Network Pharmacology and Rat Model Analysis of Yougui Pill in Carrageenan-Induced Prostatitis

Summary

This study integrates network pharmacology and in vivo experiments to investigate the potential effects of Yougui Pill in treating prostatitis. Network pharmacology analysis and YGP treatment in a rat prostatitis model suggested that YGP's therapeutic effects may be associated with the regulation of inflammatory responses, indicating that YGP exerts anti-inflammatory effects in prostatitis.

Abstract

Prostatitis is a common urological disorder with limited treatment options. Yougui Pill (YGP) has shown potential therapeutic value, particularly in inflammation-related conditions. This study aimed to evaluate its effects and explore possible underlying mechanisms. Active components of YGP were screened from TCMSP using oral bioavailability (OB ≥ 30%) and drug-likeness (DL ≥ 0.18) criteria, and corresponding targets were collected. Prostatitis-related targets were obtained from GeneCards, and overlapping targets were analyzed using protein–protein interaction (PPI) networks and Kyoto encyclopedia of genes and genomes (KEGG) enrichment. A total of 126 active compounds and 208 potential targets were identified, with 180 overlapping targets associated with prostatitis. PPI analysis identified TP53, IL6, AKT1, TNF, and IL1B as key hub genes. KEGG enrichment suggested involvement of inflammation-related pathways, including IL-17, TNF, MAPK, and PI3K-Akt signaling pathways. A rat model of prostatitis was established by intraprostatic injection of carrageenan. Histological examination and measurement of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were performed to assess therapeutic effects. YGP treatment improved prostatic histopathology and reduced TNF-α and IL-6 levels at both protein and mRNA levels. These findings suggest that YGP exerts anti-inflammatory effects in prostatitis, while the specific molecular mechanisms remain to be further validated.

Introduction

Prostatitis is a condition where the prostate gland becomes inflamed with swelling and irritation¹. It accounts for approximately 25% of global urological clinical encounters². The National Institutes of Health (NIH) stratified prostatitis into four classifications: acute bacterial, chronic bacterial, chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), and asymptomatic prostatitis³. Among these, CP/CPPS is the most prevalent form and is closely associated with persistent inflammation and immune dysregulation. Despite advances in understanding the inflammatory nature of prostatitis, current therapeutic strategies remain limited, particularly for non-bacterial subtypes^4,5. Antibiotics are commonly used in clinical practice; however, their efficacy in non-bacterial prostatitis is highly contentious⁶. Other treatments, ranging from α-blockers to non-steroidal anti-inflammatory drugs (NSAIDs), primarily target symptom relief but often show suboptimal outcomes, with non-negligible risks of gastrointestinal disturbances, orthostatic hypotension, and so on^7,8,9,10. These limitations highlight the need for alternative therapeutic approaches that can effectively modulate inflammation in prostatitis.

Traditional Chinese medicine (TCM) may offer a broader range of therapeutic benefits with a more favorable safety and tolerability profile¹¹. Yougui Pill (YGP), a classical formula documented in Jingyue Quanshu, has been empirically used for centuries to replenish kidney-yang and alleviate related disorders¹². Previous studies have reported that some of its constituents possess anti-inflammatory activities^13,14,15,16. Clinical evidence suggests that YGP can effectively alleviate the symptoms of prostatitis, although the underlying mechanism remains unclear¹⁷. Network pharmacology serves as a powerful, integrative approach that combines systems biology and pharmacology to uncover the complex mechanisms of traditional medicine¹⁸. It was hypothesized that YGP may exert anti-inflammatory effects by modulating multiple targets and inflammation-related pathways. These predictions were further evaluated using an in vivo prostatitis model, in which prostatitis was induced by carrageenan injection. Following model establishment, animals were treated with YGP. Prostatic tissues were collected for histopathological examination via hematoxylin and eosin (HE) staining, as well as for quantitative assessment of inflammatory markers by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR).

Protocol

All animal experiments were approved by the Institutional Animal Care and Use Committee of Beijing University of Chinese Medicine (BUCM-2024110107-4103).

Collection of active constituents and targets
YGP consists of Rehmannia glutinosa, Aconitum carmichaelii, Cinnamomum cassia, Dioscorea opposita, Cornus officinalis, Cuscuta chinensis, Angelica sinensis, Eucommia ulmoides, Cervi Cornus Colla, and Lycium barbarum. These herb names were input into the traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP) to retrieve their compounds. Active compounds were screened using oral bioavailability (OB ≥ 30%) and drug-likeness (DL ≥ 0.18) criteria^19,20,21. Corresponding targets of each compound were obtained from TCMSP and standardized to gene symbols using the UniProt database. Prostatitis-related targets were retrieved from the GeneCards database using "prostatitis" as the keyword, and genes with a relevance score higher than the average were selected for inclusion in the study.

Construction of the regulatory network of YGP against prostatitis
The intersection between YGP-related and prostatitis-related targets was identified and defined as a set of potential therapeutic targets. These targets were imported into Cytoscape (version 3.9.1) to construct a compound-target-disease network. Network topology analysis was performed using the CytoHubba plugin, and key components were identified based on maximal clique centrality (MCC) values²².

Protein-protein interaction (PPI) network analysis
The overlapping targets were imported into the STRING database (version 11.5), with the organism set to Homo sapiens. The minimum required interaction score was set to ≥ 0.7, and disconnected nodes were hidden²³. The interaction data were exported in TSV format and visualized in Cytoscape. Hub genes were further identified using the CytoHubba plugin based on degree centrality.

KEGG Pathway and GO enrichment analysis
The shared targets were uploaded to the Metascape platform for Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. The input and analysis species were set to Homo sapiens. KEGG pathway enrichment analysis was conducted. Pathways were further filtered based on their relevance to prostatitis, as determined by published literature and database annotations. The remaining pathways were ranked according to their Log P values. The top enriched KEGG pathways were visualized using bubble plots.

Molecular docking
The key targets were selected from the PPI network based on degree centrality, and the active compounds were identified from the YGP-prostatitis regulatory network based on MCC values for subsequent docking analysis. Protein structures were obtained from the AlphaFold Protein Structure Database in PDB format, and ligand structures were downloaded from TCMSP in mol2 format²⁴. Molecular docking was performed using the CB-Dock2 online platform²⁵. Prior to docking, protein structures were automatically preprocessed by removing water molecules, adding hydrogen atoms, and completing missing residues. Binding affinities were recorded, and heatmaps were generated using GraphPad Prism (version 10.1.2). The three-dimensional structures of ligand-protein complexes with the lowest Vina scores were visualized using ChimeraX, and two-dimensional interaction diagrams were generated using LigPlus.

YGP aqueous extract preparation
The herbal components of YGP were prepared according to the traditional formulation ratio, including 24 g of the processed root of Rehmannia glutinosa (Shudihuang), 6 g of the processed lateral root of Aconitum carmichaelii (Fuzi), 6 g of the bark of Cinnamomum cassia (Rougui), 12 g of the rhizome of Dioscorea opposite (Shanyao), 9 g of the fruit of Cornus officinalis (Shanzhuyu), 12 g of the seed of Cuscuta chinensis (Tusizi), 12 g of the processed antler gelatin of Cervi Cornus Colla (Lujiaojiao), 12 g of the fruit of Lycium barbarum (Gouqi), 9 g of the root of of Angelica sinensis (Danggui), and 12 g of the bark of of Eucommia ulmoides (Duzhong). All herbs except Aconitum carmichaelii and Cervi Cornus Colla were soaked in 6 times their total weight of distilled water for 30 min, while Aconitum carmichaelii was soaked separately in 6 times its weight of distilled water. Aconitum carmichaelii was decocted first using an electric heating device for 1 h, after which the remaining herbs, excluding Cervi Cornus Colla, were added and decocted twice for 1 h each cycle. Cervi Cornus Colla was subsequently melted in a water bath at 60–70 °C until fully dissolved and incorporated into the extract. After cooling, the decoction was filtered through double-layer gauze, aliquoted, frozen at -20 °C for 48 h, and lyophilized to obtain a powdered extract.

Animal model and experimental design
Male Sprague-Dawley rats (8 weeks old, 200–220 g) were housed under specific pathogen-free conditions with a 12 h light/dark cycle and free access to food and water. After 1 week of acclimatization, rats were randomly divided into three groups (n = 6 per group): control, model, and YGP groups. Chronic prostatitis was induced by intraprostatic injection of 3% carrageenan solution²⁶. Briefly, rats were anesthetized with isoflurane (3–4% for induction and 1.5–2% for maintenance), and a midline abdominal incision was made to expose the prostate. A total of 25 µL of 3% carrageenan solution was injected into each ventral lobe of the prostate. The control group received an intraprostatic injection of normal saline (physiological saline solution) only. Postoperative penicillin (2.5 × 10⁵ U/kg) was administered intramuscularly for 3 days.

YGP lyophilized extract powder was prepared from the decoction and reconstituted in 0.9% sterile normal saline to obtain a uniform suspension for oral gavage (10.54 g/kg/day). Rats in the YGP group received oral administration of YGP extract (10 mL/kg), while control and model groups received an equivalent volume of 0.9% sterile normal saline. After 4 weeks, rats were anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneal injection), and prostate tissues were collected after being sacrificed in accordance with ethical guidelines.

Histopathological examination
Prostate tissues were fixed in 4% paraformaldehyde for 24 h, dehydrated through graded ethanol (75% alcohol for 4 h, 85% alcohol for 2 h, 90% alcohol for 2 h, 95% alcohol for 1 h, absolute ethanol for 1h), cleared in xylene, and embedded in paraffin. The sections (4–5 µm) were then subjected to HE staining according to standard procedures²⁷. Briefly, sections were deparaffinized, rehydrated, stained with hematoxylin, differentiated, and counterstained with eosin. After dehydration and mounting, the sections were observed under a light microscope for histopathological evaluation.

Histopathological inflammation was evaluated using a semi-quantitative scoring system based on inflammatory cell infiltration, fibroblast proliferation, acinar morphology, and secretion status. Inflammatory cell infiltration was scored as 0 (no infiltration), 2 (mild infiltration), 4 (moderate infiltration), and 6 (severe infiltration). Fibroblast proliferation was graded as 0 (absent), 1 (mild), 2 (moderate), and 3 (severe). Acinar morphology was evaluated as 0 (large acinar lumen), 1 (moderately reduced lumen size), 2 (markedly reduced lumen size), and 3 (acinar occlusion or disappearance). Secretion status was scored as 0 (abundant secretion), 1 (moderate secretion), 2 (scant secretion), and 3 (absent secretion). The total histopathological score was calculated as the sum of all four parameters, with higher scores indicating more severe prostatic tissue injury and inflammation²⁸.

ELISA
Prostate tissues (100 mg) were rinsed with ice-cold phosphate-buffered saline (PBS) and homogenized in lysis buffer supplemented with protease inhibitors. The homogenates were incubated at 4 °C for 30–120 min and centrifuged at 5000 × g for 15 min at 4 °C to collect the supernatants. The protein levels of TNF-α and IL-6 were measured using ELISA kits according to the manufacturer's instructions. Standards and samples were added to 96-well plates and incubated at 37 °C, followed by sequential incubation with biotinylated antibodies and enzyme conjugates. After washing, the substrate solution was added for color development, and the reaction was terminated with the stop solution. Absorbance was measured at 450 nm using a microplate reader. Cytokine concentrations were calculated based on standard curves.

qRT-PCR
Total RNA was extracted from prostate tissues using a column-based method and quantified by measuring absorbance at 260 and 280 nm to assess purity. RNA samples were reverse-transcribed into cDNA at 42 °C for 15 min using a reverse transcription kit. qRT-PCR was performed using a thermocycler under the following conditions: initial denaturation at 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The primer sequences used in this study are listed in Supplementary Table 1. Fluorescence signals were collected during the annealing/extension step. The expression levels of TNF-α and IL-6 were normalized to the internal reference gene GAPDH. Relative gene expression was calculated using the 2^−ΔΔCt method.

Statistical analysis
All data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism software (version 10.1.2). Data distribution was assessed for normality prior to analysis. Differences among multiple groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test for multiple comparisons. A value of p < 0.05 was considered statistically significant.

Results

Identification of bioactive components and target genes
A total of 126 bioactive components in YGP meeting the criteria (OB ≥ 30% and DL ≥ 0.18) were identified. corresponding to 208 predicted target genes. The GeneCards database yielded 9,845 genes associated with prostatitis. Venn analysis revealed 180 overlapping genes between YGP's predicted targets and prostatitis-related genes, suggesting these shared targets may mediate the therapeutic effects of YGP against prostatitis (F...

Discussion

Prostatitis is pathologically defined by an increased presence of inflammatory cells within the prostatic parenchyma or mesenchyme^28,29. In a study of over 5 million Korean men followed for nine years, Kim et al. confirmed through multivariate analysis that prostatitis significantly increases the risk of developing prostate cancer³⁰. However, current clinical management remains limited, particularly for non-bacterial prostatitis, where tre...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4% paraformaldehyde	Beyotime	P0099-3L
AlphaFold Protein Structure Database	AlphaFold Protein Structure Database	https://alphafold.ebi.ac.uk/
BeyoFast SYBR Green qPCR Mix (2X)	Beyotime	D7260
Bioinformatics online platform	Bioinformatics online platform	http://www.bioinformatics.com.cn/
Carrageenan	OriLeaf	S30559
CB-DOCK2	CB-DOCK2	http://183.56.231.194:8001/cb-dock2/php/blinddock.php
Cytoscape	Cytoscape	https://cytoscape.org/
GeneCards	GeneCards	https://www.genecards.org/
Hematoxylin and Eosin (H&E) Staining Kit	Beyotime	C0105S
Metascape	Metascape	https://metascape.org/gp/index.html#/main/step1
Neutral Balsam	Servicebio	WG10004160
Paraffin Wax (for embedding)	Servicebio	RP-15
Protease Inhibitor Cocktail (100X)	Epizyme	GRF101
Rat IL-6 ELISA Kit	Beyotime	PI328
Rat TNF-α Kit	Beijing 4A Biotech Co., Ltd	CRE0003
RNAeasy Animal Long RNA Isolation Kit with Spin Column	Beyotime	R0026
STRING	STRING	https://string-db.org/
TCMSP	TCMSP	https://www.tcmsp-e.com/#/database
Uniprot	Uniprot	https://www.uniprot.org/