Integrating Network Pharmacology to Investigate Cinnamaldehyde's Protective Effect Against Oxaliplatin-induced Neurotoxicity

Summary

This study investigates the protective effects of cinnamaldehyde (CA) against oxaliplatin (OXA)-induced damage in rat dorsal root ganglion (DRG) cells. The underlying mechanism involves suppression of the JAK2/STAT3 signaling pathway and activation of the SLC7A11-GSH-GPX4 pathway, as revealed through network pharmacology and experimental validation.

Abstract

This study investigates the protective effects of cinnamaldehyde (CA) against oxaliplatin (OXA)-induced damage in rat dorsal root ganglion (DRG) cells. The pharmacological activities of CA were analyzed, and its potential targets, along with those of its metabolites, were identified using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), Integrative Pharmacology-based Network Computational Research Platform of Traditional Chinese Medicine (TCMIP), and PharmMapper platforms. The CCK-8 assay is used to determine the optimal concentrations of drugs, while malondialdehyde (MDA) assays are applied to assess the level of lipid peroxidation. Intracellular levels of Fe²⁺ and reactive oxygen species (ROS) were quantified using fluorescent probes. The impact of CA on protein expression levels, including p-STAT3, XCT, and GPX4, in OXA-treated DRG cells was examined via immunoblotting. Network pharmacology analysis identified 14 overlapping targets among "CA-neuropathic pain (NP)-ferroptosis". Experimental results demonstrated that a 24 h treatment with 4.0 µmol/L CA yielded optimal effects, significantly reducing MDA, Fe²⁺, and ROS levels in DRG cells (P < 0.05). Furthermore, CA treatment downregulated the expression of JAK2, STAT3, p-STAT3, TFRC, ErbB-1, and nuclear factor-kappa B (NF-κB) (P < 0.05) while upregulating XCT, GPX4, and FTH1 expression (P < 0.05). These findings suggest that CA mitigates OXA-induced damage in DRG cells by inhibiting the JAK2/STAT3 signaling pathway and activating the SLC7A11-GSH-GPX4 axis.

Introduction

In recent years, ferroptosis, as a non-apoptotic cell death mode, has been proven to be involved in the tissue damage induced by various chemotherapeutic drugs. The core mechanism of ferroptosis is the accumulation of iron-dependent lipid peroxidation products¹. Research on the mechanisms of cisplatin neurotoxicity reveals that this drug induces neuronal ferroptosis through multiple pathways, with this process being directly linked to the development of neurotoxicity. Specifically, cisplatin inhibits glutathione peroxidase 4 (GPX4) activity. As a critical "brake molecule" in ferroptosis, the inactivation of GPX4 prevents the clearance o....

Protocol

NOTE: The cell line was derived from rat cell lines.

1. Network pharmacology analysis

NOTE: The links can be found in the Table of Materials.

1. Finding possible targets of CA
   1. Retrieve structural information from the PubChem database and explore the potential biological mechanisms of CA. Subsequently, conduct the target prediction analysis using SwissTargetPrediction, leveraging its advanced computational capabilities.
   2. To enhance the accuracy and comprehensiveness of the analysis, cross-reference the predicted targets with Traditional Chinese Medic....

Results

Network Pharmacology and Molecular Docking
Identification of potential targets of CA: In the comprehensive investigation of CA's pharmacological activity, 60 potential targets were meticulously curated from an extensive review of relevant literature. To further expand the scope of the analysis, an additional 12 potential targets associated with CA were identified through the TCMSP database. For enhanced accuracy and thoroughness, the advanced predictive capabilities of the SwissTargetPrediction.......

Discussion

Ferroptosis, a recently identified mode of cell death, is characterized by excessive intracellular iron accumulation and lipid peroxidation. Morphologically, ferroptosis manifests as mitochondrial shrinkage, loss or reduction of cristae structures, and increased membrane density, while the cell membrane and nuclear morphology remain unaffected²⁶. Mechanistically, ferroptosis is driven by glutathione (GSH) depletion and impaired GPX4 activity, preventing the metabolism of lipid peroxides via

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Glycine	Biofroxx	1275KG001
Tris-base	Biofrooxx	1115KG001
0.5  M  Tris-HCl buffer (pH  6.8)	Biorad	1610799
1 M Tris-Hcl (PH 7.5)	Beyotime	ST775
1.5  M  Tris-HCl buffer (pH  8.8)	Biorad	1610798
APS	Beyotime	ST005
Basic electrophoresis power supply	BIO-RAD	PowerPac Basic
BCA Protein Quantification Kit	Beyotime	P0012
Beta Actin Recombinant antibody	Huabio	81115-1-RR
Carbon dioxide incubator	Thermo	150L
CCK-8 assay kit	Apexbio	K1018
Cell Activity and Cytotoxicity Assay Kit	Beyotime	C2015M
Cinnamaldehyde(CA)	Macklin	C805134	purity 98%
Decolorizing shaker	Kylin-Bell	TS-100
DMEM (Dulbecco's Modified Eagle Medium)	BasalMedia	L110KJ
DMSO	Sigma	D2650
dorsal root ganglion (DRG)	Fuheng Bio	FH165	Serial number: 20211227-1
DRG complete medium	Fuheng Bio	FH-DRG
ECL luminescent solution	Thermo	34580
ErbB-1	Proteintech	06-847	1:2000
Fetal  Bovine  Serum	Macklin	FO193
Ferrous ion detection probe - FerroOrange	Dojindo	F374
FTH1	Cell Signaling Technology	4393S	1:1000
Gel imaging system	BIO-RAD	ChemiDoc Touch
Hanks' Balanced Salt Solution(HBSS)	Solarbio	H1025
High-speed refrigerated centrifuge	Eppendorf	5804R
Hoechst 33342 Live Cell Staining Solution	Beyotime	C1028
Immunohistochemistry kit	Biosharp	BS-24-PB
Inverted fluorescence microscope system	Leica	Leica Dmi 8
JAK2 Recombinant Rabbit Monoclonal Antibody [SY0245]	Huabio	ET1607-35	1:2000
Laboratory inverted phase contrast microscope	Leica	Leica Dmi 1
Low-speed centrifuge	Eppendorf	5804
MDA Content Detection Kit	Sangon	D799762
Microplate reader	SUNRISE	TECAN
Multifunctional microplate reader	BMG	LABTECH PHERAstar FS
NF-κB p65 Polyclonal antibody	Proteintech	10745-1-AP	1:1000
Paraformaldehyde	Macklin	P804536
Ponceau staining solution	Beyotime	P0022
Protein Marker	Thermo	26617
P-STAT3 (Ser727)	Cell Signaling Technology	9134S	1:1000
P-STAT3 (Tyr705)	Cell Signaling Technology	9145S	1:2000
Recombinant Anti-Glutathione Peroxidase 4 Antibody[EPNCIR144]	abcam	ab125066	1:1000
Recombinant Anti-XCT antibody[EPR27115-64]	abcam	ab307601	1:1000
ROS Assay Kit	Dojindo	R252
SDS-PAGE Protein Loading Buffer (6X)	Beyotime	P0289
Skimmed milk powder	Biosharp	BS102-500g
Sodium dodecyl sulfate SDS	Biofrooxx	3250GR500
STAT3 (F-2)	Santa Cruz	Sc-8019	1:200
TEMED	Solarbio	T8090
TFRC	Invitrogen	13-6800	0.5 µg/mL
Tween80	Selleck	S6702
Ultra-clean workbench	HDL	DL-CJ-2NDI
Upright microscope	Olympus	CX 31
Vertical electrophoresis	BIO-RAD	Mini Protean Tetra
Wet transfer tank	BIO-RAD	Mini Trans Blot
α-Tubulin	α-Tubulin	ER130905
GraphPad Prism			Statistical analysis
ImageJ software			Obtain the gray value of the protein blot