylcholine (AChE) and gastrin (MTL) and inhibitory gastrointestinal hormone endothelin1 (ET-1) in mouse serum were measured using ELISA kits according to the manufacturer’s instructions. Briefly, blood was collected from the orbital sinus of the mice, and centrifuged at 3500 rpm for 15 min at 4°C after keeping it at room temperature for 2 h. The serum was collected and added to the 96-well plate which was pre-coated with the corresponding antibody. After washing and incubation with the second antibody, the concentrations of AChE, MTL, and ET-1 were detected with a microplate reader.

The colon tissue near the cecum was collected by 1 centimeter and fixed in 4% paraformaldehyde for 24 h. Then the tissue was embedded with paraffin, cut into 5-μm-thick slices, and stained with hematoxylin–eosin (H&E) and Alcian Blue Periodic acid Schiff (AB-PAS). Both H&E and AB-PAS sections were scanned with a light microscope for histopathological assessment.

Total RNA was extracted from colon tissue using Trizol reagent. The absorbance ratios at 260 nm and 280 nm were measured by a microspectrophotometer (TECAN Corporation) to determine the purity of RNA. The concentration of RNA was measured and quantified at 2.5 μg. cDNA was synthesized with High-Capacity cDNA Reverse Transcription Kits according to the manufacturer’s instructions (Incubation at 42°C for 35 min, 85°C for 5 min). RT-PCR reactions were performed using the Applied Biosystems 7,500 Real-time PCR System (Applied Biosystems, USA), Power SYBR GREEN PCR MASTER MIX, cDNA template as well as the relative primers under the following conditions: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 15 s, annealing at 60°C for 60 s, and extension at 60°C for 60 s, for a total of 40 cycles. The relative expressions of each gene were calculated according to the 2^-ΔΔCT method. The sample size was 6 and each sample was repeated three times. GAPDH was applied as the internal control. The sequences of the primers are displayed in Supplementary Table S1.

Total proteins extracted from colon tissues were quantified with a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA), then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 80 V, 35 min, 110 V, 80 min) and transferred to polyvinylidene fluoride membranes (Millipore). The membranes were incubated overnight at 4°C with the following primary antibodies: AQP3 (1:1000, ABclonal), β-actin (1:10000, ABclonal), PI3K (1:1000, Affinity), p-PI3K (1:1000, Affinity), AKT (1:1000, Affinity), p-AKT (1:1000, Affinity). After washing the membranes three times with Tris-buffered saline with Tween 20 (TBST, Solarbio), the membranes were incubated with the secondary antibodies for 1 h at room temperature, and then the membranes were washed three times with TBST. Finally, the bands were monitored with a chemiluminescence detection kit (Millipore), and visualized with a gel imaging analysis system (Bio-Rad). The intensity of the protein blots was calculated using ImageJ_v1.8.0.

Fecal samples were collected from each mouse at the time of defecation. Microbial DNA was extracted using the CTAB method and quantified by a Nano-Photometer® NP80 (Implen, Munich, Germany). The v3-v4 region of the bacterial 16S rRNA gene was amplified with the forward primer 341F (CCTAYGGGRBGCASCAG) and reverse primer 806R (GGACTACNNGGGTATCTAAT). DNA libraries were constructed using the Tru-Seq® DNA PCR-Free Sample Preparation Kit (Illumina, USA) according to the manufacturer’s instructions. After assessing library quality with a Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2,100 system, sequencing was performed on an Illumina NovaSeq platform to generate 250 bp paired-end reads. Following filtration of the sequencing data according to the QIIME V1.9.1 quality control pipeline, the data were clustered into operational taxonomic units (OTUs) at a 97% identity level using Uparse software V7.0.1001. Differences in community structure between groups were analyzed by PCoA. Based on the results of species annotation, the top 10 species with the highest genus-level abundance were selected for each sample and a cumulative bar chart of species relative abundance was generated to show the species with high genus-level relative abundance and their proportions for each sample. In addition, Alpha diversity of ASVs was performed with Shannon and Simpson to analyze the diversity, richness, and evenness of the sampled communities. To identify a differential representative of taxa and functional modules before and after SHTC treatment, a linear discriminant analysis effect size (LEfSe) analysis was used to identify differentially abundant taxa modules [those with linear discriminant analysis (LDA) score >4.0].

Take 50 mg of fecal samples, homogenize and mix them with 400 μL of methanol (80%), and centrifuge to obtain the supernatant. The supernatant was added to derivatization reagent (150 μL) and derivatized at 40°C for 40 min. Then supernatant (125 μL) was homogenized with 875 μL mixed internal standard solution. Finally, injected into the LC–MS/MS system for analysis. An ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS) system (Vanquish™ Flex UHPLC-TSQ Altis™, Thermo Scientific Corp., Germany) was used to quantitate SCFA. Separation was performed on a Waters ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) which was maintained at 40°C. The mobile phase, consisting of 10 mM ammonium acetate in water (solvent A) and acetonitrile: isopropanol (1:1) (solvent B), was delivered at a flow rate of 0.30 mL/min. The solvent gradient was set as follows: initial 25% B, 2.5 min; 25–30% B, 3 min; 30–35% B, 3.5 min; 35–38% B, 4 min; 38–40% B, 4.5 min; 40–45% B, 5 min; 45–50% B, 5.5 min; 50–55% B, 6.5 min; 55–58% B, 7 min; 58–70% B, 7.5 min; 70–100% B, 7.8 min; 100–25% B, 10.1 min; 25% B, 12 min. The mass spectrometer was operated in negative multiple reaction mode (MRM). Parameters were as follows: IonSpray Voltage (−4,500 V), Sheath Gas (35 psi), Ion Source Temp (550°C), Auxiliary Gas (50 psi), Collision Gas (55 psi). To further explore the relationship between the abundance of the enteric bacteria and the SCFAs, we used Pearson correlation analysis to elucidate the relationship between differential species in LEfSe and SCFAs.

IEC-6 cells (Cat No. FH0396) are rat small intestine crypt epithelial cells, provided by Shanghai Fuheng Biotechnology Co., Ltd. (Shanghai, China). IEC-6 cells were cultured in petri dishes with a complete medium consisting of DMEM, 10% fetal bovine serum, and 1% double-antibody. The cells were divided into four groups, namely normal group (Nor), sodium acetate-treated group (NaAc), PI3K inhibitor-treated group (LY294002), and sodium acetate + PI3K inhibitor-treated group (LY294002 + NaAc). When the cell density was about 80%, sodium acetate (NaAc, 5 mM) or PI3K inhibitor (LY294002, 20 μM) were added into the medium of corresponding groups, respectively. After incubation for 24 h, all the cells were collected for Western blot analysis.

The statistical analysis was performed using SPSS 25.0 statistical software (Version 25, SPSS; IBM, Armonk, NY) and GraphPad Prism 9.0 software. Data were presented as the mean ± SEM. The Shapiro–Wilk test assessed normal distribution of variables. Differences between three or more groups were evaluated using one-way ANOVA analysis, and the LSD and Dunnett’s tests were used for post hoc evaluations. Independent t-tests were used to compare two independent samples. Spearman’s rank correlation analysis was applied to assess the correlations between environmental factors and gut microbiota, as well as the correlations between gut microbiota abundances and SCFAs levels. p < 0.05 was considered statistically significant (In the figures, #, ## and ### indicate comparisons between the Model group and the Normal group/FMT-Normal group: ^#p < 0.05, ^##p < 0.01, ^###p < 0.001; *, ** and *** indicate comparisons between each administration group and the Model group/FMT-Model group: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001).

The concentrations of 2,3,5,4′-tetrahydroxyl diphenylethylene-2-o-glucoside, Naringin, and Aloin were analyzed using UPLC. The representative chromatograms of the mixed three standards and SHTC extracted solution are shown in Supplementary Figure S1. The concentrations of the components are as follows: 11.28 mg/g for 2,3,5,4′-tetrahydroxyl diphenylethylene-2-o-glucoside, 24.7 mg/g for Naringin, 58.74 mg/g for Aloin in the SHTC (Supplementary Table S2).

The fecal characters were important symptoms for evaluating the successful establishment of a constipation model. Therefore, during the establishment of 5-FU induced constipation mice model, we observed and recorded the size, amount, shape and texture of the fecal (Supplementary Table S3). Mice in the normal group had smooth, oval shaped feces with a slightly moist surface. After the second dose of 5-FU, the mice in the model group exhibited diarrhoea-like symptoms, as evidenced by an increase in the frequency of defecation and unformed, watery stools. As the dosage of 5-FU increased, mice in the model group gradually showed signs of constipation, such as difficulty in defecation, reduced frequency of excretion, dryness and reduced volume of feces. These observations demonstrated that 5-FU could induce constipation in mice under our experimental scheme.

To assess the role of SHTC in 5-FU-induced constipation, three different dosages (100, 200, 400 mg/kg) of SHTC were given to the mice, and the body weight, food, and water intake were recorded. The experimental design is shown in Figure 1A. SHTC slightly restored the body weight of mice, but has no effect on food and water consumption (Supplementary Figure S2). The number of defecations, fecal water content, and defecation time were key indicators in the assessment of constipation. As shown in Figures 1B–D, 5-FU treatment significantly reduced the number of defecation and fecal water content, and increased the time of the first black stool defecation, while SHTC markedly improved these symptoms. Furthermore, SHTC treatment significantly improved 5-FU-induced inhibition of small intestine peristalsis and increased gastrointestinal propulsion rate (Figure 1E). Histological changes were observed by HE and AB/PAS staining of mouse colon tissues (Figure 1F). The colon tissues from the normal group showed healthy and integrated structures with abundant goblet cells and smooth crypt surfaces. The colon tissues from the model group showed severe loss of goblet cells. However, these histological damages were found to be significantly improved after SHTC administration. These results indicated that SHTC has the potential to improve 5-FU-induced constipation.

Studies have reported that AChE, MTL and ET-1 were important gastrointestinal regulation-related peptides involved in the regulation of gastrointestinal motility (Zhang et al., 2021). In this study, the results of ELISA analysis showed that the serum levels of AChE and MTL in the mice of the model group were significantly lower than those in the normal group. SHTC treatment obviously recovered the decrease of AChE and MTL (Figure 2A). The levels of ET-1 were significantly increased in the constipation model mice compared with the normal mice. Nevertheless, this alteration was reversed after SHTC administration (Figure 2A).

Aquaporins (AQPs) are a family of transmembrane proteins expressed in the gastrointestinal tract mainly responsible for water transport. The abnormal expression of AQPs is closely related to the occurrence of gastrointestinal diseases including constipation (Chu and Cai, 2023). In this study, we have observed that the mRNA levels of Aqp3 in the colon of constipated mice were lower than those of normal mice, while the mRNA levels of Aqp4 and Aqp8 in the colon of constipated mice were higher than those of normal mice. SHTC administration significantly increased the expressions of Aqp3 and decreased the expressions of Aqp4 and Aqp8 in the colon of constipated mice (Figure 2B).

To explore the effect of SHTC on the expressions of inflammatory factors, the levels of several cytokines were detected. As shown in Figure 2C, compared with the normal group, the mRNA expression levels of anti-inflammatory factors, Il-4 and Il-10, were significantly decreased in the model group, while the expression levels of Tnf-α, Nf-κb, and iNOS were all significantly increased. After administration of SHTC, the levels of Il-4 and Il-10 were increased, Tnf-α, Nf-κb, and iNOS were decreased. These results suggest that SHTC can affect the levels of intestinal motility regulatory factors, modulate intestinal inflammation, control the expression of AQPs, and alleviate constipation symptoms.

A significant body of evidence has highlighted the role of gut microbiota disturbance in the development of constipation. In addition, studies have reported that gut microbiota disorders are a common adverse effect of patients receiving 5-FU treatment (Alexander et al., 2017). For these reasons, we further carried out 16S rRNA analysis to investigate the effect of SHTC on the homeostasis of the gut microbial community. As shown in Figure 3A, in the PcoA analysis, the intestinal flora composition of mice in the model group was markedly different from that of mice in the normal group; the flora composition of the SHTC-treated group was closer to that of the normal group, which indicated that the intestinal flora composition of mice after SHTC treatment was similar to that of the normal group. In addition, the distributions of the bacteria were analyzed. As shown in Figure 3B and Supplementary Figure S3A, which display the top 10 genera in terms of abundance, 5-FU treatment markedly increased the abundance of harmful bacteria, including Bacteroides and Parabacteroides whereas decreased that of beneficial bacteria, including Muribaculum, Dubosiella, and Faecalibaculum. With the treatment of SHTC, the relative abundance of these bacteria was regulated to levels close to those of the normal group. Notably, these beneficial bacteria are closely associated with increased production of SCFAs. Among them, Muribaculum primarily generates acetic acid and small amounts of propionic acid through the fermentation of carbohydrates (Ayimbila et al., 2025). Studies on Dubosiella have shown that strains within this genus can promote butyric acid production via specific metabolic pathways (Gao et al., 2024). Additionally, the study found that Faecalibaculum can significantly promote the biosynthesis of butyric acid in the colon, which regulates G protein-coupled receptor 109A (GPR109A) to improve ulcerative colitis (Song et al., 2025). Under physiological conditions, the ratio between Firmicutes and Bacteroides (F/B) is relatively stable, whereas a disruption in their ratio may lead to abnormal conditions such as colitis and constipation (Qi et al., 2023). In our study, we observed that the ratio of F/B was significantly lower in the 5-FU model group compared to the normal group, whereas the ratio of F/B was elevated in both the positive drug and the SHTC administration groups (Figure 3C). The Alpha diversity indices of each fecal sample were also calculated to analyze the diversity and richness of the gut microbiota. In the 5-FU model group, Shannon and Simpson indices were decreased. However, the administration of SHTC increased the Shannon and Simpson index (Figures 3D,E). LEfSe analysis (LDA score>4) indicated that the abundance of pernicious bacteria Bacteroides was higher in the model group, while the beneficial bacteria (Lachnoclostridium, Bifidobacteriaceae, Faecalibaculum, Dubosiella) were more abundant in SHTC administration group (Figure 3F). Among them, Lachnoclostridium has been reported to be a core producer of butyric acid in the gut (He et al., 2025). Bifidobacterium could metabolize oligosaccharides into acetate. Enrichment of Bifidobacterium significantly elevates gut acetate concentrations, reduces intestinal pH, and thereby suppresses the proliferation of pathogenic bacteria (Ayimbila et al., 2025).

To investigate the correlation between gut microorganisms and environmental factors, we analyzed the correlation between the top 10 bacteria in terms of abundance (genus level) and inflammatory factors, intestinal motility regulatory factors, and AQPs. As shown in Figure 3G, the results revealed that intestinal bacteria with different functions have various relationships with environmental factors. For example, the abundance of Bacteroides and Parabacteroides was positively correlated with the levels of pro-inflammatory factors but negatively correlated with the levels of anti-inflammatory factors Il-4 and Il-10. Meanwhile, these bacteria have a negative relationship with MTL, AChE, Aqp4, and Aqp8, while have a positive relationship with ET-1 and Aqp3. These observations suggested that SHTC may alleviate intestinal inflammation, alter levels of intestinal motility regulatory biomarkers, and increase intestinal water content by modulating the intestinal microbiota.

To confirm whether the effect of SHTC on constipation is related to gut microbiota, we further conducted FMT experiments (Figure 4A). The results showed that the mice received microbiota from SHTC-treated mice had no alterations in body weight, food intake, and water intake (Supplementary Figure S4). However, in these mice, the number of defecations, fecal water content, defecation time, and intestinal propulsion rate were restored to a healthy state (Figures 4B–E). The results of ELISA showed that the mice in the FMT-SH group significantly restored the level of AChE (Figure 4F) and decreased the level of ET-1 (Figure 4G). The protein expression of AQP3 in the colonic tissues of recipient mice showed that AQP3 in the mice of the FMT-Mod group was lower than that of the FMT-Nor group. However, the expression of AQP3 was significantly increased in the FMT-SH group (Figure 4H). These results confirmed that the therapeutic effect of SHTC on 5-FU-induced constipation may be related to its ability to regulate the gut microbiota.

SCFAs are major microbial metabolites produced by the gut microbiota through glycolysis. A previous study suggested that the level of SCFAs in the feces is related to the development of constipation (Zhuang et al., 2019). In this study, we used HPLC-MS to detect the content of SCFAs in feces and investigated whether SHTC has a regulatory effect on SCFAs. The results were shown in Figure 5A and S3B, the levels of acetic acid, butyric acid, and valeric acid were significantly decreased in the constipated mice compared with those in the Nor group. The levels of acetic acid, butyric acid, and valeric acid were elevated in the SHTC-treated group, with acetic acid being the most significantly increased one. Moreover, the result of Pearson correlation analysis showed that the level of acetic acid was positively correlated with Erysipelotrichales and Firmicutes (Figure 5B). The above results suggested that SHTC may play a regulatory role in SCFAs by regulating intestinal bacteria.

Recent studies have found that SHTC had an activating effect on PI3K/AKT pathway in the liver (Wang et al., 2024). Its main components, naringin and aloin, can also regulate the PI3K/AKT pathway to exert their effects (Lu et al., 2024; He et al., 2021). It has also been shown that activation of PI3K/AKT pathway could promote the expression of AQP3 (Zhang et al., 2022; Park et al., 2022). In addition, considering that SHTC significantly increases the level of acetic acid, we query whether SHTC plays a regulatory role in the activation of PI3K/AKT/AQP3 pathway via alternating the production of acetic acid, which further improved 5-FU induced constipation. To find the answer, we first detected the changes in the protein levels of PI3K/AKT and AQP3 in mouse colon tissues, as shown in Figures 6A 5-FU significantly reduced the phosphorylation of PI3K, AKT, and the protein expression of AQP3 in the colon tissues. After treatment with SHTC, the expression of p-PI3K, p-AKT, and AQP3 increased significantly. The dose-dependent upregulation of AQP3 aligns with the action of SHTC on p-PI3K and p-AKT. This association between dosage and functional proteins underscores the necessity for meticulous dose optimization in future studies.

In addition to in vivo experiments, we treated IEC-6 cells with sodium acetate to simulate the microenvironment of the mice after SHTC administration. We found that sodium acetate significantly promotes the phosphorylation of PI3K and AKT and the expression of AQP3 in Figure 6B. Furthermore, we exposed the cells to the inhibitors of PI3K. After the experimental treatment, the expression of AQP3 was detected. As shown in Figure 6C, sodium acetate significantly promoted the phosphorylation of PI3K and AKT and up-regulated the expression of AQP3. However, when PI3K was inhibited, sodium acetate treatment did not further upregulate the level of AQP3. These results suggest that SHTC may alleviate 5-FU-induced constipation by increasing acetic acid production and activating PI3K/AKT signaling pathway, thereby promoting the expression of AQP3, improving intestinal water content, and alleviating constipation symptoms.