Therapeutic Effects of Shengdu Pingmu Formula on Loperamide-Induced Constipation in Rats via PI3K/AKT Signaling and Gut Microbiota Regulation

Summary

This study demonstrates that Shengdu Pingmu Formula alleviates constipation in rats by increasing fecal water content and intestinal propulsion. The effects are associated with activation of the PI3K/AKT pathway, upregulation of AQP3 and AQP8, and modulation of gut microbiota composition.

Abstract

Constipation is a common gastrointestinal disorder, and current treatments often have limitations. Shengdu Pingmu Formula (SDPF), a modified formulation derived from Traditional Chinese Medicine, has been used clinically to treat constipation, but its mechanism of action remains unclear. This study employed UPLC-Q/TOF-MS analysis to identify 188 compounds in SDPF, of which 50 were conclusively confirmed. In animal experiments, SDPF treatment significantly enhanced intestinal propulsion, increased fecal water content, and shortened the time to first black stool. Network pharmacology and molecular docking analyses suggested the involvement of the PI3K/AKT pathway, which was subsequently validated by observed upregulation of this pathway and increased expression of AQP3 and AQP8 in the rectum. Furthermore, 16S rRNA gene sequencing revealed that SDPF restored gut microbiota homeostasis by elevating beneficial bacteria such as Lactobacillus and Bacteroides while reducing bacteria associated with inflammation. Collectively, SDPF effectively alleviates constipation through mechanisms that regulate the PI3K/AKT pathway and modulate gut microbiota.

Introduction

Constipation is a widespread gastrointestinal disorder characterized by infrequent bowel movements, straining, and hard stools, affecting approximately 20% of the global population with significant socioeconomic implications¹. Chronic constipation not only causes physical complications such as hemorrhoids, anal fissures, and fecal impaction but also contributes to psychological distress, including anxiety and depression, severely compromising patients' quality of life². Conventional therapies, including osmotic laxatives and dietary fiber supplementation, often yield suboptimal outcomes due to limited efficacy, potential side effects, and risk of dependency³. Traditional Chinese Medicine (TCM) offers a holistic approach, utilizing herbal formulations and acupuncture to regulate intestinal motility by restoring Qi flow and visceral homeostasis⁴. However, the lack of standardized protocols and mechanistic clarity limits broader clinical adoption. Emerging research highlights the potential of novel interventions, such as gut microbiota modulation and bioactive TCM-derived compounds, which may synergistically enhance intestinal function with minimal adverse effects⁵. Consequently, the development of an evidence-based integrative therapy combining TCM principles with modern pharmacotherapy holds promise for revolutionizing the management of constipation.

Slow transit constipation (STC) is a vital pathological model in basic research, effectively simulating the hallmark motility disorder of human constipation—significantly delayed colonic transit⁶. It serves as a "decoder" for elucidating the molecular mechanisms of constipation, enabling researchers to directly examine pathological changes in rectal tissue⁷, the enteric nervous system⁸, interstitial cells of Cajal⁹, and key signaling pathways¹⁰, thereby bridging macroscopic symptoms with cellular and molecular abnormalities beyond the reach of clinical studies¹¹. Internationally, established methods for developing STC rat models primarily include chemically induced approaches¹², notably using loperamide—a peripheral opioid receptor agonist that suppresses neuronal activity and acetylcholine release to consistently induce delayed transit within 5–7 days—and etiology-mimicking strategies¹³, such as long-term, high-dose stimulant laxative administration, which leads to colonic nerve impairment, melanosis coli, and subsequent laxative-dependent colonic inertia. Thus, the STC rat model not only offers a window into the complex etiology of constipation but also serves as a crucial bridge connecting basic mechanistic insights to clinical applications.

Shengdu Pingmu Formula (SDPF), comprising Radix Paeoniae Alba (Baishao), Curcuma longa (Jianghuang), Rhizoma cibotii (Tanggouji), Siegsbeckia orientalis L. (Xixiancao), and Clematis chinensis Osbeck (Weilingxian), is a modified hospital preparation derived from Traditional Chinese Medicine theory and has long been used in our department for related disorders. Preliminary clinical observations from our hospital suggested that SDPF may alleviate constipation symptoms (unpublished data). Research has demonstrated that the PI3K/AKT signaling pathway is involved in the regulation of colonic motility¹⁴, in which gut microbiota and their metabolites may play a critical role by modulating enteric neural and smooth muscle activities¹⁵. Nevertheless, the precise regulatory mechanisms remain incompletely understood. We propose that the PI3K/AKT pathway and the gut microbiota together form an essential axis for regulating colonic motility. It is hypothesized that the therapeutic effects of TCM formulations on colonic dysmotility may be associated with modulation of the PI3K/AKT pathway and rebalancing of the microbiota-gut axis. This study addresses this gap by proposing and testing a novel, integrated hypothesis: that the PI3K/AKT pathway and the gut microbiota function as a critical, interdependent axis for colonic motility. The primary objective of this work is to determine whether the therapeutic effect of SDPF on slow-transit constipation is mediated through the dual modulation of this axis. Specifically, using a rat model, we investigated the effects of SDPF on key PI3K/AKT signaling proteins, shifts in gut microbiota composition, and colonic motility function. By elucidating this combined mechanism, this research provides, to our knowledge, the first experimental evidence for a microbiota-PI3K/AKT axis as a unified target of TCM-based intervention, thereby offering a more comprehensive molecular basis for the treatment of colonic motility disorders.

Protocol

All experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, revised 2011) and were approved by the Ethics Review Board of the Shandong University of Traditional Chinese Medicine (Welfare Ethics License number: SDUTCM20250421238).

Preparation of SDPF
The herbal mixture consists of Radix Paeoniae Alba (Baishao) 20 g, Curcuma longa (Jianghuang) 10 g, Rhizoma Cibotii (Tanggouji) 10 g, Siegsbeckia orientalis L. (Xixiancao) 30 g, and Clematis chinensis Osbeck (Weilingxian) 30 g. This combination ratio was determined based on clinical experience. The formula was extracted by first soaking in deionized water at a solid-to-liquid ratio of 1:9 (g/mL) for 30 min, followed by boiling for 60 min. The resulting solution was immediately filtered through gauze and centrifuged at 4,500 × g for 5 min to collect the supernatant. The residue was then re-extracted twice under the same conditions (1:9 ratio, deionized water) with a reduced boiling time of 30 min for each subsequent extraction. The supernatants from all three extractions were combined, concentrated, and vacuum freeze-dried for 5–7 days to obtain the SDPF powder, which was stored under refrigeration for further use.

Constituent analysis of SDPF
To prepare solid samples, a suitable quantity of homogenized material was transferred into a 2 mL centrifuge tube, combined with 1 mL of 70% methanol in water and 3 mm stainless steel beads, then processed in an automated grinder for 3 min, followed by 10 min of vortexing. Liquid specimens were diluted with pure methanol and vortexed for 10 min. Subsequently, all samples were centrifuged at 12,000 × g for 10 min at 4 °C, and the resulting supernatants were filtered through a 0.22 µm filter. Before instrumental analysis, 2-chlorophenylalanine (internal standard, 100 µg/mL) was incorporated into the filtrate to achieve a final concentration of 1 mg/L. Chromatographic separation was performed on an LC-MS system equipped with a C18 column (1.8 µm × 2.1 mm × 100 mm), maintained at 30 °C, with a flow rate of 0.3 mL/min. The mobile phase consisted of 0.1% formic acid in water (A) and neat acetonitrile (B). The injection volume was set to 2 µL, and the autosampler was kept at 4 °C. Mass spectrometry detection was performed in both positive and negative ionization modes under the following conditions: heater temperature at 325 °C, sheath gas at 45 arbitrary units, auxiliary gas at 15 arb, sweep gas at 1 arb, electrospray voltage at 3.5 kV, capillary temperature at 330 °C, and S-Lens RF level at 55%. Data were acquired across an m/z range of 100–1500 in full-scan MS mode at a resolution of 120,000, while MS/MS fragmentation was conducted in data-dependent acquisition mode (dd-MS², TopN=5) at 60,000 resolution using higher-energy collisional dissociation.

Animal experiment
Seventy-two male Sprague-Dawley rats (aged 6 weeks, initial body weight 180–200 g; RRID: IMSR_JAX:000664) were housed under SPF conditions (24 ± 1 °C, 12 h light/dark cycle) with free access to food and water. After one week of acclimatization, they were randomly divided into six groups (n=12/group) based on body weight: (1) blank control (normal saline), (2) constipation model group (10 mg/kg loperamide hydrochloride), (3) low(SDPF-L)-, (4) medium(SDPF-M)-, and (5) high(SDPF-H)-dose SDPF groups (6, 9, and 12 g/kg, respectively), and (6) positive control (2 mg/kg mosapride citrate). SDPF was freshly prepared as a suspension in distilled water at concentrations of 3, 4.5, and 6 g/mL and administered by oral gavage at a fixed volume of 2 mL/kg, corresponding to doses of 6, 9, and 12 g/kg, respectively. Loperamide hydrochloride was freshly prepared as a suspension in distilled water at a concentration of 5 mg/mL and administered by oral gavage at a fixed volume of 2 mL/kg (equivalent to 10 mg/kg). After one week of acclimatization, constipation was induced in groups (2) – (6) by oral gavage of loperamide hydrochloride once daily for one week. The blank control group received an equivalent volume of normal saline. Subsequently, for the following week, rats in groups (3)–(5) received SDPF treatment via oral gavage once daily, the positive control group (6) additionally received mosapride citrate (2 mg/kg) concurrently, while rats in groups (2) – (6) continued to receive loperamide hydrochloride (10 mg/kg) once daily to maintain the constipation model. The blank control group continued to receive normal saline. At the end of the treatment period, all rats were fasted overnight with free access to water. Animals were then anesthetized with an intraperitoneal injection of sodium pentobarbital (40 mg/kg body weight). Following deep anesthesia confirmed by the absence of pedal reflexes, rats were euthanized by cervical dislocation. Immediately thereafter, rectal tissues were rapidly harvested. For histological and immunohistochemical analyses, tissue segments were fixed in 4% paraformaldehyde. For protein expression analysis, tissue samples were snap-frozen in liquid nitrogen and stored at -80 °C until further use.

First black stool
The time to excretion of the first black stool was recorded for three rats per group. Prior to the experiment, all rats were fasted for 12 h with free access to water. Each animal then received an intragastric administration of a prepared ink suspension (5% activated charcoal and 5% gum Arabic) at a dose of 10 mL/kg body weight, with the exact time of administration recorded. Following the gavage, the rats were individually housed in separate cages with ad libitum access to food and water. The appearance of the first black stool from each rat was carefully observed and recorded to calculate the intestinal transit time.

Fecal water content
Following successful model establishment and at the end of treatment, fresh fecal samples excreted within 2 h were collected from each group of rats (n = 6) and placed into pre-weighed dry EP tubes. The wet weight of the feces was immediately recorded. Subsequently, the samples were transferred to a drying oven and dehydrated at 60 °C for 36 h. After the initial drying period, the samples were weighed and returned to the oven, with weight measurements repeated every 2 h until constant weight was achieved (indicating complete dehydration). The fecal water content percentage was then calculated using the following formula:

Fecal water content (%) = [(Wet weight - Dry weight) / Wet weight] × 100%

Intestinal propulsion rate
Following a 12 h fasting period (with free access to water), rats from each group (n = 6) were administered an intragastric dose (10 mL/kg) of ink suspension containing 5% activated charcoal and 5% gum Arabic. Twenty-five minutes later, the animals were anesthetized via intraperitoneal injection of 5% chloral hydrate. The abdominal region was disinfected by wiping with 75% ethanol before tissue collection, and the limbs were then pinned to a dissection board. The abdominal cavity was carefully exposed with surgical scissors, and the intestinal tract from the pylorus to the ileocecal junction was excised. The isolated intestine was straightened on sterile filter paper, and two measurements were recorded: (1) the total length of the small intestine, and (2) the distance traveled by the ink front. The intestinal propulsion ratio was then calculated using the following formula:

Intestinal propulsion rate (%) = (Ink migration distance / Total intestinal length) × 100%

HE staining
For the control, model, SDPF-M, and Mos groups (n = 3 per group), rectal specimens were collected and processed for hematoxylin and eosin (HE) staining. The specimens were fixed in 4% paraformaldehyde, dehydrated in graded ethanol, and embedded in paraffin blocks. Thin sections, measuring 4–5 µm in thickness, were prepared and affixed to glass slides prior to undergoing conventional hematoxylin and eosin staining. The procedure commenced with deparaffinization using xylene, followed by rehydration through a series of descending ethanol concentrations. Nuclei were stained by exposing sections to hematoxylin for 5–8 min. Following differentiation in 1% acid alcohol and bluing in 0.2% ammonia water, the cytoplasm and extracellular matrix were counterstained with eosin for 1–2 min. Finally, the stained sections were dehydrated, cleared, and mounted with coverslips for microscopic observation. This approach enabled a detailed assessment of intestinal mucosal morphology, including the structure of villi and crypts, the condition of epithelial cells, and the presence of inflammatory infiltrates within the lamina propria.

KEGG pathway and GO enrichment analysis
Leveraging the previously established chemical profile of SDPF, silico predictions of protein targets for each constituent were performed using the SwissTargetPrediction platform. Putative targets relevant to constipation were filtered and subjected to functional annotation using the DAVID 6.8 database, with a significance cutoff of P < 0.05. The resulting gene set was subsequently subjected to GO classification to elucidate its roles in biological processes, molecular functions, and cellular components. To further elucidate their involvement in disease pathogenesis, these targets were subsequently mapped to KEGG pathways, revealing several signaling cascades that were significantly enriched and potentially implicated in the onset and development of constipation.

Construction of the "Component-Target-Pathway" network
A "Component-Target-Pathway" network was assembled in Cytoscape (version 3.6.1) by systematically integrating the chemical constituents of SDPF, their corresponding therapeutic targets for constipation, and the relevant enriched pathways. Within this graphical model, different node geometries were employed to represent the three data types: circles for compounds, triangles for targets, and rectangles for pathways. The strength of connections between these nodes was quantified through edge weighting, providing a holistic view of the intricate, multi-layered pharmacological interactions.

Molecular docking
For the molecular docking analysis, four bioactive compounds—namely Cyclocurcumin, Albiflorin, Chlorogenic acid, and Quercetin—were selected as ligand molecules, with their structures downloaded from the PubChem database using corresponding CAS registry numbers. To prepare the protein receptors, the UniProt knowledgebase was consulted to confirm the target proteins PI3K and AKT, and their three-dimensional structures were subsequently retrieved from the RCSB Protein Data Bank. These protein and compound structures were then processed in Discovery Studio 2019, where hydrogen atoms were added, charges were assigned, and atom types were defined; additionally, potential binding pockets on the receptors were identified. The prepared ligands and receptors were subsequently saved in PDBQT format for docking simulations. The docking procedure was carried out with the following configuration: a pose cluster radius of 0.5, generation of 10 random ligand conformations, and 10 refinement orientations. Following completion of the docking runs, candidate complexes were evaluated and ranked according to their predicted binding free energies. The most energetically favorable binding poses were ultimately selected and visualized using PyMOL to illustrate key intermolecular interactions.

Immunohistochemistry
Paraffin-embedded rectal specimens, cut to a thickness of 4 µm and obtained from the Control, Model, and SDPF-M treatment cohorts, were first cleared of paraffin and progressively hydrated. Antigenic sites were unmasked by heating the slides in citrate buffer adjusted to pH 6.0. To quench endogenous peroxidases, the tissues were treated with a 3% hydrogen peroxide solution, followed by incubation with normal goat serum to reduce non-specific antibody binding. The sections were then incubated overnight at 4 °C with primary antibodies targeting phosphorylated PI3K and AKT; parallel sections incubated with PBS served as negative controls. After thorough washing, horseradish peroxidase-conjugated secondary antibodies were applied, and immunostaining was developed using a DAB chromogen under microscopic observation to control reaction time. Finally, the tissues were lightly counterstained with hematoxylin, dehydrated through graded alcohols, cleared, and mounted. The expression levels of p‑PI3K and p‑AKT were qualitatively assessed by two independent investigators blinded to the experimental groups, and representative images were selected to illustrate differences among the groups.

Western blotting
Rectal tissue samples from rats were rinsed twice with chilled phosphate-buffered saline before being homogenized on ice for 10 min in a suitable volume of RIPA lysis buffer. Following homogenization, the mixture was centrifuged at 15,000 × g, and the supernatant was retained for analysis. Protein quantification was performed utilizing the bicinchoninic acid method. Subsequently, the samples were combined with SDS loading buffer and denatured via incubation at 95 °C for 5 min. Equivalent protein quantities were then resolved through SDS-polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene difluoride membranes with a pore size of 0.45 µm. After transfer, the membranes were allowed to air-dry before being blocked with 5% non-fat dry milk in TBST for 1 h at ambient temperature. Following this step, the membranes were rinsed with TBST and then incubated overnight at 4 °C with primary antibodies targeting AQP3 (RRID: AB_2837708) and AQP8 (RRID: AB_605941). The next day, after additional washes with TBST, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Immunoreactive bands were ultimately detected and captured using an imaging system.

Immunofluorescence
The immunofluorescence staining procedure was performed on rectal tissue sections from Control, Model, and SDPF-M-treated rats as follows: after cryosectioning, tissues were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with a suitable blocking buffer to prevent non-specific binding. The sections were then incubated overnight at 4 °C with a mixture of primary antibodies against AQP3 and AQP8, after which unbound antibodies were washed away and the tissues were incubated for 1 h at room temperature in the dark with a mixture of secondary antibodies—specifically, goat anti-rabbit IgG conjugated to Alexa Fluor 594 (red) to label AQP3 and goat anti-mouse IgG conjugated to Alexa Fluor 488 (green) to label AQP8. Following secondary antibody incubation and subsequent washes, cell nuclei were counterstained with DAPI, and the slides were coverslipped with an aqueous mounting medium prior to visualization and image acquisition using a fluorescence or confocal microscope.

16S rRNA sequencing of fecal microbiota
For each experimental group (n = 8), bacterial genomic DNA was isolated from rat fecal material using a commercial Soil DNA Extraction Kit, with all procedures strictly following the supplier's protocol. PCR amplification targeting the V3–V4 hypervariable regions of the 16S rRNA gene was performed on a thermal cycler using the primer pair 338F/806R. Amplicon sequencing was performed on the Illumina MiSeq platform according to the manufacturer’s guidelines, generating sufficient sequencing depth to achieve rarefaction curve saturation and ensure reliable microbial diversity analysis. Upon completion of sequencing, the raw reads were quality-filtered, adapter-removed, and merged using Trimmomatic and FLASH. High-quality sequences were subsequently clustered into operational taxonomic units (OTUs) at a 97% similarity threshold using UPARSE, and potential chimeric sequences were identified and removed using UCHIME. Taxonomic assignment of representative sequences was performed using the RDP classifier against the SILVA reference database, with a confidence threshold of 70%. To evaluate alpha diversity within microbial communities, the Chao1 richness estimator and Shannon diversity index were calculated using the Mothur software package (v.1.30.2). For beta-diversity analysis, principal coordinate analysis was performed to visualize compositional dissimilarities between groups. A Venn diagram was constructed to illustrate the distribution of unique and shared OTUs across different experimental conditions. Identification of differentially abundant taxa between cohorts was performed using linear discriminant analysis effect size. Lastly, predictive profiling of microbial community functions was performed using PICRUSt2, which inferred functional potential from KEGG orthology annotations.

Statistics
All statistical analyses were performed using SPSS 23.0 and GraphPad Prism 8.0. Data are presented as mean ± standard deviation (SD). The normality of data distribution was assessed using the Shapiro–Wilk test. For comparisons between two groups, the independent-samples Student's t‑test was used for normally distributed data, and the Mann–Whitney U test was used for non-normally distributed data. For comparisons involving three or more groups, one-way analysis of variance (ANOVA) followed by Tukey's post‑hoc test was employed for normally distributed data, whereas the Kruskal–Wallis test followed by Dunn's post‑hoc test was used for non-normally distributed data. For microbiome analyses, differentially abundant taxa were identified using linear discriminant analysis effect size (LEfSe) with the default threshold (LDA score > 2.0), and multiple testing correction was applied using the Benjamini–Hochberg false discovery rate (FDR) method. Microbiome statistical analyses were performed using Mothur (v.1.30.2), LEfSe (online tool, Huttenhower lab), and PICRUSt2 (v.2.5.0). A p‑value of less than 0.05 was considered statistically significant.

Results

SDPF extraction and lyophilization
SDPF is a multi-herbal formulation comprising five medicinal plants: Radix Paeoniae Alba (Baishao), Curcuma longa (Jianghuang), Rhizoma cibotii (Tanggouji), Siegesbeckia orientalis L. (Xixiancao), and Clematis chinensis Osbeck (Weilingxian) (Figure 1A). The mixture was extracted with purified water and lyophilized to a powder for further analysis (Figure 1B).

...

Discussion

Chronic constipation is a multifactorial disorder involving complex interactions among gut microbiota dysbiosis, impaired neuromuscular function, and altered intestinal secretion. These manifestations persist for more than six months and significantly impair patients' quality of life¹⁹. Constipation can be categorized into several subtypes, including slow-transit, normal-transit, defecation disorder, and mixed types²⁰. Although the exact pathogenesis of chronic constipa...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Automated grinder	High-throughput tissue homogenizer for sample grinding	JXFSTPRP-48
LC-MS system	Thermo Fisher Scientific	Q Exactive HF LC-MS system	Ultra-high-performance liquid chromatography–mass spectrometry system
Mass spectrometer	Thermo Fisher Scientific	Q Exactive HF	High-resolution Orbitrap mass spectrometer
C18 column	Agilent Technologies	Zorbax Eclipse Plus C18 (2.1 × 100 mm, 1.8 μm)	Reverse-phase column for compound separation
Imaging scanning system	GE Healthcare	ImageQuant LAS 4000	Gel and blot imaging system
Thermocycler	Applied Biosystems (Thermo Fisher Scientific)	GeneAmp PCR System 9700	PCR amplification instrument
Soil DNA Extraction Kit	Omega Bio-Tek	D5625	Kit for microbial DNA extraction from fecal/soil samples