Abstract
Aim of the Study
Zhizhu Decoction (ZZD), a classical traditional Chinese herbal formula composed of Atractylodis Macrocephalae Rhizoma and Aurantii Fructus Immaturus, has long been used to treat gastrointestinal disorders resembling constipation-predominant irritable bowel syndrome (IBS-C). This study aimed to elucidate the potential therapeutic mechanisms of ZZD in IBS-C by integrating network pharmacology, molecular docking, and in vivo experimental validation.
Materials and Methods
Active compounds and targets of ZZD were identified using network pharmacology, followed by molecular docking to evaluate compound-target interactions. An IBS-C rat model was established to assess the therapeutic effects of ZZD. H&E and AB-PAS staining were used to observe histological lesions in colonic tissues. TUNEL staining was performed to detect apoptotic epithelial cells in colon sections. Immunohistochemistry (IHC) was used to assess the spatial distribution of TPH1 and tight junction proteins ZO-1 and Claudin-1 in colon tissues. RT-qPCR combined with western blotting was used to quantify the expression of ZO-1 and Claudin-1. Serum 5-HT concentrations were determined by ELISA, and liquid chromatography-mass spectrometry (LC–MS) was used to measure fecal short-chain fatty acid (SCFA) levels.
Results
Network pharmacology analysis: ZZD contains 23 bioactive compounds. This herbal formula mainly acts on core targets including AKT1, BCL-2, Caspase-3, and EGFR, with prominent enrichment of PI3K/AKT, apoptosis, TNF and NF-κB signaling. Molecular docking results further confirmed the favorable binding affinity of its main active ingredients (luteolin, naringenin, 3β-acetoxyatractylone, and 8β-ethoxyatractylenolide III) to these targets. In vivo experiments: In IBS-C model rats, ZZD intervention alleviated weight loss, elevated 24-h fecal output, fecal water content and Bristol stool scale scores, accelerated small intestinal carbon powder transit, and lowered visceral hypersensitivity as indicated by reduced AWR scores (P < 0.05), and it recovered goblet cell abundance, improved crypt architecture and thickened colonic mucus and muscular layers. ZZD markedly alleviated colonic injury and maintained intestinal barrier and tight junction integrity, as evidenced by upregulation of mRNA and protein levels of tight junction proteins ZO-1 and Claudin-1 (P < 0.05), as well as restoration of their continuous distribution in rat colonic tissues. ZZD suppressed epithelial apoptosis by downregulating Caspase-3 and upregulating BCL-2 and c-JUN (P < 0.05). In addition, IHC staining for TPH1, a specific marker of EC cells, revealed that ZZD treatment significantly increased the number of TPH1‑positive cells in the colonic epithelium (P < 0.0001), accompanied by elevated serum 5‑HT levels (P < 0.0001), suggesting recovery of the EC cell population and intestinal 5‑HT synthetic capacity. Moreover, ZZD increased the phosphorylation levels of colonic EGFR, PI3K and AKT (P < 0.05). It also significantly raised fecal SCFA levels (P < 0.05), implying improved gut microbial metabolism as a potential protective factor.
Conclusion
ZZD exerts therapeutic effects against IBS-C that involve multiple mechanisms, including inhibition of intestinal epithelial apoptosis, preservation of tight-junction protein expression and distribution (ZO-1 and Claudin-1), restoration of EC cell integrity (as indicated by increased TPH1-positive cell abundance on IHC), and modulation of short-chain fatty acid metabolism. In addition, increased phosphorylation of EGFR, PI3K, and AKT was observed in association with these protective effects, suggesting potential involvement of this pathway in the underlying mechanisms.
Graphical Abstract
Supplementary Information
The online version contains supplementary material available at https://doi.org/10.1186/s12575-026-00353-1.
Keywords: Zhizhu Decoction1, IBS-C2, Intestinal Barrier Integrity3, Apoptosis4, EGFR/PI3K/AKT Pathway5, Network Pharmacology6
Introduction
Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder characterized by recurrent abdominal pain associated with defecation and alterations in bowel habits, including constipation, diarrhea, or mixed patterns. Among its subtypes defined by the Rome IV criteria, constipation-predominant IBS (IBS-C) represents a substantial proportion of patients and is associated with persistent symptoms and marked impairment in quality of life [1]. Despite the absence of overt structural abnormalities, IBS-C poses a significant clinical challenge due to its chronic course, symptom recurrence, and limited effectiveness of currently available treatments. In the United States, IBS imposes a substantial economic burden, with recent estimates from a large cohort study showing that IBS patients incur mean annual all-cause healthcare costs of $13,288 per patient, substantially higher than matched non-IBS controls ($5,999). Notably, IBS-C patients bore the highest costs among all IBS subtypes, with median total healthcare expenditures reaching $16,005 per patient annually [2].
The pathophysiology of IBS-C is complex and multifactorial, involving dysregulation of the gut–brain axis, visceral hypersensitivity, altered gastrointestinal motility, low-grade mucosal inflammation, microbial imbalance, and aberrant central processing of visceral signals [3–5]. In recent years, increasing attention has been directed toward the role of intestinal epithelial barrier dysfunction in IBS-C. The intestinal epithelial barrier, composed of epithelial cells, tight junction proteins, mucus, and secretory factors, is essential for maintaining intestinal homeostasis [6]. The balance between apoptosis and the proliferation of intestinal epithelial cells plays a significant role in maintaining intestinal homeostasis. Excessive apoptosis of epithelial cells can disrupt the epithelial defense system, potentially leading to impaired epithelial barrier function and increased intestinal permeability [7–9]. Thus, impairment of epithelial integrity represents a critical yet insufficiently explored pathological feature of IBS-C.
Current therapeutic strategies for IBS-C are largely symptom-oriented and primarily target single pathways, such as intestinal motility or secretion. While these agents may provide partial relief, their long-term efficacy is limited and adverse effects remain a concern [10]. In contrast, traditional Chinese medicine (TCM) adopts a holistic and syndrome-differentiated approach, emphasizing the regulation of functional imbalance rather than isolated symptoms. According to TCM theory, IBS-C is commonly attributed to disharmony between the spleen and stomach, resulting in impaired qi movement and weakened intestinal propulsion. In TCM, qi is one of the fundamental substances of the human body, providing energy and vitality. Qi disorders manifest in various forms, with qi stagnation and qi deficiency being the most frequently observed. Qi stagnation refers to the sluggish, impaired, or even blocked flow of qi in specific parts of the body, internal organs, or meridians. Qi deficiency, on the other hand, refers to a deficiency of qi in the internal organs or throughout the body, resulting in an inability to supply sufficient energy and vitality for physical and mental activities [11]. Spleen deficiency is traditionally characterized by weakened digestive and absorptive functions, which may contribute to delayed intestinal transit and constipation. With the modernization of research methods for TCM syndromes, combined with the application of animal models of these syndromes, the biological essence of spleen deficiency syndrome has been gradually elucidated. Centered on the digestive system, this understanding encompasses phenotypic characteristics, such as gastrointestinal motility, secretion, immunity, and the microbiome, as well as molecular mechanisms involving multiple genes and proteins. Zhizhu Decoction (ZZD), a classical two-herb formula recorded in Jin Gui Yao Lue (Synopsis of the Golden Chamber, ca. third century AD), consists of Atractylodis Macrocephalae Rhizoma (Baizhu, BZ) and Aurantii Fructus Immaturus (Zhishi, ZS). Traditionally, ZZD has been prescribed for abdominal distension, fullness, food stagnation, and constipation [12, 13]. Baizhu is used to tonify spleen qi, strengthen digestive function, and alleviate abdominal distension [14], whereas Zhishi promotes qi movement, relieves food stagnation, and facilitates intestinal transit [15]. From a TCM perspective, this herb pair addresses spleen deficiency-related impaired intestinal propulsion and qi stagnation, which conceptually aligns with dysmotility and altered visceral function observed in IBS-C.
Recent experimental studies on ZZD and related formulations have demonstrated beneficial effects in models of functional or slow-transit constipation, including enhanced intestinal motility, increased fecal water content, and modulation of gut microbiota composition [16–19]. However, IBS-C is a distinct functional disorder in which constipation is accompanied by abdominal pain and visceral hypersensitivity, and the precise molecular mechanisms by which ZZD may exert protective effects in this context remain unclear. In particular, whether ZZD can improve IBS-C by preserving intestinal epithelial integrity and restoring intestinal homeostasis has not been systematically investigated.
To address this knowledge gap, the present study investigated the therapeutic effects of ZZD in an IBS-C rat model, with a specific focus on intestinal epithelial barrier integrity and epithelial cell survival. An integrative strategy combining network pharmacology, molecular docking, and in vivo experimental validation was employed to explore the potential mechanisms underlying ZZD action. By bridging classical ethnopharmacological theory with modern experimental evidence, this study aims to provide a mechanistic basis for the traditional use of ZZD and to support its rational development as a complementary therapeutic option for IBS-C.
Materials and Methods
Reagents and Instruments
Mosapride Citrate Capsules were purchased from Shanghai Sine Pharmaceutical Co., Ltd. (Shanghai, China). EGFR Monoclonal antibody (66,455–1-Ig, Proteintech); Phospho-EGFR Polyclonal antibody (30,277–1-AP, Proteintech); PI3K p85 alpha Monoclonal Antibody (MA1-74,183, Thermofisher); Phospho-PI3K p85 alpha Polyclonal Antibody (PA5-104,853, Thermofisher); AKT Monoclonal antibody (60,203–2-Ig, Proteintech); Phospho-AKT Monoclonal antibody (66,444–1-Ig, Proteintech); TPH1 Polyclonal Antibody (PA1-777, Thermofisher); c-Jun Monoclonal Antibody (MA5-15,881, Thermofisher); Bcl-2 Monoclonal Antibody (MA5-11,757, Thermofisher); Caspase-3 Monoclonal Antibody (43–7800, Thermofisher); ZO-1 Polyclonal Antibody(33–9100, Thermofisher); Claudin-1 Polyclonal Antibody (51–9000, Thermofisher); GAPDH Monoclonal Antibody (60,004–1-Ig, Proteintech); Beta Actin Polyclonal Antibody (380,624, ZENBIO); 5-hydroxytryptamine (5-HT) ELISA Kit (E-EL-0033c, Elabscience); TUNEL Apoptosis Detection Kit (PF00006, Proteintech); Concentrated DAB Kit (K135925C, ZSGB-BIO Co., Ltd.); Spectrometer (μQuant, BioTek); Microtome (Leica-2016, Germany); Tissue Dehydrator (Changzhou Zhongwei Electronic Instrument Co., Ltd.); Embedding Machine (Changzhou Suburbs Zhongwei Electronic Instrument Factory); Digital Trinocular Camera Microscope (BA400Digital, Motic Instruments Group Co., Ltd.); ACQUITY UPLC H-Class (Waters, Corporation, USA); Q Exactive Orbitrap Mass Spectrometer (Thermo Scientific, USA).
Network Pharmacology
Collection of Active Components of ZZD and Prediction of the Targets
Used the Traditional Chinese Medicine Systems Pharmacology (TCMSP, https://www.tcmsp-e.com/tcmsp.php) database to find out the active chemical compositions of BZ and ZS and keeping chemical components that have an oral bioavailability (OB) of at least 30% and a drug-likeness (DL) of at least 0.18 based on pharmacokinetic parameters. The TCMSP platform was used to collect the corresponding target information of the active chemical composition in the aforementioned medicine.
IBS-C Disease Target Screening
The GeneCards database (https://www.genecards.org/) was used to identify disease-associated gene targets by employing the keywords “irritable bowel syndrome with predominant constipation,” “constipation predominant irritable bowel syndrome,” or “IBS-C.”
Construction of Protein–Protein Interaction (PPI) Networks
Once the predicted drug and disease targets intersected, the intersecting targets were imported into the STRING database (https://cn.string-db.org/). The species “Homo sapiens” was chosen, the screening condition “medium confidence (≥ 0.400)” was applied, and disconnected nodes were eliminated to create a PPI network. A “drug-target-disease” composite network was then created using Cytoscape 3.7.1 software.
Target Gene Function and Pathway Enrichment
Target genes were imported into the DAVID database (https://davidbioinformatics.nih.gov/) for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Perform a visual analysis and sort the findings based on p-values.
Molecular Docking of the Core Components of Drugs with the Core Targets of Diseases
The mol2 structure files of the core drug components (luteolin, naringenin, 3β-acetoxyatractylone, and 8β-ethoxy atractylenolide III), were downloaded from the TCMSP database. Proteins with higher degree values related to IBS-C pathways in the KEGG enrichment analysis were selected, and the crystal structures of the core targets were downloaded from the PDB database (https://www.rcsb.org/). The co-crystallized ligands, metal ions, and water molecules present in the crystal structures were removed using the PyMOL software. The potential ligand-binding pockets of the core target proteins were predicted using CavityPlus2022 (http://pkumdl.cn:8000/cavityplus/index.php#/), and the top-scoring binding pocket, along with the coordinates and size information of the docking box, were selected. The prediction results are presented in Table S1. The target proteins and core drug components were subjected to semi-flexible docking validation using AutoDock Vina 1.1.2 software to predict the interactions between the key protein and the key small-molecule compound. Ligand-target binding was visualized using PyMOL software.
Medication Preparation
A total of 12 g of ZS slices and 6 g of BZ slices were prepared according to the ratio specified in the Synopsis of the Golden Chamber [12], supplied by Beijing Kangrentang Pharmaceutical Co., Ltd., and verified by Prof. Xuegui Tang from Chengdu University of TCM (Table S2). The herbs were soaked in six volumes of distilled water for 60 min and then decocted twice (30 min each) with six and four volumes of water. The combined filtrates were concentrated to 1 g/mL (crude drug) using a rotary evaporator, bottled under sterile conditions, and stored at 4 °C. Dilutions were performed as required.
UPLC-MS Analysis of ZZD
Following vacuum freeze-drying of the filtered medicinal liquid, 2 g of the lyophilized powder was dissolved in 10 mL of 70% methanol and vortexed to a paste-like consistency. The mixture was ultrasonicated for 1 h and then centrifuged at 13,000 rpm for 10 min. The supernatant was collected for subsequent analysis. Chromatographic separation was achieved using an ACQUITY UPLC® BEH C18 column (2.1 × 100 mm, 1.7 µm) with a gradient mobile phase consisting of 0.1% aqueous formic acid and 0.1% formic acid in acetonitrile, at a flow rate of 0.3 mL/min and a column temperature of 30 °C. Detection utilized an electrospray ionization (ESI) source operating in positive and negative ion modes. The spray voltage was set to 3.5 kV (positive) and 3.0 kV (negative), with an ion source temperature of 350 °C. The sheath and auxiliary gas flows were maintained at 40 and 15 arb, respectively, and the ion transfer tube temperature was set to 350 °C. Data were acquired in Full MS/dd-MS2 mode with resolutions of 35,000 (MS1) and 17,500 (MS2), scanning an m/z range of 100–1500 with collision energy settings of 20, 40, and 60 eV (Fig. 1).
Fig. 1.
Detailed Experimental Timeline and Schematic Diagram of in Vivo Study Design
In Vivo Experiments
Laboratory Animals
Forty-eight male SPF-grade SD rats, aged 6–7 weeks and weighing 180–190 g, were obtained from the Experimental Animal Center of North Sichuan Medical College (NSMC) under License No. SYXK (Chuan) 2023–0076. Rats were maintained in a controlled environment (23 ± 1 °C, 55 ± 5% humidity, 12-h light/dark cycle) with unrestricted access to food and water. All procedures were approved by the NSMC Ethics Committee (approval no. 2024077).
Animal Grouping and Treatment
After one week of acclimatization, 48 male SD rats were randomly divided into six groups using a random number table, with eight rats per group: control, IBS-C model, ZZD low-dose (ZZD-L, 0.90 g/kg), ZZD medium-dose (ZZD-M, 1.80 g/kg), ZZD high-dose (ZZD-H, 2.70 g/kg), and mosapride (1.57 mg/kg) groups. The ZZD doses for rats were converted from the human clinical dose of 18 g crude drug per day for a 60 kg adult, based on the standard body surface area conversion method described in the Pharmacological Experimental Methodology [20]. According to this method, the equivalent rat dose is six times the human dose per kilogram of body weight.
Induction of IBS-C Model
Rats in the control group were gavaged with distilled water at room temperature (1 mL/100 g body weight), whereas all other rats were gavaged with ice-cold distilled water (0–4°C) (1 mL/100 g body weight) twice daily for 14 consecutive days to establish the IBS‑C model [21]. According to recent literature, cold water (0–4 °C) intragastric stimulation leads to a significant decrease in fecal pellet numbers and fecal moisture, a delayed intestinal transit rate, and notably, the development of marked visceral hypersensitivity [22, 23]. In clinical settings, the ingestion of cold food/beverages or exposure to cold environments is a major, highly prevalent trigger for symptom exacerbation in patients with a significant subtype of IBS [24, 25]. Cold stimulation directly activates temperature-sensitive transient receptor potential (TRP) channels (such as TRPA1 and TRPM8) abundantly expressed in enteric neurons and mucosal epithelial cells, inducing low-grade mucosal inflammation, barrier disruption, and smooth muscle dysmotility [26–28]. More importantly, from the perspective of TCM, ice-water gavage perfectly mimics the etiology of “spleen deficiency and cold-accumulation” or “qi stagnation”. Since the therapeutic agent evaluated in this study possesses properties aimed at invigorating the spleen, resolving dampness, and regulating gastrointestinal qi, employing a model induced by cold stress provides a more appropriate “disease-syndrome combination” model than purely psychological stress models. The successful establishment of the model was confirmed by assessing the abdominal withdrawal reflex (AWR), fecal pellet count, and fecal-water content.
Drug Administration
Following confirmation of model establishment, all groups received their respective treatments once daily by intragastric gavage for 14 days. Rats in the control and IBS-C model groups received an equal volume of distilled water. The ZZD‑L, ZZD‑M, ZZD‑H, and mosapride groups were administered the corresponding doses, as indicated above.
Detection of Fecal Quantity, Fecal Consistency Score, and Moisture Content in Rats Over a 24-h Period
After the final dose was administered, the rats were individually housed in wire-bottom cages equipped with fecal collection trays in a controlled, quiet environment. Feces, excluding urine-contaminated samples, were collected and counted for 24 h. Stool consistency was evaluated using the Bristol Stool Scale [29]. Feces were measured for wet weight (W1), subsequently dried in a thermostatic oven, and remeasured for dry weight (W2). Fecal water content was calculated as follows: [(W1-W2)/W1] × 100%.
AWR Cores
Following feces collection, AWR testing was conducted to evaluate the visceral sensitivity in rats. Prior to testing, the rats were subjected to 12-h food deprivation with free access to water. The animals were placed in transparent restraint cages. A balloon catheter lubricated with liquid paraffin was inserted 5 cm deep into the rectum and fixed to the tail. After the rats had acclimated to the environment, 1.0 mL of air was slowly injected into the balloon via a 1 mL syringe within 20 s to induce colorectal distension. Behavioral responses were recorded and scored according to the AWR scale (0–4). Each rat was tested three times, and the mean score was used for subsequent analysis. AWR scoring criteria: 0 points, the rat is generally calm with no behavioral response; 1 point, the rat’s head remains still for a brief moment; 2 points: the rat exhibits mild abdominal muscle contractions, but the abdomen does not lift off the table; 3 points: the rat exhibits strong abdominal muscle contractions, and the abdomen lifts off the table; 4 points: the rat exhibits tonic abdominal contractions; the body arches, and the abdomen, pelvis, and perineum lift off the table.
Determination of Carbon Powder Propulsion Rate in the Small Intestine
Following the AWR testing, the rats were subjected to an additional 12-h fasting period (a total of 24 h of fasting) with unrestricted access to water, followed by gavage with a 10% activated carbon suspension at a dosage of 10 mL/kg. After 45 min, the rats were anesthetized with 0.3% sodium pentobarbital and prepared for surgical interventions. Blood was obtained from the abdominal aorta, and the small intestine, extending from the pylorus to the ileocecal junction, was excised. The overall intestinal length (L1) and distance traversed by the carbon marker (L2) were quantified. The intestinal propulsion rate was determined using the formula (L2/L1) × 100%.
Histopathological Observation of Rat Colon Tissue
The distal colon tissues were rinsed with saline, fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin blocks, and sectioned. Hematoxylin–eosin (H&E) and Alcian blue-periodic acid-Schiff (AB-PAS) staining were conducted to assess goblet cell abundance, crypt morphology, and the overall appearance of the mucus and muscular layers. Histological evaluations were performed qualitatively by visual inspection of representative sections under a light microscope by two independent observers blinded to the groups.
Detection of Apoptosis in Rat Colonic Epithelial cells by TUNEL Assay
Colon tissues preserved in paraffin were sliced into 5 μm thick sections and underwent dewaxing and rehydration. These sections were treated with 50 μL of TUNEL reaction mixture at 37 °C for an hour, followed by a 10-min counterstaining with DAPI. Apoptotic cells were observed under a light microscope, and the apoptosis rate was calculated using the formula: (TUNEL-positive cells/total cells) × 100%.
Western Blotting (WB)
WB was performed to detect the expression of ZO-1, Claudin-1, BCL-2, Caspase-3, c-Jun, p-EGFR, p-PI3K, p-AKT, EGFR, PI3K, and AKT.
RNA Extraction and RT-qPCR Detection of mRNA Expression of ZO-1 and Claudin-1 Proteins
Total RNA was extracted using an Animal Total RNA Isolation Kit (Foregene, NO. RE-03014). Reverse transcription and amplification were performed using 5 × All-In-One MasterMix (with AccuRT Genomic DNA Removal kit) (abm, G492) and EvaGreen Express 2 × qPCR MasterMix-No Dye (abm, MasterMix-ES). The relative mRNA expression was calculated using the 2−△△ct method. The primer sequences are shown in Table S3 and were provided by Sangon Biotech (Shanghai) Co., Ltd.
Immunohistochemical (IHC) Detection of ZO-1, Claudin-1 Proteins and Gut Enterochromaffin (EC) Cells
Proximal colon tissues were rinsed with saline and fixed with 4% paraformaldehyde solution. Following paraffin embedding and sectioning, antigen retrieval and blocking were performed on the samples. The sections were incubated overnight at 4 °C with the primary antibody, followed by secondary antibody incubation and DAB staining. After mounting, five non-overlapping high-power fields per section were imaged, and the average number of positive cells was quantified using ImageJ software.
Enzyme-linked Immunosorbent Assay (ELISA) for 5-HT Content in Rat Serum
Blood samples (5 mL) were obtained from the abdominal aorta of rats in each group. Following a 30-min incubation at room temperature, the blood was subjected to low-speed centrifugation to isolate the serum, which was subsequently stored in cryotubes. The experimental procedures adhered to the manufacturer’s instructions, including sample processing, BAC quantification, ELISA detection, and data analysis.
Fecal Short-Chain Fatty Acids (SCFAs) Content
500μL of methanol was combined with fecal samples in EP tubes. The mixtures were vortexed to create suspensions after standing for ten minutes. Sulfuric acid was used to reduce the pH to 2–3, and then there was another 10-min rest and mixing. The samples were centrifuged for 20 min at 5000 rpm, and the supernatants were collected for liquid chromatograph-mass spectrometer (LC–MS) to quantify SCFAs.
Statistical Analysis
Data analysis was conducted using GraphPad Prism 8.0, and the results are expressed as mean ± SD. Comparisons between the two groups were performed using Student’s t test. For multiple group comparisons, one-way ANOVA was employed, followed by LSD or Dunnett’s T3 test in cases of unequal variances. Statistical significance was set at P < 0.05.
Result
Network Pharmacology Analysis
Active Chemical Components and Targets Information of ZZD
Using the TCMSP database, four active chemical components were identified for BZ and nineteen for ZS (Table S4). After deduplication, 128 unique targets were obtained. The drug-component-target network (Fig. 2A) consists of 153 nodes and 352 edges, with hexagons representing the active components and circles representing the targets.
Fig. 2.
Network pharmacology analysis and functional enrichment analysis of ZZD (ZS-BZ) for the treatment of constipation-predominant irritable bowel syndrome (IBS-C). A Network diagram illustrating the interactions among traditional Chinese medicine (TCM) active ingredients, overlapping gene targets, and the disease. Note: Blue diamonds represent TCM compounds or active ingredients, green ellipses represent target proteins, where darker shading and larger node size indicate greater importance. B Venn diagram of targets shared between ZZD and IBS-C. C The top 13 targets ranked by degree value. D Protein–protein interaction (PPI) network. E Biological process (BP) analysis of targets for ZZD in the treatment of IBS-C (top 20 terms). Note: the horizontal axis represents the gene ratio, and the vertical axis represents the enriched BP terms; bars are sorted from smallest to largest P-value. F Cellular component (CC) analysis of targets for ZZD in the treatment of IBS-C (top 20 terms). Note: the horizontal axis represents the gene ratio, and the vertical axis represents the enriched CC terms; bars are sorted from smallest to largest P-value. G Molecular function (MF) analysis of targets for ZZD in the treatment of IBS-C (top 20 terms). H Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of targets for ZZD in the treatment of IBS-C (top 19 terms). Note: the horizontal axis represents the gene ratio, and the vertical axis represents the enriched KEGG pathways; bars are sorted from smallest to largest P-value
Intersection Targets of ZZD in IBS-C
After deduplication, 2,426 disease-related targets were identified. The intersection of these targets with those of ZZD yielded 100 common targets. A Venn diagram was generated for visualization using Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/index.html) (Fig. 2B).
Construction of PPI Network
Key targets were analyzed using the STRING database (https://cn.string-db.org/) to construct a PPI network, which was visualized using Cytoscape 3.7.1 (Fig. 2D). The top 13 targets, ranked by degree, are shown in Fig. 2C.
Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Enrichment Analysis
GO enrichment analysis identified 2,157 biological processes (BP), 108 cellular components (CC), and 163 molecular functions (MF), with the top 20 entries for each category displayed in Fig. 2E–G. Key BPs included responses to oxidative stress, inflammatory regulation, small-molecule metabolism, and apoptotic signaling. CCs were primarily associated with membrane regions, the nuclear envelope, and the collagen-containing extracellular matrix. MFs involved transcription factor binding and receptor activator activity. To investigate the therapeutic mechanisms of ZZD in IBS-C, KEGG pathway enrichment analysis was performed on the 100 common targets. Using a threshold of P < 0.01, 179 pathways were identified. The top 19 most relevant pathways, which primarily involved PI3K/AKT, apoptosis, TNF, and NF-κB signaling, are shown in Fig. 2H.
Molecular Docking
A “drug-target-disease” network was constructed and analyzed using Cytoscape 3.7.1 to identify key active components of ZZD and their potential targets. Components with higher degree values interact with multiple targets, suggesting their potential importance in the treatment of IBS-C. Based on the network pharmacology results, four key target proteins (AKT1, BCL2, Caspase-3, and EGFR) and four representative active compounds from ZZD (luteolin, naringenin, 3β-acetoxyatractylone, and 8β-ethoxy atractylenolide III) were selected for molecular docking analysis. Docking results indicated that binding energies below 0 kcal/mol represent spontaneous binding, whereas values below −5.0 kcal/mol indicate strong interactions. As shown in Table S5, the binding affinities varied among the compounds, with lower binding energies reflecting stronger binding capacities [30]. The docking models and interaction patterns presented in Fig. 3A–D demonstrated that all four compounds exhibited strong binding affinity toward the selected targets, with binding energies lower than −5.0 kcal/mol. Among these compounds, luteolin exhibited the strongest binding affinity with EGFR, with a binding energy of −8.6 kcal/mol. The remaining compounds also formed stable hydrogen bonds with key amino acid residues of their respective target proteins. These findings suggest that multiple bioactive compounds in ZZD may simultaneously interact with IBS-C–related targets, supporting the multi-target pharmacological characteristics of this herbal formula.
Fig. 3.
Molecular docking. A-D Molecular docking diagram of compounds with AKT1, BCL2, Caspase-3, and EGFR
Identification of the Chemical Constituents in ZZD by UPLC-MS
The total ion chromatograms of ZZD in both negative and positive ion modes are presented in Fig. 4A–B. We identified 22 compounds, including synephrine, chlorogenic acid, narirutin, and atractylenolide III in ZZD. Detailed information on the identified compounds is presented in Table 1.
Fig. 4.
Diagram of the total ion flow of the traditional chinese medicine ZZD in ion modes. A Positive ion mode. B Negative ion mode
Table 1.
ChemicaL Constituents of ZZD Identified by UPLC-MS Analysis
| No | RT (min) | Component name | Formula | m/z | ppm | Adducts | MS fragmentation |
|---|---|---|---|---|---|---|---|
| 1 | 1.372 | Synephrine | C9H13NO2 | 168.10217 | 1.60616 | [M + H]+ | 150.09183, 135.06836, |
| 2 | 4.274 | Chlorogenic acid | C16H18O9 | 353.08856 | 2.15243 | [M-H]− | 353.08881, 191.05591, 179.03464 |
| 3 | 4.522 | Neochlorogenic acid | C16H18O9 | 353.08871 | 2.57726 | [M-H]− | 353.08908, 191.05592, 173.04520, 135.04454 |
| 4 | 4.881 | Caffeic acid | C9H8O4 | 179.03477 | −1.17295 | [M-H]− | 135.04453, 117.03394 |
| 5 | 5.509 | Atractylone | C15H20O | 271.15953 | 3.86813 | [M + H]+ | 199.14814, 145.10119 |
| 6 | 6.885 | 7-Hydroxycoumarine | C9H6O3 | 163.03954 | 3.49609 | [M + H]+ | 163.03946, 159.39348, 145.02901, 135.04459 |
| 7 | 7.146 | (E)-Ferulic acid | C10H10O4 | 193.05066 | 0.15539 | [M-H]− | 178.02682, 134.03671 |
| 8 | 7.223 | Atractylenolide III | C15H20O3 | 249.14906 | 2.16738 | [M + H]+ | 231.13796, 187.14823 |
| 9 | 7.853 | Narirutin | C27H32O14 | 579.17242 | 0.86330 | [M-H]− | 579.17261, 271.06165 |
| 10 | 7.855 | Rhoifolin | C27H30O14 | 577.15717 | 1.55936 | [M-H]− | 577.15747, 269.04593 |
| 11 | 7.857 | Naringenin chalcone | C15H12O5 | 237.07635 | 2.19719 | [M + H]+ | 273.07669, 153.01878, 147.04460 |
| 12 | 8.034 | Naringin | C27H32O14 | 581.18805 | 2.70137 | [M + H]+ | 273.07657, 153.01865 |
| 13 | 8.44 | Hesperetin | C16H14O6 | 303.08694 | 2.07861 | [M + H]+ | 303.08713, 153.01868 |
| 14 | 8.463 | Neohesperidin | C28H34O15 | 609.18365 | 1.90419 | [M-H]− | 609.18237, 301.07224 |
| 15 | 8.486 | Isochlorogenic acid | C25H24O12 | 515.11993 | 0.85417 | [M-H]− | 353.08441, 335.07761, 191.05588 |
| 16 | 9.369 | Hesperidin | C28H34O15 | 609.18414 | 2.70854 | [M-H]− | 463.12445, 286.04830 |
| 17 | 10.571 | Heraclenin | C16H14O5 | 287.09225 | 2.96072 | [M + H]+ | 287.09222, 203.07069, 153.01877 |
| 18 | 11.873 | Naringenin | C15H12O5 | 271.06189 | 2.58244 | [M-H]− | 271.06201, 151.00320, 119.04953 |
| 19 | 14.922 | Limonin | C26H30O8 | 469.18771 | 1.96083 | [M-H]− | 469.18719, 229.12395 |
| 20 | 16.807 | 6-Gingerol | C17H26O4 | 293.17642 | 2.01244 | [M-H]− | 273.17066, 177.09151 |
| 21 | 21.165 | 13(S)-HODE | C18H32O3 | 295.22836 | 1.69360 | [M-H]− | 277.21674, 195.13957, 178.09665 |
| 22 | 22.488 | Atractylenolide I | C15H18O2 | 231.13889 | 4.06683 | [M + H]+ | 231.13885, 213.12808, 185.13333 |
In Vivo Experimental Results
Comparison of General Condition, 24-h Fecal Output, Fecal Water Content, Bristol Stool Scale Score, AWR Score, and Small Intestine Carbon Powder Propulsion Rate in Rats
Prior to modeling, all rats displayed normal behavior, smooth fur, and well-formed feces. After successful induction of IBS-C, the IBS-C model group exhibited irritability, lethargy, dull fur, reduced activity, decreased food intake, and dry feces (Fig. 5A & C). At the experiment’s conclusion, the IBS-C model group had a significantly lower body weight than the control group (P < 0.0001), whereas the mosapride and ZZD treatment groups significantly attenuated this weight loss (compared with the IBS-C model group, P < 0.05) (Fig. 5B). Compared to the IBS-C model group, both mosapride and all ZZD dose groups showed improvements in fecal output, fecal water content, Bristol stool scores, small intestinal transit rate, and a reduction in AWR scores (P < 0.01). These improvements occurred in a dose-dependent manner (Fig. 5D–H).
Fig. 5.
ZZD ameliorated the symptoms in a rat model of IBS-C. A Representative images comparing body size between the control and IBS-C model groups. B Effect of ZZD on body weight. C Representative stool images from the different experimental groups. D Effect of ZZD on the 24-h fecal pellet count. E Effect of ZZD on fecal water content. F Effect of ZZD on the Bristol stool scale score. G Effect of ZZD on the abdominal withdrawal reflex (AWR) score. H Effect of ZZD on intestinal transit rate. Note: Data were represented as mean ± SD, n = 8; data are compared with the IBS-C model group (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)
Morphological Manifestations of Rat Colon Tissue
Histological changes in the colon were assessed qualitatively by visual inspection of H&E- and AB-PAS-stained representative sections. In the control group, colonic tissue displayed intact architecture, characterized by orderly arranged columnar epithelial cells, abundant goblet cells, well-organized crypts with an intact basement membrane, and an absence of inflammatory infiltration. Conversely, the IBS-C model group exhibited evident histopathological disruption, including disorganized epithelial structure, reduced goblet cell populations and diminished acidic mucin deposition, irregular crypt branching, and submucosal inflammatory infiltration. Treatment with mosapride and varying doses of ZZD alleviated these pathological alterations to different extents. Representative images from the ZZD-treated groups showed partial recovery of goblet cell populations, improved crypt structural regularity, and increased mucus and muscular layer thickness relative to the IBS-C model group (Fig. 6A & B).
Fig. 6.
ZZD ameliorated colonic pathological damage and restored the abundance of goblet cells in rats. A Representative images of H&E staining of colon tissue sections. B Representative images of AB-PAS staining of colon tissue sections. Note: Low-magnification micrograph at 40 × objective lens (scale bar, 2 mm), with a higher magnification inset at 100 × objective lens (scale bar, 1 mm)
ZZD Upregulated the mRNA and Protein Levels of Tight Junction Proteins (ZO-1, Claudin-1) and Restored their Continuous Distribution in the Colon of Rats
In the present study, multi-dimensional techniques were employed to evaluate alterations in intestinal tight junctions in rats. RT-qPCR analysis showed that the mRNA expression levels of ZO-1 and Claudin-1 in the IBS-C model group were significantly lower than those in the control group (P < 0.0001), whereas treatment with high-dose ZZD (ZZD-H) markedly increased their mRNA levels (compared with the IBS-C model group, P < 0.0001) (Fig. 7D & E). Consistent with these transcriptional changes, western blot analysis revealed that ZZD-H treatment significantly upregulated the protein expression levels of ZO-1 and Claudin-1 compared with the IBS-C model group (P < 0.05) (Fig. 7G), confirming the quantitative changes at the translational level. Immunohistochemical staining further revealed changes in the spatial distribution of these proteins. The intensity of ZO-1 and Claudin-1 protein positive staining (brown) on the intestinal epithelial cell membrane of the IBS-C model group decreased (compared with the control group, P < 0.0001) and the continuity was interrupted, indicating the destruction of tight junction structures. In contrast, in the ZZD-H groups, the intensity of ZO-1 and Claudin-1 protein positive staining was enhanced (compared with the IBS-C model group, P < 0.0001) and exhibited continuous distribution along the epithelial villus margins of the colon (Fig. 7A & B). Collectively, these findings indicate that ZZD not only upregulates the mRNA and protein levels of tight junction proteins, but also restores their normal localization within the colonic epithelium, thereby contributing to the maintenance of tight junction structure.
Fig. 7.
ZZD upregulated the mRNA and protein levels of tight junction proteins (ZO-1, Claudin-1), restored their continuous distribution, increased EC cells numbers and serum 5-hydroxytryptamine (5-HT) levels. A Representative immunohistochemical (IHC) staining for colonic ZO-1, original magnification, × 200; scale bar, 100 μm. B Representative IHC staining for colonic Claudin-1, original magnification, × 200; scale bar, 100 μm. C Representative IHC staining of colonic TPH1-positive enterochromaffin (EC) cells, original magnification, × 200; scale bar, 100 μm. D mRNA expression of ZO-1. E mRNA expression of Claudin-1. F Quantification of serum 5-HT content. G Protein expressions of ZO-1 and Claudin-1 determined by western blotting assay. Note: Data were represented as mean ± SD, n = 8; data are compared with the IBS-C model group (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)
Apoptosis of Rat Intestinal Epithelial Cells
As shown in Fig. 8A & B, TUNEL staining revealed increased apoptosis in the colonic tissues of the IBS-C model group (compared with the control group, P < 0.0001). Treatment with ZZD at all doses significantly reduced the apoptosis rates (compared with the IBS-C model group, P < 0.05), with the greatest effect observed in the ZZD-H group. Western blot analysis showed elevated expression of the pro-apoptotic protein Caspase-3 (compared with the control group, P < 0.05) and reduced expression of the anti-apoptotic proteins Bcl-2 and c-JUN (compared with the control group, P < 0.05) in the IBS-C model group (Fig. 9A). These alterations were reversed in the mosapride and ZZD-H groups (compared with the IBS-C model group, P < 0.05), indicating that ZZD treatment effectively inhibited intestinal epithelial cell apoptosis and exerted a protective effect against apoptosis-related mucosal barrier injury.
Fig. 8.
ZZD reduced apoptosis in intestinal epithelial cells. A Quantification of epithelial cell apoptosis across experimental groups, original magnification, × 200; scale bar, 50 μm. B Effect of ZZD on epithelial cell apoptosis. Note: Data were represented as mean ± SD, n = 8; data are compared with the IBS-C model group (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)
Fig. 9.
Western blot analysis of apoptosis-associated proteins and phosphorylated EGFR/PI3K/AKT expression in colon from IBS-C rats treated with ZZD-H or mosapride. A Representative western blot bands of Caspase-3, c-Jun, Bcl-2, total EGFR, p-EGFR, total PI3K, p-PI3K, total AKT, p-AKT, and the internal reference GAPDH. Quantification of relative protein expression and phosphorylation ratio (p-EGFR/EGFR, p-PI3K/PI3K, p-AKT/AKT) are shown in the corresponding bar graphs. Note: Data were represented as mean ± SD, n = 3; data are compared with the IBS-C model group (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)
Colonic EC Cell Abundance and Serum 5-HT Levels
TPH1 immunohistochemical staining was used to identify EC cells, which appeared yellow–brown and were predominantly distributed at the epithelial base or between glands. As shown in Fig. 7C, the number of TPH1-positive EC cells was significantly lower in the IBS-C model group than in the control group (P < 0.0001), whereas treatment with mosapride and ZZD at all doses significantly increased TPH1-positive EC cell abundance compared with the IBS-C model group (P < 0.05). Consistently, serum 5-HT levels were significantly decreased in the model group relative to the control group (P < 0.0001) and were markedly restored by mosapride and ZZD treatment (P < 0.05) (Fig. 7F).
Effects of ZZD on EGFR/PI3K/AKT Phosphorylation in Rat Intestinal Tissues
KEGG enrichment analysis revealed that the target genes of ZZD against IBS-C were significantly enriched in multiple signaling pathways. The PI3K/AKT signaling cascade ranked first in terms of the degree of enrichment, followed by the apoptosis pathway. These enrichment results implied that the PI3K/AKT pathway and apoptosis process may jointly mediate the therapeutic effects of ZZD on intestinal injury in IBS-C. Based on the key targets identified via network pharmacology, the expression of EGFR, PI3K, and AKT pathway proteins in rat colon tissue was assessed. As shown in Fig. 9A, the phosphorylation levels of EGFR, PI3K, and AKT were significantly decreased in the IBS-C model group (compared with the control group, P < 0.05) but were markedly elevated after ZZD treatment (compared with the IBS-C model group, P < 0.05).
Fecal SCFAs Content
Fecal concentrations of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and caproic acid were significantly lower in the IBS-C model group than in the control group (P < 0.05) (Fig. 10A–H). Z-score distribution of fecal SCFAs across groups (Fig. 10I), compared with IBS-C model group, ZZD-H group shows a general rightward shift, indicating increased levels of most SCFAs. Notably, isovaleric acid, isobutyric acid, valeric acid, propionic acid, butyric acid and acetic acid exhibit higher Z-scores, suggesting significant metabolic restoration. ZZD treatment significantly restored SCFAs profiles.
Fig. 10.
ZZD increased fecal short-chain fatty acids (SCFAs) levels. A Heatmap of SCFAs in rats of each group. B-H Comparison of SCFAs in rat feces. I Z-Score of SCFAs in rats of each group. Note: Data were represented as mean ± SD, n = 8; data are compared with the IBS-C model group (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)
Discussion
Zhizhu Decoction (ZZD) is a classical traditional Chinese herbal formula composed of Atractylodis Macrocephalae Rhizoma and Aurantii Fructus Immaturus, which has long been used to treat symptoms resembling those of constipation-predominant irritable bowel syndrome (IBS-C). In the present study, we combined network pharmacology, molecular docking, and in vivo validation to explore the potential therapeutic mechanisms of ZZD. IBS-C is a functional gastrointestinal disorder characterized by impaired colonic motility, epithelial barrier dysfunction, and dysregulated gut-brain communication [10, 31]. In the present study, ZZD markedly alleviated colonic pathological damage in the IBS‑C rat model. It upregulated mRNA and protein expression of tight‑junction proteins ZO‑1 and Claudin‑1 and recovered their continuous distribution along the colonic epithelium. Meanwhile, ZZD suppressed intestinal epithelial apoptosis, restored EC cell integrity as indicated by increased colonic TPH1‑positive cell abundance, and increased circulating 5‑HT concentrations. In addition, ZZD increased fecal SCFA content and enhanced the phosphorylation levels of EGFR, PI3K, and AKT. Collectively, these results suggest that ZZD protects intestinal epithelial integrity, attenuates epithelial apoptosis, modulates the serotonergic system, increases phosphorylation of the PI3K/AKT pathway, and influences gut microbial metabolism, which may collectively contribute to its protective effects against IBS‑C.
Network pharmacology analysis indicated that volatile oils, flavonoids, alkaloids, and lactones are major bioactive constituents of ZZD, reflecting the multi-component nature typical of traditional herbal prescriptions. Molecular docking further demonstrated that representative compounds of ZZD, including luteolin, naringenin, 3β-acetoxyatractylone, and 8β-ethoxy atractylenolide III, could interact with IBS-related targets such as AKT1, BCL2, Caspase-3, and EGFR, with binding energies below −5 kcal/mol, suggesting favorable binding affinity. These major bioactive constituents of ZZD, including atractylenolides from Atractylodis Macrocephalae Rhizoma and flavonoids such as Naringenin and Luteolin from Aurantii Fructus Immaturus, have also been reported to exert anti-inflammatory, anti-apoptotic, and gastrointestinal protective effects [32–35], suggesting that the therapeutic effects of ZZD likely arise from synergistic interactions among multiple phytochemicals acting on different biological targets, a pharmacological characteristic commonly observed in traditional Chinese herbal formulas. Further pharmacological studies using isolated compounds and combination experiments are required to clarify the potential synergistic or additive interactions among major constituents of ZZD.
KEGG enrichment analysis suggested that apoptosis and the PI3K/AKT signaling pathway may play important roles in the therapeutic action of ZZD. The epidermal growth factor receptor (EGFR), a receptor tyrosine kinase widely expressed in epithelial tissues, regulates cell proliferation, tissue repair, and mucosal immune responses. Upon ligand binding, EGFR undergoes dimerization and autophosphorylation, triggering downstream signaling cascades such as the PI3K/AKT pathway. Activation of this pathway promotes cell survival by suppressing pro-apoptotic signaling and enhancing anti-apoptotic factors [36, 37]. Previous studies have reported reduced PI3K and AKT expression in models of slow-transit constipation and intestinal injury [38]. In the present study, ZZD significantly increased the phosphorylation levels of EGFR, PI3K, and AKT in colonic tissues, which is consistent with the possibility that activation of this pathway contributes to the epithelial protective effects of ZZD. However, since pathway-specific inhibitors were not used, a causal relationship between these phosphorylation changes and the therapeutic effects cannot be conclusively established and requires further investigation.
The intestinal epithelial barrier plays a crucial role in maintaining gut homeostasis. Tight junction proteins such as ZO-1 and Claudin-1 are essential for barrier integrity, and their disruption has been widely observed in IBS patients [5]. Excessive apoptosis of intestinal epithelial cells can further compromise barrier function and exacerbate intestinal permeability [9], representing both a pathological feature of IBS-C and a potential therapeutic target. In this study, ZZD treatment decreased the expression of the pro-apoptotic protein Caspase-3 and increased the levels of the anti-apoptotic proteins Bcl-2 and c-Jun, indicating reduced epithelial apoptosis and enhanced mucosal repair. Together with the restoration of colonic morphology, goblet cell preservation, and increased expression of the tight junction proteins ZO-1 and Claudin-1, these findings suggest that ZZD exerts a protective effect on intestinal barrier integrity. However, direct functional assessment of intestinal permeability was not performed,therefore, the present results should be interpreted as evidence of improved barrier-associated structural integrity rather than definitive proof of enhanced barrier function.
EC cells play a central role in gut-brain communication by producing the majority of intestinal 5-HT, which is a key mediator of gastrointestinal motility and visceral sensitivity. Alterations in EC cell numbers and 5-HT levels have been implicated in the pathophysiology of IBS, with reduced levels commonly observed in IBS-C [39]. In this study, the IBS-C model rats exhibited increased epithelial apoptosis, reduced TPH1-positive EC cell abundance, and decreased serum 5-HT levels. ZZD treatment significantly restored EC cell abundance (as indicated by increased colonic TPH1-positive cell abundance on IHC) and elevated serum 5-HT levels while concurrently reducing epithelial apoptosis. These findings suggest that the protection of epithelial cells may contribute to the preservation of EC cell populations. Nevertheless, the concurrent reduction in epithelial apoptosis and preservation of EC cells suggest that inhibiting cell death may be the predominant mechanism underlying EC cell recovery. However, whether ZZD directly regulates EC cell differentiation or indirectly influences EC cells through generalized epithelial protection remains unclear. In addition, as we did not directly measure colonic 5-HT levels, the local serotonergic effects of ZZD warrant further investigation.
A pivotal finding of this study is that ZZD significantly suppressed colonic epithelial cell apoptosis (as evidenced by decreased TUNEL-positive cells, downregulated Caspase-3, and upregulated BCL-2) and concurrently alleviated IBS-C symptoms, including intestinal dysmotility, visceral hypersensitivity, and defecation barriers. Although our experimental design did not establish a direct causal relationship between apoptosis inhibition and these functional outcomes, the multidimensional data obtained in this study allowed us to propose a plausible mechanistic framework. First, excessive apoptosis of colonic epithelial cells disrupts the structural integrity of the mucosal lining, triggering a cascade that drives visceral hypersensitivity. In our study, the inhibition of apoptosis by ZZD directly correlated with the robust preservation of tight junction proteins (ZO-1 and Claudin-1, as confirmed by WB, RT-qPCR, and IHC). A compromised epithelial barrier (leaky gut) allows luminal antigens, bacterial products, and cold gavage irritants to penetrate the lamina propria. This exposure initiates low-grade neuroinflammation, activates mast cells, and releases chemical mediators that sensitize adjacent nociceptors (such as TRPV1), thereby lowering the pain threshold and increasing the AWR score. Therefore, ZZD may effectively block this neuroimmune sensitization pathway by inhibiting epithelial cell apoptosis and sealing the physical barrier, ultimately alleviating visceral hyperalgesia. Second, epithelial apoptosis regulation is structurally intertwined with neuroendocrine signaling that controls intestinal motility. EC cells embedded within the colonic epithelium act as essential mechanoreceptors and chemoreceptors that synthesize and secrete serotonin, a master regulator of the enteric nervous system driving peristalsis. Massive apoptosis of colonic epithelial cells can lead to mucosal atrophy, accompanied by depletion of EC cells and insufficient 5-HT secretion, resulting in intestinal motility disorders and visceral pain hypersensitivity. Our data demonstrated that ZZD normalized EC cell abundance and serum 5-HT levels while maintaining mucosal and crypt architecture (H&E and AB-PAS staining). This homeostatic restoration of 5-HT signaling re-establishes coordinated gastrointestinal smooth muscle contractions, directly explaining the accelerated small intestinal carbon powder propulsion rate and the subsequent normalization of 24-h fecal quantity, Bristol stool consistency scores, and fecal water content. Third, as discussed in the following section, the concurrent increase in fecal SCFA levels may act synergistically by reinforcing epithelial barrier function and modulating EC cell function, further amplifying functional benefits. Collectively, these interconnected pathways suggest that the anti-apoptotic action of ZZD serves as an upstream event that contributes to the normalization of intestinal motility, visceral sensitivity, and defecation patterns in IBS C, through the preservation of epithelial and EC cell integrity.
Growing evidence also supports an important role for gut microbiota-derived metabolites in maintaining intestinal homeostasis. Short-chain fatty acids, particularly butyrate, are essential for epithelial energy metabolism, regulation of tight junction proteins, and modulation of immune responses and visceral sensitivity [40–44]. As noted above, the observed increase in fecal SCFAs may also act synergistically with the anti-apoptotic and serotonergic effects of ZZD. In the present study, IBS-C rats exhibited marked reductions in fecal SCFAs, indicating microbial metabolic dysfunction. ZZD treatment significantly restored SCFAs profiles, including acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid. These metabolic changes may contribute to improvements in epithelial survival and enteroendocrine function by supporting colonocyte energy metabolism, enhancing barrier integrity, and promoting serotonin biosynthesis in EC cells, potentially acting in parallel with intracellular survival signaling pathways such as EGFR/PI3K/AKT. Notably, SCFAs have been shown to promote 5-HT synthesis in EC cells and to enhance barrier integrity [45, 46], providing a plausible link between microbial metabolism and the observed epithelial and neuroendocrine effects. However, since the present study quantified only SCFAs levels without directly analyzing microbial community composition, the regulatory relationship between ZZD, gut microbiota, and SCFAs production cannot be fully established. Therefore, although the observed increase in SCFAs may contribute to preserved epithelial structure, EC cell function, and elevated serum 5-HT levels, the underlying microbial mechanisms remain to be elucidated. Future studies combining 16S rRNA sequencing, metagenomic analysis, and microbial transplantation experiments would help clarify whether ZZD reshapes gut microbiota composition and establishes a regulatory chain linking microbiota-SCFAs-intestinal homeostasis.
Several limitations should be acknowledged. First, our mechanistic conclusions are based on relevant molecular evidence, including changes in protein phosphorylation and expression, but have not been functionally validated using pathway inhibitors, intestinal permeability assays, or proliferation markers (such as Ki-67 and PCNA). Second, although serum 5-HT levels and colonic TPH1 staining were evaluated, TPH1 serves only as an indirect indicator of local serotonin biosynthesis, and colonic 5-HT levels and downstream serotonergic signalling were not directly assessed. In addition, although alterations in fecal SCFAs suggest modulation of gut microbial metabolism, the gut microbiota composition was not characterized. Therefore, the contributions of local serotonergic regulation and microbiota-mediated effects require further investigation. Despite these limitations, this study provides a mechanistic framework that supports the traditional use of ZZD and highlights its potential as a complementary treatment strategy for IBS-C.
Conclusion
In conclusion, ZZD alleviates IBS-C symptoms in a rat model, which is associated with reduced intestinal epithelial apoptosis, improved the expression and distribution of tight junction proteins (ZO-1 and Claudin-1), restored EC cell integrity (as indicated by colonic TPH1 staining), and elevated serum 5-HT levels. These effects may be related to synergistic interactions among multiple herbal constituents, potentially involving phosphorylation of the EGFR/PI3K/AKT signaling pathway and modulation of SCFAs metabolism (Fig. 11). However, further studies, including pathway specific inhibition experiments and gut microbial composition analysis, are required to confirm these causal mechanisms.
Fig. 11.
Summary of the multi-target effects of ZZD in IBS-C model rats: inhibition of epithelial apoptosis, preservation of tight-junction proteins (ZO-1 and Claudin-1), restoration of EC cell integrity and serum 5-HT, modulation of SCFAs, and increased EGFR/PI3K/AKT phosphorylation
Supplementary Information
Authors’ Contributions
Zhibin zhang: Writing-original draft, Software, Project administration, Methodology, Formal analysis, Data curation, Conceptualization. Rong Wu: Data curation, Investigation, Methodology. Fang Liu: Validation, Supervision, Project administration. Simin Chen: Software, Investigation, Formal analysis. Lina Guan: Visualization, Supervision, Software. Yu Zhan: Data curation, Investigation. Yong Wen: Data curation, Investigation. Xuegui Tang: Writing-review & editing, Supervision, Project administration, Funding acquisition.
Funding
This work was supported by the North Sichuan Medical College University-level Research Project (No. CBY24-QNA24), the Youth Program of National Natural Science Foundation of China (No. 82004173) and the National Natural Science Foundation of China (No. 82474516).
Data Availability
The data from the current study can be provided based on the request from the corresponding author.
Declarations
Ethics Approval and Consent to Participate
All animal experiments were conducted in accordance with the guidelines for the care and use of laboratory animals and were approved by the Ethics Committee of North Sichuan Medical College (approval no. 2024077).
Consent for Publication
Not applicable.
Competing Interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Data Availability Statement
The data from the current study can be provided based on the request from the corresponding author.