Unveiling Xuanshen decoction: A novel approach to combat slow transit constipation

Abstract

BACKGROUND

Xuanshen decoction (XSD) is a traditional Chinese medicine formulation that is often applied in treating slow transit constipation (STC). However, its specific therapeutic mechanism remains to be characterized.

AIM

To investigate the mechanism of XSD for STC, we combined network pharmacology prediction, molecular docking analysis, and in vivo studies.

METHODS

The therapeutic effects of XSD on loperamide-induced STC in rats were assessed through 24-hour fecal number, fecal moisture content, and intestinal propelling rate. Hematoxylin–eosin and Alcian blue/periodic acid-Schiff staining were applied to analyze colonic mucosa for histopathological presentation and mucin production. Next, the mechanism of action of XSD for STC was elucidated through network pharmacology and molecular docking analyses, and the findings were validated by the animal experiments.

RESULTS

XSD significantly alleviated the symptoms of STC in rats. Relative to the STC rats, in the medium-dose XSD and high-dose XSD rats, stem cell factor, C-kit, phospho-phosphoinositide 3-kinase/phosphoinositide 3-kinase, phospho-protein kinase B/protein kinase B, catalase, and superoxide dismutase were substantially upregulated (P < 0.01); nuclear factor erythroid 2-related factor 2 (nuclear/cytoplasmic) and B-cell lymphoma 2 (Bcl-2) were increased (P < 0.05), while cleaved caspase-3, Bcl-2-associated X protein (Bax)/Bcl-2, and malondialdehyde were significantly reduced (P < 0.01). Heme oxygenase-1 and glutathione peroxidase in the high-dose XSD group were significantly increased (P < 0.01), and Bax was statistically lowered (P < 0.01); glutathione peroxidase in the medium-dose XSD group was increased (P < 0.05), while Bax was reduced (P < 0.05).

CONCLUSION

XSD may inhibit oxidative-stress-induced apoptosis in interstitial cells of Cajal by stimulating the phosphoinositide 3-kinase/protein kinase B/nuclear factor erythroid 2-related factor 2 pathway, thereby effectively treating STC.

Key Words: Slow transit constipation; Oxidative stress; Network pharmacology; Experimental verification; Molecular docking

Core Tip: There are limited drugs available to treat slow transit constipation (STC). Xuanshen decoction is a traditional Chinese medicine formulation that can effectively treat STC. In this study, Xuanshen decoction significantly alleviated the symptoms of STC in rats. Xuanshen decoction promoted the expression of key proteins in the phosphoinositide 3-kinase/protein kinase B/nuclear factor erythroid 2-related factor 2 signaling pathway, improved oxidative stress-related parameters, and suppressed the apoptosis of interstitial cells of Cajal.

INTRODUCTION

Slow transit constipation (STC) is a disorder manifesting as decreased defecatory propulsion and increased colonic transit time and can cause symptoms such as abdominal pain, bloating, difficulty defecating, perianal disease, and an increased risk of colon cancer or cerebrovascular disorders[1]. The global prevalence of STC has been estimated at 14%[2], with significant detrimental effects on both health outcomes and socioeconomic stability in affected populations[3,4]. The pathogenesis of STC is complicated, with its core pathological mechanisms remaining poorly characterized. However, the condition is predominantly associated with the enteric nervous system (ENS), interstitial cells of Cajal (ICC), neurotransmitters, and intestinal smooth muscle[5,6].

Pharmacotherapy remains the primary strategy for the management of STC and mainly comprises volumetric laxatives, irritant laxatives, osmotic laxatives, and prokinetic agents. Although these drugs show transient efficacy, their discontinuation may lead to recurrence of STC. Consequently, the development of novel STC therapies with enhanced safety and effectiveness is urgently required. Contemporary research has confirmed that traditional Chinese medicine (TCM) can treat STC by regulating ICC, changing neurotransmitter concentrations within the ENS, and regulating gastrointestinal hormone levels, intestinal flora, and anti-inflammatory activity[7,8]. Chinese medicines such as Shouhui Tongbian capsules and Xiaochengqi decoction may treat STC by inhibiting intestinal inflammation and regulating expression of ICC[9-11]. Maren Pills can treat STC by regulating the levels of neurotransmitters[12]. Although these prescriptions are effective in the treatment of STC, they contain anthraquinone laxatives such as Rhei Radix et Rhizoma and Aloe vera (L.) Burm.f., which hinders their promotion and application. Xuanshen decoction (XSD) is based on Zengye decoction[13,14], a traditional prescription for treating constipation. Professor Xiang-Dong Yang is one of the top TCM professors in Sichuan Province, China. With extensive clinical experience exceeding 30 years, he has established himself as an expert in TCM interventions for constipation. XSD is derived from Professor Yang’s modification of Zengye decoction, and it has a remarkable clinical effect in treating constipation. XSD consists of Scrophularia ningpoensis Hemsl. (Xuanshen), Sophora flavescens Aiton (Ku Shen), Cimicifuga heracleifolia Kom. (Sheng Ma), Atractylodes lancea (Thunb.) DC. (Cang Zhu), Dioscorea polystachya Turcz. (Shan Yao), Sanguisorba officinalis L. (Di Yu), Glycyrrhiza uralensis Fisch. (Gan Cao), and according to the TCM theory, it can nourish yin, moisten intestines, and promote the flow of bodily fluids. More significantly, XSD does not contain anthraquinone laxatives.

Previous clinical randomized controlled studies have shown that XSD can successfully treat constipation[15,16]. While clinically effective, XSD’s mechanistic basis requires systematic investigation. Our strategy combined computational network pharmacology with structure-based molecular docking to identify XSD’s bioactive compounds and their putative targets against STC. The results were verified in rats with loperamide (LOP)-induced STC to further explore the pharmacological mechanism of XSD in STC. These findings provide novel mechanistic insights and support XSD’s further development as a constipation treatment.

MATERIALS AND METHODS

XSD preparation methodology

The XSD prescription comprised Scrophularia ningpoensis 50 g, Sophora flavescens 20 g, Cimicifuga heracleifolia 15 g, Atractylodes lancea 15 g, Dioscorea polystachya 30 g, Sanguisorba officinalis 20 g, and Glycyrrhiza uralensis 15 g. The First Affiliated Hospital of Guizhou University of TCM (Guizhou Province, China) supplied all the herbs authenticated by pharmacists. All herbs were prepared according to prescription, using the traditional decoction method. The herbal mixture underwent 30 minutes hydration (10 × the herb volume) followed by 1.5 hours decoction. The aqueous extract was filtered utilizing gauze, and the residue obtained was subjected to a second decoction using water (8 × herb volume) for 1 hour. It was also filtered with gauze. The filtrate mixture were evaporated to 5 g/mL, and placed in a 4 °C refrigerator for later use.

Reagents

LOP and prucalopride (PRU) were sourced from Xi’an Janssen Pharmaceutical (H10910085) and Jiangsu Hausen Pharmaceutical Group (H20183481), respectively. All drugs were dissolved in normal saline (0.9%) before administering them to rats. The following assay kits were used: The malondialdehyde (MDA) from Shanghai Biyuntian (S0131S, China), the superoxide dismutase (SOD) from Suzhou Mengxi (M0102A, China), and the glutathione peroxidase (GSH-PX) from Nanjing Jiancheng (A005-1-2, China).

Rat model of STC induced by LOP

The LOP-induced STC rat model was developed as reported previously[17]. The Experimental Animal Research Institute of Guizhou University of TCM permission [No. SYXK-(qian) 2021-0003] supplied specific pathogen-free, Sprague-Dawley rats (n = 90; male: 45; weight: 200 ± 20 g). All rats were maintained in a specific pathogen-free-grade vivarium with sterilized feed and water available ad libitum. Following 1 week of acclimation on a basal diet, all rats were divided randomly into the control, model, PRU, low-dose XSD (L-XSD), medium-dose XSD (M-XSD), and high-dose XSD groups (H-XSD) (n = 15 each).

To establish the STC model, LOP (10 mg/kg) was administered by oral gavage daily for 4 weeks to both model and experimental groups. If the fecal moisture content, the number of fecal particles in 24 hours, and time to excretion of the first black feces in each group were different from those in the control group (P < 0.05), the modeling was determined to be successful[18-21].

The body weight of all experimental animals was monitored and collected. Following successful model induction, the L-XSD, M-XSD, and H-XSD groups received 10, 20, and 40 g/kg/day XSD, respectively. The PRU group received 0.2 mg/kg/day PRU. Drugs were delivered via daily intragastric gavage for 4 weeks. On day 29, rats were euthanized with serum and colon tissues collected for analysis. Except for paraformaldehyde-fixed samples, all others were maintained at -80 °C until analysis.

Time of first black stool discharge

After a 24-hour fast on day 28 of modeling, all rats received an ink containing 10% activated carbon in acacia via oral gavage. The time of the first black stool discharge of all rats was observed, gathered, and compared.

24-hour defecation and fecal water content

Following treatment, rats were housed in metabolic cages for 24 hours for fecal collection, with fecal number and weight measurements. Samples were then oven-dried at 60 °C for 12 hours. The formula denoted below was utilized to measure the fecal water content: Fecal moisture content (%) = (fresh weight of feces - dry stool weight)/fresh weight of feces × 100%.

Intestinal propelling rate

The assessment of intestinal movement in rats was conducted by the intestinal propelling of ink. After a 24-hour fast at the end of the trial, all rats received an ink containing 10% activated carbon in acacia via oral gavage. Approximately 3 hours later, all rats were anesthetized and killed by bleeding from the abdominal aorta. The abdominal cavity was dissected, and the intestine from the pylorus to the end of the rectum was collected and placed on a sterile gauze moistened with saline. Photographs recorded the propelling length of carbon (activated carbon pushes the distance in the intestine) and the total length of the intestine. The ink propelling rate in each group was calculated as follows: Intestinal propelling rate (%) = activated carbon pushes the distance in the intestine/total length of intestinal length × 100%.

Alcian blue/periodic acid-Schiff and hematoxylin and eosin staining of colon tissue

Colonic segments were immersion-fixed in 4% paraformaldehyde, processed through xylene clearing and paraffin embedding, and sectioned for histological examination. Tissue morphology was assessed by hematoxylin and eosin staining. Alcian blue/periodic acid-Schiff staining was applied to measure mucin in the colon. Quantitative data (e.g., mucosal layer thickness, muscle layer thickness, goblet cell number, mucin-positive area, and lymphocyte infiltration rate) were analyzed using Image J and CaseViewer v2.4.0.

Network pharmacology profiling

The TCM Systems Pharmacology Database and Analysis Platform and Symmap databases were utilized to identify the bioactive constituents of XSD[22]. The potential active components with an oral bioavailability (OB) of ≥ 30% and drug-likeness (DL) ≥ 0.18 were chosen for the next target prediction. Additionally, the structural data of compounds, based on the Simplified Molecular Input Line Entry System format, was downloaded from the PubChem database. Potential component targets were identified using the SwissTargetPrediction database by querying computed identifiers or Simplified Molecular Input Line Entry System structures[23], with a probability threshold of 0.1. Simultaneously, PharmGkb, Online Mendelian Inheritance in Man, GeneCards, Drugbank, DisGeNET, and Therapeutic Target Database databases were searched to identify STC-related targets[24,25]. After eliminating duplicate targets, potential targets related to STC were obtained. To identify the intersection of XSD and STC targets, Venn diagrams were constructed.

To unambiguously screen the functional interactions among the possible proteins, a protein-protein interaction network was constructed utilizing the Search Tool for the Retrieval of Interacting Genes/Proteins database before inputting it into Cytoscape 3.8.2 software for analyzing the topology of the intersection network. In addition, “degree” was utilized to screen key network targets. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted using the Metascape database[26]. For functional annotation clustering, terms with adjusted P < 0.05 were chosen.

Molecular docking

The chemical structures of XSD active compounds were retrieved from PubChem, while protein structures were derived from Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB); Core regulatory targets of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway were docked to primary compounds using AutoDockTool 1.5.7 software to obtain molecular docking scores. Based on binding affinity rankings, the optimal docking pose was identified and subsequently rendered in PyMOL 2.5 software.

Determination of serum levels of oxidative stress parameters

Serum oxidative stress parameters (MDA, SOD, GSH-PX) were quantified using standardized biochemical assays.

Immunofluorescence analysis

To analyze the expression of Nrf2 and C-kit proteins, immunofluorescence staining was performed by the primary antibodies specific to the protein. Primary antibody incubation (anti-Nrf2, No.: 16396-1-AP, Proteintech, Wuhan, China, 1:100; anti-c-kit, No. 18696-1-AP, Proteintech, 1:100) proceeded at 4 °C overnight, followed by 37 °C secondary antibody (1:100) incubation for 30 minutes. The sections were then photographed (Olympus, Japan) for analysis.

Western blotting

The total protein obtained from the colon tissues utilizing RadioImmunoPrecipitation Assay ysis buffer (Boster, Wuhan, Hubei Province, China) underwent quantification via the bicinchoninic acid protein assay kit (Boster). The extraction of nucleoprotein was carried out strictly according to the kit instructions (Abmart, Shanghai, China).

The antibodies used were as follows: C-Kit (1 500; Proteintech), stem cell factor (SCF) (1 1200; Proteintech), PI3K (1:500; ABclonal, China), Akt (1:1000; Servicebio), phospho (P)-PI3K (1:500; ABclonal), P-Akt (1:1000; Abclonal), heme oxygenase 1 (HO-1)(1:1000; Abclonal), Nrf2-C (1:800; Servicebio), and Nrf2-N (1:800; ABclonal), cleaved caspase-3 (1:1000; Cell Signaling Technology, Danvers, MA, United States), B-cell lymphoma 2 (Bcl-2) -associated X protein (Bax) (1:1500; Boster), Bcl-2 (1: 1000; Boster), β-actin (1: 1000; Servicebio), β-tubulin (1:800; ABclonal), Lamin B1 (1:800; ABclonal), glyceraldehyde-3-phosphate dehydrogenase (1:5000; ABclonal). The membranes were washed thrice before 2 hours room-temperature incubation with secondary antibodies. Ultimately, the chemiluminescence detection system detected the samples, whereas Image J quantified the band intensity.

Statistical analysis

Statistical analyses were performed using GraphPad Prism (version 9.5.1), with all data presented as mean ± SD. Data were analyzed using one-way analysis of variance and Student’s t-test. Results with P < 0.05 denoted statistical significance.

RESULTS

XSD alleviated LOP-induced constipation symptoms

Four weeks after intragastric administration of LOP, compared with the control group, the 24-h fecal particle count, fecal moisture content, and first black stool excretion time of the rats were strongly reduced (Figure 1) (P < 0.01). These results confirmed successful establishment of the STC animal model (Figure 1A, D, and E).

Figure 1 Improvement in slow transit constipation symptoms with Xuanshen decoction treatment. A: Time of first black stool discharge (week 4, n = 15); B: Intestinal propelling rate (week 8, n = 15); C: Intestinal propelling length of carbon: The yellow arrow marks the distance to the end of the carbon; D: Fecal moisture content (n = 15); E: Fecal number (n = 15); F: Body weight (n = 15). Data were presented as mean ± SD. ^bP < 0.01 vs control, ^cP < 0.05 vs model, and ^dP < 0.01 vs model. PRU: Prucalopride; L-XSD: Low-dose Xuanshen decoction; M-XSD: Medium-dose Xuanshen decoction; H-XSD: High-dose Xuanshen decoction.

In contrast to the normal rats, the model rats exhibited markedly reduced fecal moisture content (P < 0.01). XSD treatment increased this parameter (P < 0.01) (Figure 1D). The intestinal propelling rate was markedly lower in the model rats and was apparently higher in the M-XSD and H-XSD rats (P < 0.01) (Figure 1B and C). Compared with the normal rats, the number of stools collected at 24 h in the model rats was markedly decreased (P < 0.01). In the M-XSD and H-XSD rats, the number of stools collected was higher than in the model rats (P < 0.05) (Figure 1E). The model rats had lower body weight than the control rats (P < 0.01). STC rats showed significant weight gain following XSD therapy (P < 0.05) (Figure 1F).

XSD ameliorated the histopathology and secretory function of the intestinal mucosa

Hematoxylin and eosin staining showed that compared with the control animals, the mucosal and muscle layers in the colon tissue of the STC rats were strongly thinner (P < 0.01), and the proportion of infiltrated lymphocytes in the lamina propria of colon mucosa was markedly increased (P < 0.01). The number of goblet cells significantly decreased (P < 0.01). After treatment with M-XSD and H-XSD, the mucosal and muscle layers in colon tissue were thickened (P < 0.01), the proportion of infiltrating lymphocytes in the lamina propria of colon mucosa decreased (P < 0.01), and the number of goblet cells markedly increased (P < 0.01) (Figure 2).

Figure 2 Hematoxylin and eosin and Alcian blue/periodic acid-Schiff staining results. A: Hematoxylin and eosin staining; B: AB-PAS staining; C: Detection parameters (n = 3). ^bP < 0.01 vs control, and ^dP < 0.01 vs model. PRU: Prucalopride; L-XSD: Low-dose Xuanshen decoction; M-XSD: Medium-dose Xuanshen decoction; H-XSD: High-dose Xuanshen decoction.

Alcian blue/periodic acid-Schiff staining showed that compared with the normal rats, the level of mucin in the colon tissue of the model rats decreased (P < 0.01). After treatment with PRU, M-XSD or H-XSD, the level of mucin increased (P < 0.01). There was no significant change in the level of mucin in the L-XSD rats (P > 0.05) (Figure 2B and C).

Network pharmacology profiling

The herb-compound-target network comprised 1200 nodes and 10447 edges (Figure 3, Supplementary Table 1). According to OB and DL values, the top five ranked active ingredients in XSD were diosgenin, glyceollin, shinpterocarpin, phaseolin, and glycyrol (Table 1).

Figure 3 Herb-compound-target interaction network. The yellow-green circle nodes symbolized the seven herbs of Xuanshen decoction; the triangular and hexagonal nodes symbolized the components; the hexagonal nodes symbolized the shared components of the herbs; the blue quadrilateral nodes denoted the target. MOL000422 and MOL000211 were the common compounds of Glycyrrhiza uralensis and Sanguisorba officinalis; MOL000392, MOL000417, and MOL004941 were the common compounds of Glycyrrhiza uralensis and Sophora flavescens; MOL001924 was the component of Glycyrrhiza uralensis and Cimicifuga heracleifolia; MOL000098 was the common component of Glycyrrhiza uralensis, Sanguisorba officinalis, Sophora flavescens; MOL000359 was the common component of Glycyrrhiza uralensis, Cimicifuga heracleifolia, and Scrophularia ningpoensis; MOL000449 was the common compound of Cimicifuga heracleifolia, Dioscorea polystachya, and Atractylodes lancea; MOL000358 was the common component of Glycyrrhiza uralensis, Sanguisorba officinalis, Dioscorea polystachya, and Scrophularia ningpoensis. XS: Scrophularia ningpoensis, KS: Sophora flavescens; CZ: Atractylodes lancea; DY: Sanguisorba officinalis; SM: Cimicifuga heracleifolia; SY: Dioscorea polystachya; GC: Glycyrrhiza uralensis; XSD: Xuanshen decoction.

Table 1 Top five active ingredients in Xuanshen decoction for slow transit constipation.

MOL ID	Ingredients	CAS ID	OB (%)	DL
MOL000546	Diosgenin	512-04-9	80.8779	0.81
MOL006596	Glyceollin	57103-57-8	97.2747	0.76
MOL004891	Shinpterocarpin	157414-04-5	80.2953	0.73
MOL000456	Phaseolin	22090-93-3	78.2006	0.73
MOL002311	Glycyrol	23013-84-5	90.7758	0.67

MOL: Prefix for molecule entries in the TCMSP database; CAS: Chemical Abstracts Service registry number; OB: Oral bioavailability; DL: Drug-likeness.

The protein-protein interaction network of 370 nodes and 2992 edges was constructed utilizing the Search Tool for the Retrieval of Interacting Genes/Proteins database (Figure 4A). According to the degree value, 11 key proteins were screened out, including phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha, epidermal growth factor receptor, Bcl-2, Akt1, catenin 1, hypoxia-inducible factor 1, signal transducer and activator of transcription 3, tumor necrosis factor, heat shock protein 90AA1, SRC proto-oncogene, and estrogen receptor 1 (Figure 4A).

Figure 4 Network pharmacology flowchart. A: Protein-protein interaction network construction and detection of Xuanshen decoction-slow transit constipation target genes; B: Venn diagram demonstrating the shared targets of Xuanshen decoction and slow transit constipation; C: Enrichment analysis. XSD: Xuanshen decoction; STC: Slow transit constipation.

A total of 2476 targets relevant to STC treatment were retrieved from PharmGkb, GeneCards, Online Mendelian Inheritance in Man, Drugbank, DisGeNET, and Therapeutic Target Database. Three hundred and eighty potential target genes for STC treated with XSD were identified through the Sangerbox website (Figure 4B). As is shown in Figure 4C, Biological Process involved response to reactive oxygen species, response to oxidative stress, regulation of oxidative stress-induced intrinsic apoptotic signaling pathway, execution phase of apoptosis, regulation of execution phase of apoptosis, and reactive oxygen species metabolic process, etc. Cellular component (CC) involved Bcl-2 family protein complex, membrane raft, and membrane microdomain. Molecular function included protein kinase activity, protein serine/threonine kinase activity, and nuclear receptor activity. Kyoto Encyclopedia of Genes and Genomes pathways included the PI3K/Akt signaling pathway, and apoptosis (Figure 4C).

According to the results of network pharmacology, the mechanism of XSD in treating STC may be related to cell apoptosis, oxidative stress, and PI3K/Akt signaling pathway. Molecular docking was applied to evaluate the important active components in XSD and the key targets in the PI3K/Akt/Nrf2 pathway, apoptosis targets, and oxidative stress targets to further evaluate the accuracy and reliability of this prediction results.

Molecular docking

Targets of PI3K, AKT, Nrf2, Bcl-2, Bax, catalase (CAT), and SOD were sequentially aligned with the active ingredients of diosgenin, glyceollin, shinpterocarpin, phaseolin, and glycyrol for molecular docking. All labeled compounds have low free energies of binding to their receptors (Figure 5). These results showed that the related compounds of XSD (diosgenin, glyceollin, shinpterocarpin, phaseolin and glycyrol) had strong affinity with PI3K, Akt, Nrf2, CAT, SOD, Bcl-2 and Bax proteins. The results of target proteins-active molecules with lower binding energies were visualized with PyMoL software (Figure 6).

Figure 5 Schematic diagram of the binding energy of components and targets. Mode: The coordination mode; Affinity: The binding energy; Akt: Protein kinase B; PI3K: Phosphoinositide 3-kinase; Nrf2: Nuclear factor erythroid 2-related factor 2; CAT: Catalase; SOD: Superoxide dismutase; BAX: B-cell lymphoma 2-associated X protein; Bcl2: B-cell lymphoma 2.

Figure 6 Molecular docking and visualization. A: Phosphoinositide 3-kinase and diosgenin; B: Superoxide dismutase and diosgenin; C: Nuclear factor erythroid 2-related factor 2 (Nrf2) and glyceollin; D: Catalase and glyceollin; E: Nrf2 and shinpterocarpin; F: B-cell lymphoma 2 and shinpterocarpin; G: Protein kinase B and phaseolin; H: Nrf2 and phaseolin; I: Nrf2 and glycyrol; J: B-cell lymphoma 2-associated X protein and glycyrol.

In summary, it can be seen that the predictive results of network pharmacology are reliable. XSD may activate the body’s antioxidant capacity through the PI3K/Akt/Nrf2 pathway, inhibit ICC apoptosis, and effectively treat STC. We verified this conclusion in animal experiments.

Effects of XSD on oxidative stress

The results confirmed significantly upregulated MDA levels in STC rats vs controls (P < 0.01). In contrast, SOD and GSH-PX were markedly lower than in the healthy animals (P < 0.01) (Figure 7). Post-intervention biochemical analyses revealed distinct oxidative stress modulation across treatment groups. Both M-XSD and H-XSD groups exhibited significant reductions in MDA levels (P < 0.01), accompanied by elevated SOD activity (P < 0.01). The M-XSD rats additionally demonstrated elevated GSH-PX levels (P < 0.05). Notably, the L-XSD rats demonstrated no significant alterations in these markers (P > 0.05) (Figure 7).

Figure 7 Impact of Xuanshen decoction on oxidative stress and phosphoinositide 3-kinase/protein kinase B/nuclear factor erythroid 2-related factor 2 pathway in slow transit constipation Rats. A: The oxidative stress level (n = 15); B: Western blotting (n = 6). ^bP < 0.01 vs control, ^cP < 0.05 vs model, and ^dP < 0.01 vs model. L-XSD: Low-dose Xuanshen decoction; M-XSD: Medium-dose Xuanshen decoction; H-XSD: High-dose Xuanshen decoction; GSH-PX: Glutathione peroxidase; C-KIT: Tyrosine-protein kinase Kit; SCF: Stem cell factor; HO-1: Heme oxygenase-1; P-PI3K: Phospho-phosphoinositide 3-kinase; PI3K: Phosphoinositide 3-kinase; P-Akt: Phospho-Protein kinase B; Akt:‌ Protein kinase B; Nrf2: Nuclear factor erythroid 2-related factor 2; Nrf2-N: Nuclear-nuclear factor erythroid 2-related factor 2; Nrf2-C: Cytoplasmic nuclear factor erythroid 2-related factor 2.

Influences of XSD on the expression of PI3K/AKT/Nrf2 pathway

Among the STC rats, the protein levels of C-kit, SCF, P-PI3K/PI3K, P-Akt/Akt, HO-1, and Nrf2 (nuclear/cytoplasmic) were strongly decreased compared to the healthy rats (Figure 7) (P < 0.01). After the intervention, the protein levels of C-kit, SCF, P-PI3K/PI3K, P-Akt/Akt in the H-XSD and M-XSD groups were greatly increased (P < 0.01). HO-1 expression in the H-XSD rats was strongly increased (P < 0.01); Nrf2 (N/C) expression in the H-XSD and M-XSD groups was increased (P < 0.05) (Figure 7). Immunofluorescence analysis further revealed significantly reduced C-kit and Nrf2 expression levels in model animals relative to controls. XSD treatment significantly upregulated C-kit and Nrf2 protein expression, with the most pronounced effects observed in M-XSD and H-XSD groups (Figure 8).

Figure 8 Immunofluorescence staining. DAPI: 4’,6-diamidino-2-phenylindole; Nrf2: Nuclear factor erythroid 2-related factor 2; L-XSD: Low-dose Xuanshen decoction; M-XSD: Medium-dose Xuanshen decoction; H-XSD: High-dose Xuanshen decoction.

Effect of XSD on expression of apoptosis-related proteins

In contrast to the control rats, expression of Bax protein in the model rats was markedly upregulated (P < 0.01), while expression of Bcl-2 protein was downregulated (P < 0.05). Relative to the STC rats, the M-XSD and H-XSD rats exhibited elevated Bcl-2 protein expression (P < 0.05). Conversely, Bax protein expression was reduced in M-XSD rats (P < 0.05) and markedly downregulated in H-XSD rats (P < 0.01). No statistically significant alterations in either Bcl-2 or Bax expression were observed in the L-XSD rats (P > 0.05) (Figure 9).

Figure 9 Apoptosis-related proteins in colonic tissues (n = 6). ^aP < 0.05 vs control, ^bP < 0.01 vs control, ^cP < 0.05 vs model, and ^dP < 0.01 vs model. L-XSD: Low-dose Xuanshen decoction; M-XSD: Medium-dose Xuanshen decoction; H-XSD: High-dose Xuanshen decoction; Bax: B-cell lymphoma 2-associated X protein; Bcl-2: B-cell lymphoma 2; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

In contrast to the control rats, expression of Bax/Bcl-2 and cleaved caspase-3 in the model rats was markedly upregulated (P < 0.01). Relative to the STC rats, expression of cleaved caspase-3 in the L-XSD, M-XSD, and H-XSD rats was markedly reduced (P < 0.01); the Bax/Bcl-2 protein expression ratio in L-XSD rats was downregulated (P < 0.05), while Bax/Bcl-2 protein expression ratio in M-XSD and H-XSD rats was significantly reduced (P < 0.01) (Figure 9).

DISCUSSION

The study evaluated the laxative effect of XSD in rats with LOP-induced STC. Network pharmacology analysis suggested that the mechanism of XSD in treating STC may involve the PI3K/Akt signaling pathway, oxidative stress, and cell apoptosis. Molecular docking results showed that the main active ingredients in XSD (diosgenin, glyceollin, shinpterocarpin, phaseolin, and glycyrol) bind well to PI3K, AKT, Nrf2, Bcl-2, Bax, CAT, and SOD targets. Subsequently, through animal experiments, we concluded that XSD might effectively treat STC by activating the PI3K/Akt/Nrf2 pathway, thereby inhibiting oxidative stress-induced apoptosis of ICCs.

STC, one of the most important types of constipation, arises from poor colonic motility and delayed colonic transit, resulting in dry, hard stools and low stool volume[27]. In this research, M-XSD and H-XSD significantly accelerated the colonic propelling rate and improved almost all other indicators. L-XSD effectively increased the body weight and stool moisture in STC rats. L-XSD demonstrated efficacy against STC and M-XSD and H-XSD had a greater effect. Assessment of colonic tissue showed that XSD reduced inflammatory infiltration, repaired LOP-induced pathological damage and increased the number of goblet cells, increasing mucin levels, as indicated in prior studies[17,20,28-30].

It has been verified that only herbal components with appropriate pharmacokinetic profiles can reach the therapeutic target organs to express their biological efficacy[24]. Natural compounds with OB ≥ 30% and DL ≥ 0.18 are likely to be assimilated and dispersed in the body and are therefore regarded to have pharmacokinetic activity[31,32]. Thus, we assumed that XSD compounds with higher OB and DL values demonstrate efficacy in STC treatment. According to network pharmacology, diosgenin was the predominant active compound in XSD, followed by glyceollin, shinpterocarpin, phaseolin, and glycyrol. Emerging evidence indicates that diosgenin ameliorates oxidative stress through activation of the PI3K/Akt signaling pathway, which consequently mitigates apoptotic cell death[33]. Diosgenin can reduce the level of MDA, enhance antioxidant enzyme activity, and activate the Nrf2 pathway[34,35]. Glyceollin can suppress NO formation and has anti-inflammatory effects[36]. Shinpterocarpin can enhance immunity and mediates an antioxidant effect by reducing ROS production[37,38]. The accumulated evidence has demonstrated the antioxidant as well as the anti-inflammatory effects of phaseolin[39]. Glycyrol can regulate autoimmune and inflammatory responses as well as reduce the level of ROS, thus mediating an antioxidant effect[40,41].

Studies have verified that ICC in the colon of STC patients exhibit functional abnormalities and reduced numbers[42-44]. Additional research identified significant ICC loss in both clinical STC patients and experimental STC models induced by LOP[45,46]. Furthermore, ICC apoptosis was observed in STC patients’ colonic tissues[47], establishing a critical association between ICC apoptosis and STC pathogenesis[48,49]. These findings suggest that reducing ICC apoptosis may represent a therapeutic target for STC[25]. Studies have shown that, C-kit protein serves as the most important identification marker for ICC[50], with weakened C-kit immunofluorescence intensity observed in STC model colons[25], consistent with our experimental findings. XSD treatment enhanced C-kit fluorescence intensity. Western blotting revealed that XSD upregulated Bcl-2 expression while suppressing Bax and cleaved caspase-3 Levels in STC rat colons, normalizing the Bax/Bcl-2 ratio. This indicates the antiapoptotic effects of XSD on ICC, thereby improving intestinal motility.

Pharmacological activation of the PI3K/Akt pathway has been reported to alleviate STC symptoms by inhibiting ICC apoptosis[25,51]. Our experiments demonstrated reduced P-PI3K/PI3K and P-Akt/Akt ratios in STC rats, which XSD treatment reversed, confirming its therapeutic mechanism via PI3K/Akt pathway activation. Substantial evidence confirms that PI3K/Akt exerts antioxidant effects through Nrf2 regulation[52-55]. Oxidative stress, closely associated with ICC apoptosis[56,57], serves as a key driver in STC pathogenesis. Notably, the Nrf2/HO-1 pathway has been verified to attenuate oxidative-stress-induced apoptosis[58], with multiple studies confirming the critical involvement of oxidative stress in STC development[25,45,59,60]. We therefore performed molecular docking between key targets in the PI3K/Akt/Nrf2 pathway and active components of XSD. Oxidative stress results from disrupted homeostasis between ROS production and antioxidant capacity. Critical antioxidant enzymes including SOD and GSH-PX play essential roles in ROS scavenging[59,61], while MDA serves as a lipid peroxidation biomarker. Our experiments demonstrated reduced SOD and GSH-PX activities alongside elevated MDA levels in STC rats; all of which were normalized by XSD treatment, confirming its antioxidant capacity. Western blotting further showed that XSD increased the Nrf2 (N/C) ratio and upregulated HO-1 expression in colonic tissues, suggesting enhanced Nrf2 nuclear translocation and subsequent activation of antioxidant enzymes (SOD, GSH-PX, and HO-1)[62-64].

These findings collectively indicate that XSD may inhibit oxidative-stress-induced ICC apoptosis via PI3K/Akt/Nrf2 pathway activation, thereby exerting therapeutic effects against STC. In this study, network pharmacology identified PI3K, Akt, Nrf2, Bcl-2, Bax, CAT, and SOD as key therapeutic targets, with molecular docking confirming stable binding between the primary active components of XSD (diosgenin, glyceollin, shinpterocarpin, phaseolin, and glycyrol) and these targets. Some limitations of this study should be acknowledged. First, many proteins, particularly novel disease-associated targets, lack experimentally resolved 3D structures. Second, static crystal structure prediction, particularly the AlphaFold model[65,66], provide revolutionary solutions to these challenges. Through its exceptional predictive capabilities, AlphaFold generates high-accuracy 3D structural models for potential targets, enabling deeper exploration of structure-function relationships[65]. Compared to conventional methods like X-ray crystallography and cryo-electron microscopy, the deep learning-based approach of AlphaFold achieves faster and more precise structural predictions, significantly enhancing research efficiency and operational feasibility[66]. TCM typically treats STC through multicomponent, multitarget mechanisms[7]. While this study investigated XSD’s therapeutic modulation of the PI3K/Akt/Nrf2 pathway, oxidative stress, and cell apoptosis, future investigations should integrate AlphaFold to explore other potential targets and their interactions. Combining AlphaFold could effectively address critical bottlenecks in network pharmacology (target ambiguity) and molecular docking (static structure dependency). Its capacity to rapidly generate dynamic, multiscale structural models holds significant potential for elucidating multicomponent, multitarget therapeutic mechanisms of XSD against STC. Therefore, AlphaFold implementation will be essential in advancing research on TCM formulations for STC treatment.

CONCLUSION

This study confirmed the laxative efficacy of XSD on LOP-induced STC in rats and provided evidence for the underlying therapeutic mechanism of XSD in STC. In other words, XSD may exert its antioxidative stress effect through the PI3K/Akt/Nrf2 pathway and inhibit ICC apoptosis to treat STC. Therefore, this study has identified a novel herbal formula candidate for STC treatment.