Network Pharmacology and Rat Caudal-disc Puncture Validation of Yi Qi Huo Xue Decoction For Lumbar Disc Degeneration

Summary

This study used network pharmacology, molecular docking, and a rat caudal-disc puncture model to investigate the potential preclinical effects of Yi Qi Huo Xue Decoction on lumbar disc degeneration, with a focus on candidate targets, including AKT1, BCL2, and STAT3, and pathways such as PI3K-AKT signaling.

Abstract

In this study, network pharmacology, molecular docking, molecular dynamics simulation, and experimental validation were used to investigate the pharmacological basis and potential molecular mechanisms of Yi Qi Huo Xue Decoction (YQHXD) in lumbar disc degeneration (LDD). A total of 678 active compound-related targets of YQHXD and 296 potential LDD-associated therapeutic targets were identified. Protein-protein interaction network analysis and topological analysis identified several candidate targets, including AKT1, IL6, TNF, BCL2, and STAT3. Enrichment analyses using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes indicated that the candidate targets were linked to functions including protein binding and ATP binding, with pathway enrichment that included PI3K-AKT signaling. Molecular docking predicted interactions between selected active compounds, such as quercetin, Tetraneurin A, and isorhamnetin, and the core targets. Molecular dynamics simulations further suggested that the lowest-energy complexes remained stable. Seventy male Sprague-Dawley rats were randomly assigned to the control, sham-operated, model, positive control, and low-, medium-, and high-dose YQHXD groups. After the establishment of a coccygeal intervertebral disc puncture model in the model, positive control, and YQHXD groups, rats were administered YQHXD or celecoxib by oral gavage for 8 weeks, whereas the control and sham-operated groups received normal saline. Histopathological evaluation showed that YQHXD treatment was associated with reduced intervertebral disc degeneration in this model. Enzyme-linked immunosorbent assay, polymerase chain reaction, and western blot analyses showed changes in the mRNA and protein expression levels of selected targets, including AKT1, BCL2, and STAT3. These findings suggest that YQHXD may exert preclinical protective effects in LDD through multi-component and multi-target regulation involving inflammation- and apoptosis-related mechanisms. This study provides preliminary preclinical evidence supporting further investigation of YQHXD for LDD.

Introduction

Lumbar disc degeneration (LDD) is a common age-related orthopedic condition characterized by late onset, difficulty of treatment, high recurrence, and disability. LDD involves degenerative pathological changes associated with an imbalance between catabolic and anabolic metabolism of the extracellular matrix (ECM) in the intervertebral disc (IVD) and is closely associated with low back pain (LBP)¹. Epidemiological studies show that LBP is one of the leading causes of disability in clinical practice².

From 1990 to 2017, the number of global cases increased from 42.5 million to 64.9 million

Protocol

All experiments were approved by the Institutional Review Board of Nanjing University of Chinese Medicine (Protocol Code: 202501A018) and were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. All experimental procedures adhered to the ARRIVE guidelines.

Experimental drugs
The YQHXD sample solution contained 20 g of Astragalus membranaceus root, 20 g of Angelica sinensis root, 15 g of Ligusticum chuanxiong rhizome, 6 g of Stephania tetrandra root, 15 g of Chaenomeles speciosa fruit, and 10 g of Sinapis....

Results

Conclusions derived from the study
This study integrated network pharmacology, molecular docking, molecular dynamics simulations, and in vivo experiments to investigate the potential protective effects of YQHXD against LDD. The results suggest that YQHXD may act, at least in part, through regulation of the AKT1/STAT3/BCL-2 signaling axis, characterized by reduced STAT3 expression and increased AKT1 and Bcl-2 expression. Histological and molecular analyses showed that YQHXD treatment was asso.......

Discussion

Traditional Chinese herbal medicines have been reported to exert pharmacological effects including anti-inflammatory, analgesic, and blood circulation–promoting activities¹⁹. Previous studies have suggested that Xiaosui Huahe Decoction, the precursor formula of YQHXD, may promote resorption of massive IVD herniation by affecting nucleus pulposus (NP) resorption and IVD absorption²⁰; however, the underlying molecular mechanisms remain unclear. In the present study, net.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5% sodium carboxymethylcellulose (CMC-Na)	Not specified		Vehicle used to prepare the celecoxib suspension for gavage.
21 G needle	Not specified		Used for caudal intervertebral disc puncture.
2× Universal Blue SYBR Green qPCR Master Mix	Servicebio Technology Co., Ltd., Wuhan	G3326	qPCR amplification reagent.
30% acrylamide/bis-acrylamide gel solution (29:1)	Solarbio Co., Ltd.		Gel preparation solution for SDS-PAGE; listed in original materials table as 30%/29:1 gel solution.
3D frozen grinding machine	Servicebio Technology Co., Ltd., Wuhan		Used for tissue homogenization/grinding.
4× SDS-PAGE concentrating gel buffer (pH 6.8)	Solarbio Co., Ltd.		Western blot gel preparation reagent.
Acidic ethanol	Not specified		Used for histological staining differentiation.
ACPYPE	ACPYPE project		Used to convert ligand topology files into a GROMACS-compatible format.
AKT1 ELISA kit	Hangzhou UpingBio Technology Co., Ltd.	SYP-R1928	Used to measure serum AKT1 levels; detection range 0.625–40 ng/mL.
AM1-BCC charge method	Antechamber/AMBER Tools		Used for ligand atomic charge calculation.
Amber14SB force field	AMBER force field		Protein force field used in molecular dynamics simulation.
Angelica sinensis root	Jianshou Pharmacy, Nanning, Guangxi, China	Batch No. 20250503	YQHXD botanical component; 20 g per prescription.
Anhydrous ethanol	Tianjin Aopusheng Chemical Co., Ltd.		Used in tissue processing/dehydration.
Antechamber	AMBER Tools		Used for ligand charge calculation and atom typing.
Anti-AKT1 antibody	Wuhan Sanying Co., Ltd.		Primary antibody for Western blotting; dilution 1:5,000.
Anti-Bcl-2 antibody	Wuhan Sanying Co., Ltd.		Primary antibody for Western blotting; dilution 1:1,500.
Anti-STAT3 antibody	Wuhan Sanying Co., Ltd.	10253-2-AP	Primary antibody for Western blotting; dilution 1:6,000.
APS reagent	Beyotime/Biyuntian Co.		Western blot gel polymerization reagent.
Astragalus membranaceus root	Jianshou Pharmacy, Nanning, Guangxi, China	Batch No. 20250405	YQHXD botanical component; 20 g per prescription.
Autoclave	SANYO, Japan		Sterilization equipment.
AutoDock Vina engine	AutoDock/Vina project		Docking engine used within CB-Dock2.
BCA protein assay kit	Solarbio Co., Ltd.		Used to determine protein concentration before Western blotting.
Bcl-2 ELISA kit	Wuhan Fine Biotech Co., Ltd.	PMK1545	Used to measure serum Bcl-2 levels; detection range 0.16–10 ng/mL.
Bupivacaine	Not specified		Local infiltration anesthetic used preoperatively.
CB-Dock2	CB-Dock2 web server		Automated blind molecular docking platform.
Celecoxib	Sichuan New Ston Pharmaceutical Co., Ltd.	H20233911	Positive-control drug; 20 mg/kg/day by gavage.
Chaenomeles speciosa fruit	Jianshou Pharmacy, Nanning, Guangxi, China	Batch No. 231001	YQHXD botanical component; 15 g per prescription.
Cytoscape	Cytoscape Consortium	Version 3.9.0	Used to construct and analyze network diagrams.
DAB chromogenic reagent kit (20×)	Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.		Listed in existing materials table.
DAVID	National Cancer Institute	Version 6.8	Database for Annotation, Visualization and Integrated Discovery; used for GO and KEGG enrichment analysis.
Decalcification solution	Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.		Used for decalcification of caudal vertebral IVD tissue.
Decolorization shaker	Liu Yi Biotechnology Co., Ltd., Beijing		Laboratory shaker.
DEPC water	Wuhan Sanying Co., Ltd.		RNA work/reagent preparation.
Digital radiography imaging system	Not specified		Used for postoperative radiographic model validation.
Distilled water	Not specified		Vehicle/control gavage and YQHXD decoction preparation.
ECL chemiluminescent substrate	Biosharp Co.		Western blot signal detection reagent.
EDTA antigen retrieval solution (pH 8.0)	Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.		Listed in existing materials table.
Electrophoresis apparatus	Not specified		Used for SDS-PAGE electrophoresis.
Electrophoresis buffer	Servicebio Co.		Western blot electrophoresis solution.
FM40 ice maker	Changliu Scientific Instruments Co., Ltd., Beijing		Ice maker.
Formaldehyde	Not specified		Fixative used for caudal vertebral IVD tissue.
GAFF2 atom types	AMBER Tools		Ligand atom types used for molecular dynamics simulations.
Gel imaging system	Tianen Scientific, Shanghai		Used to capture Western blot images.
GeneCards	Weizmann Institute of Science	Version 5.26	Human gene database used to retrieve disease-associated targets.
gmx_MMPBSA	gmx_MMPBSA project		Used for MM-PBSA binding free-energy estimation where applicable.
GraphPad Prism	GraphPad Software	Version 8.0	Statistical/graphing software listed in the authors' earlier manuscript version.
Grinding beads	Servicebio Technology Co., Ltd., Wuhan		Used for tissue grinding/homogenization.
GROMACS	GROMACS development team	Version 2022.2	Used for molecular dynamics simulations.
Hematoxylin and eosin staining reagents	Not specified		Used for HE staining of caudal IVD sections.
High-speed centrifuge	Eppendorf, Germany		Laboratory centrifuge.
High-speed low-temperature tissue grinder	Servicebio, China		Used for low-temperature tissue grinding.
Horizontal shaker	Chirinbell Company		Laboratory shaker.
HRP-labeled goat anti-rabbit antibody	Servicebio Co.		Secondary antibody for Western blotting; dilution 1:5,000.
HyperScript III RT SuperMix for qPCR with gDNA Remover	Servicebio Technology Co., Ltd., Wuhan		Reverse transcription reagent listed in existing materials table.
Ice maker	SIMAG, China		Ice maker.
ImageJ	National Institutes of Health		Used for Western blot band-intensity quantification.
Inverted biological microscope	Olympus, Japan		Used for microscopy of stained sections.
Isoflurane	Not specified		Inhalation anesthetic/euthanasia agent.
Isopropanol reagent	China National Pharmaceutical Group Chemical Reagent Co., Ltd.		RNA extraction reagent.
Joung-Cheatham ion parameters	Not applicable		Ion parameter set used for molecular dynamics simulations.
Ligusticum chuanxiong rhizome	Jianshou Pharmacy, Nanning, Guangxi, China	Batch No. 20250301	YQHXD botanical component; 15 g per prescription.
Liquid nitrogen	Not specified		Used for tissue grinding before RNA extraction.
Low-speed centrifuge	Eppendorf, Germany		Used for serum separation and other low-speed centrifugation steps.
Masson's trichrome staining solution	Not specified		Used for Masson staining of caudal IVD sections.
Microplate reader (TECAN)	TECAN, Switzerland		Used for absorbance measurement at 450 nm; listed in existing materials table.
Microplate reader (Thermo Fisher Scientific)	Thermo Fisher Scientific, USA		Multifunctional microplate reader listed in existing materials table.
Microsoft Excel/spreadsheet software	Microsoft		Used to remove duplicate target entries and manage tabular data.
NanoDrop 2000 spectrophotometer	Thermo Fisher Scientific, USA		Used to measure RNA concentration and purity.
Non-enzymatic lysis buffer	Not specified		Used for tissue lysis before RNA extraction.
OMIM	Johns Hopkins University School of Medicine		Online Mendelian Inheritance in Man; used to retrieve disease-associated targets.
Online bioinformatics plotting platform	Not specified		Used to generate GO and KEGG bar plots.
PAGE gel polymerization agent	Solarbio Co., Ltd.		Western blot gel preparation reagent.
Paraffin	Not specified		Embedding medium for histological sections.
PCR primers for Akt1, Bcl-2, Stat3, and Gapdh	Wuhan Servicebio Technology Co., Ltd.		Primer sequences are listed in Table 1.
PDB	RCSB PDB		Protein Data Bank; used to obtain 3D structures of target proteins.
Phosphorylated/protease inhibitors	Servicebio Co.		Added to RIPA lysis buffer for protein extraction.
Phosphotungstic acid	Not specified		Used in Masson staining.
Picrosirius Red	Not specified		Used in Masson staining.
Polyvinylidene difluoride membrane (0.45 μm)	Servicebio Co.		PVDF membrane used for Western blot transfer.
Positive-control drug vehicle	Not specified		0.5% CMC-Na suspension vehicle for celecoxib.
Povidone-iodine	Not specified		Used for surgical site disinfection.
Protein marker	Thermo Fisher Scientific, USA		Molecular weight marker for Western blotting.
Protein transfer apparatus	Bio-Rad, USA		Wet transfer equipment for Western blotting.
Protein transfer module (wet transfer)	Bio-Rad, USA		Western blot wet transfer module.
PubChem	National Center for Biotechnology Information (NCBI)		Used to obtain SMILES/SDF files of compound structures.
Pure water preparation system	Le Feng Biotechnology Co., Ltd., Shanghai		Used for pure water preparation.
PyMOL	Schrödinger, LLC		Used for visualization and analysis of molecular dynamics/docking outputs.
qPCR real-time PCR system	Applied Biosystems QuantStudio, USA		Used for quantitative PCR amplification.
RIPA tissue/cell lysis buffer	Solarbio Co., Ltd.		Used for total protein extraction.
RNA dissolution solution	Servicebio Technology Co., Ltd., Wuhan		RNA handling reagent.
Rotary evaporator	Not specified		Used to concentrate the combined YQHXD filtrates under reduced pressure.
Sinapis alba mature seeds	Jianshou Pharmacy, Nanning, Guangxi, China	Batch No. 20240801	YQHXD botanical component; 10 g per prescription.
Sprague-Dawley rats	Shanghai Slack Experimental Animal Co., Ltd.	SCXK (Shanghai) 2022-0004	Seventy male rats, aged 3–8 weeks, used for the caudal-disc puncture model.
SPSS	IBM, USA	Version 21.0	Statistical software listed in the authors' earlier manuscript version.
STAT3 ELISA kit	Hangzhou UpingBio Technology Co., Ltd.	SYP-R1555	Used to measure serum STAT3 levels; detection range 31.25–1000 pg/mL.
Stephania tetrandra root	Jianshou Pharmacy, Nanning, Guangxi, China	Batch No. 230301	YQHXD botanical component; 6 g per prescription.
Sterile gauze	Not specified		Used to protect the puncture site after modeling.
STRING	STRING Consortium	Version 12.0	Protein-protein interaction network platform.
SweScript All-in-One RT SuperMix for qPCR	Servicebio Technology Co., Ltd., Wuhan	G3337	Reverse transcription reagent listed in existing materials table.
SwissTargetPrediction	SIB Swiss Institute of Bioinformatics		Used to predict potential targets of compounds.
TBST buffer	Not specified		Used for membrane washing during Western blotting.
TCMSP	Center for Bioinformatics, College of Life Science, Northwest A&F University	Version 2.3	Traditional Chinese Medicine Systems Pharmacology Database; used to obtain active ingredients of Chinese herbal compounds.
TIP3P water model	Not applicable		Water model used in molecular dynamics simulations.
Toluidine blue	Not specified		Used in Masson staining.
Transfer buffer	Servicebio Co.		Western blot transfer buffer.
TriQuick reagent	Servicebio Technology Co., Ltd., Wuhan		RNA extraction reagent.
TRIzol reagent	Not specified		Total RNA extraction reagent mentioned in the manuscript.
TTD	The Chinese University of Hong Kong (IDRBLab)		Therapeutic Target Database; used to retrieve disease-associated targets.
Venny	BioinfoGP, CNB-CSIC	Version 2.1.0	Used to identify overlapping targets between YQHXD and LDD.
Verlet cutoff scheme	GROMACS		Non-bonded interaction scheme used in molecular dynamics simulations.
Vertical electrophoresis apparatus	Bio-Rad, USA		Used for SDS-PAGE.
VMD	University of Illinois at Urbana-Champaign		Used for visualization and analysis of molecular dynamics outputs.
Wet transfer system	Bio-Rad, USA		Used for protein transfer in Western blotting.
X-ray fluoroscopy system	Not specified		Used to identify caudal IVD levels during puncture modeling.
Xylene	Not specified		Used for deparaffinization/clearing during histology.
β-actin primary antibody	Wuhan Sanying Co., Ltd.		Loading-control primary antibody for Western blotting; dilution 1:4,000.