Gegen Qinlian Decoction Attenuates Ulcerative Colitis by Inhibiting PANoptosis: A Multi-Omics and Experimental Study

Summary

Here, we present a protocol integrating multi-omics analysis with in vivo validation to investigate how Gegen Qinlian Decoction alleviates ulcerative colitis by regulating PANoptosis-related targets and inflammatory signaling pathways.

Abstract

We present an integrated bioinformatics and network pharmacology protocol to clarify the therapeutic mechanisms of Gegen Qinlian Decoction (GQD) in ulcerative colitis (UC), with a particular focus on PANoptosis-related regulation. This protocol is designed to systematically identify potential bioactive compounds, therapeutic targets, signaling pathways, and immune-related mechanisms through multi-database mining and transcriptomic analysis. Gene expression profiles from UC cohorts, including GSE87466 and GSE92415 from the GEO database, are analyzed using differential expression analysis and weighted gene co-expression network analysis to identify disease-related genes. Candidate targets of GQD are collected from public pharmacology databases and intersected with UC- and PANoptosis-associated genes to determine core therapeutic targets. Protein-protein interaction network construction, enrichment analyses, and compound-target-pathway network modeling are then performed to reveal the molecular basis of GQD activity. In addition, immune infiltration analysis is incorporated to explore the relationship between key genes and the intestinal immune microenvironment. Molecular docking is further used to evaluate the binding potential between major active compounds and hub targets. This protocol provides a reproducible, systematic framework for investigating how GQD alleviates UC by modulating PANoptosis and related inflammatory pathways, and may serve as a useful reference for mechanistic studies of other traditional Chinese medicine formulas in complex inflammatory diseases.

Introduction

Ulcerative colitis (UC), one of the major forms of inflammatory bowel disease, is a chronic relapsing inflammatory disorder that primarily involves the colonic mucosa and typically begins in the rectum with continuous proximal extension. Its pathogenesis is complex and involves aberrant mucosal immune activation, epithelial barrier dysfunction, microbial perturbation, and dysregulated host-environment interactions, together imposing a substantial and growing global health burden^1,2. Persistent mucosal inflammation not only causes recurrent symptoms such as abdominal pain, diarrhea, and hematochezia, but also i....

Protocol

All animal procedures were conducted in accordance with institutional guidelines and were approved before study initiation by the Ethical Committee of Chengdu University of Traditional Chinese Medicine (Approval No. 2022-126). Only publicly available GEO and GWAS datasets were used for the bioinformatics analyses; therefore, no additional informed consent or ethics approval was required for the computational component of this study. All procedures involving hazardous chemicals, biological samples, and animal waste were performed in accordance with institutional laboratory safety regulations and institutional procedures for hazardous waste disposal.

Results

GEO dataset acquisition and UC-Related gene identification

To investigate gene expression changes in UC, two transcriptomic datasets-GSE87466 and GSE92415-were obtained from the GEO database using “ulcerative colitis” as the search term, with selection criteria including dataset type, clear disease/control annotation, and sufficient sample size. These datasets encompassed 140 UC cases and 42 healthy controls. Samples lacking unambiguous phenotype labels or valid expression pro.......

Discussion

Ulcerative colitis (UC) is a chronic, idiopathic inflammatory bowel condition primarily affecting the colonic mucosa, characterized by persistent or episodic inflammation. The disease significantly diminishes patient quality of life and elevates the risk of developing colorectal carcinoma. Although its global prevalence is steadily rising, the precise etiology and underlying mechanisms remain only partially understood. Current treatment options often yield suboptimal outcomes and are frequently accompanied by adverse eff.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Puerariae Lobatae Radix	Sichuan Neautus Traditional Chinese Medicine Co., Ltd.	NTC-001	Herbal material for GQD preparation
Scutellariae Radix	Sichuan Neautus Traditional Chinese Medicine Co., Ltd.	NTC-002	Herbal material for GQD preparation
Coptidis Rhizoma	Sichuan Neautus Traditional Chinese Medicine Co., Ltd.	NTC-003	Herbal material for GQD preparation
Glycyrrhizae Radix et Rhizoma	Sichuan Neautus Traditional Chinese Medicine Co., Ltd.	NTC-004	Herbal material for GQD preparation
BALB/c mice, male, 8 weeks old	Beijing HFK Bioscience Co., Ltd.	N/A	Experimental animals
Dextran sulfate sodium (DSS), MW 36-50 kDa	MP Biomedicals	160110	Colitis-inducing reagent
Sodium carboxymethyl cellulose	Sigma-Aldrich, St. Louis, MO, USA	C5678	Vehicle for oral gavage
Paraformaldehyde	Sigma-Aldrich, St. Louis, MO, USA	158127	Tissue fixative
Hematoxylin	Sigma-Aldrich, St. Louis, MO, USA	H9627	Nuclear stain
Eosin	Sigma-Aldrich, St. Louis, MO, USA	E4009	Counterstain for histology
Bright-field microscope	Nikon	Eclipse 80i	Histological observation
Slide-scanning system	3DHISTECH Ltd.	Panoramic 250	Digital whole-slide image acquisition
Trypsin	Gibco, Carlsbad, CA, USA	25200-056	Antigen retrieval reagent
Hydrogen peroxide	Sigma-Aldrich, St. Louis, MO, USA	H1009	Blocking endogenous peroxidase activity
Bovine serum albumin (BSA)	Sigma-Aldrich, St. Louis, MO, USA	A7906	Antibody diluent / blocking reagent
Anti-STAT3 primary antibody, rabbit	Cell Signaling Technology	9139	Primary antibody for IHC/WB
Anti-TIMP1 primary antibody, rabbit	Abcam	ab211925	Primary antibody for IHC/WB
Anti-SPHK2 primary antibody, rabbit	Abcam	ab187030	Primary antibody for IHC/WB
Anti-HSPA5 primary antibody, rabbit	Cell Signaling Technology	3177	Primary antibody for IHC/WB
Anti-GAPDH primary antibody, rabbit	Abcam	ab9485	Loading control for WB
Goat anti-rabbit secondary antibody	Yeasen Biotechnology	33201	Secondary antibody for IHC/WB
Chromogenic detection reagent	Beyotime Biotechnology [Shanghai] Co., Ltd., Shanghai, China	P0202	Signal development for immunohistochemistry
Image-analysis software	Media Cybernetics	Image-Pro Plus 7.0	Quantification of staining and band intensity
Phenol-based RNA extraction reagent	Invitrogen	TRIzol Reagent, 15596026	Total RNA extraction
First-strand cDNA synthesis kit	Beyotime Biotechnology	DRR047A	Reverse transcription
Real-time PCR system	Applied Biosystems	ABI Prism 7300	Quantitative PCR amplification
SYBR Green qPCR mix	Beyotime Biotechnology	DRR420A	qPCR detection reagent
Primer-design software	Premier Biosoft International, Palo Alto, CA, USA	Primer 5.0	Primer design
RIPA lysis buffer	Beyotime Biotechnology	P0013B	Protein extraction reagent
Protease inhibitor (PMSF)	Beyotime Biotechnology	ST506	Protein protection during lysis
BCA protein assay kit	Beyotime Biotechnology	P0010	Protein quantification
PVDF membrane	Merck Millipore	IPVH00010	Protein transfer membrane
Chemiluminescent detection reagent / system	Thermo Fisher Scientific	32106	Signal detection for western blot
Statistical software	IBM	SPSS 23.0	Statistical analysis