The Mechanism of Ermiao Powder In Treating Prostatitis: A Study Based on Network Pharmacology and Experimental Verification

Summary

Employing a network pharmacology approach, this study revealed that Ermiao Powder exerts its therapeutic effects against prostatitis by inhibiting inflammatory factors and associated signaling pathways. Subsequent in vitro experiments further demonstrated that its aqueous extract significantly suppressed the expression of key inflammatory cytokines in an LPS-induced model of prostate epithelial cells.

Abstract

This protocol presents a combined network pharmacology and experimental validation approach to investigate the mechanism of action of Ermiao Powder (EMP) in the treatment of prostatitis. The workflow follows a stepwise design: (i) database-driven screening of active compounds and targets, (ii) construction and analysis of interaction networks, and (iii) in vitro experimental validation. First, active ingredients of EMP and their corresponding targets were retrieved from the TCMSP database, while prostatitis-related targets were obtained from the GeneCards database. The intersection of EMP targets and disease targets was used to identify key therapeutic targets. Second, a “Drug–Active Ingredient–Target–Disease” network was constructed using Cytoscape. Protein–protein interaction (PPI) analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment were performed on the key targets, revealing that inflammatory factors such as IL6 and TNF served as core targets, and pathways including PI3K/AKT and MAPK were significantly enriched. Finally, in vitro validation was conducted using an LPS-induced inflammatory model of prostate epithelial cells. Treatment with EMP significantly reduced the release of pro-inflammatory factors IL-6 and TNF-α compared to the LPS-treated group (p < 0.05), confirming the anti-inflammatory efficacy of EMP. This integrative approach provides a reproducible framework that can be adapted to investigate other multi-component therapeutic systems. In conclusion, this study preliminarily elucidates that EMP exerts its anti-prostatitis effects through a multi-component, multi-target, and multi-pathway mechanism, closely associated with the suppression of inflammatory responses, thereby offering a scientific basis for its clinical application.

Introduction

Prostatitis, particularly chronic non-bacterial prostatitis, represents a prevalent urological disorder characterized by complex etiology and a lack of highly effective therapies, imposing a significant burden on patients' quality of life^1,2,3. Current clinical management of prostatitis employs an integrated approach including antibiotics, α-receptor blockers, and non-steroidal anti-inflammatory drugs^4,5,6. The principal challenges include the absence of etiology-specific th....

Protocol

Ethical approval was not required for this study, as it did not involve human participants or clinical samples. All procedures involving cell lines were conducted in accordance with institutional biosafety guidelines and regulations. The prostate epithelial cell line used in this study was obtained from a certified commercial supplier.

NOTE: All reagents, instruments, and software used in this study are listed in the Table of Materials. The flowchart of this study is presented in Figure 1.

1. Screening of active ingredients

1. Access the TCM....

Results

Identification of active compounds and target prediction

These results demonstrate the effectiveness of our integrated network pharmacology and in vitro approach for identifying and validating EMP’s therapeutic targets in prostatitis. A search of the TCMSP database identified 46 bioactive components from the constituent herbs of EMP, corresponding to 178 potential targets. From the Genecards database, 13,960 prostatitis-related targets were acquired. The 162 targets c.......

Discussion

This protocol integrates network pharmacology with experimental validation to provide a systematic framework for investigating multi-component therapeutic mechanisms. Critical steps include accurate screening of active compounds using defined thresholds (OB ≥ 30%, DL ≥ 0.18), reliable network construction, and appropriate selection of experimental validation models. Careful parameter selection in bioinformatics analyses and strict control of experimental conditions are essential to ensure reproducibility.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Human IL-6 ELISA KIT	4Abio	CHE0009
Human TNF-α ELISA KIT	4Abio	CHE0019
K-SFM supplements	Procell	CM-0200
Lipopolysaccharides	Solarbio	L8880
MTS	Promega	G1111
Reverse Transcription System	Promega	A3500
RWPE-1	Procell	CL-0200	RRID:CVCL_EQ24
RWPE-1 Cell Complete Medium	Procell	CM-0200
SYBR Green Realtime PCR Master Mix	Toyobo	QPK-201
Trizol reagent	Thermo Fisher Scientific (Invitrogen)	15596026
trypsin-EDTA	Gibco (Thermo Fisher)	25200072