Potential Role of the PGE2-EP4-Ca2+ Signaling Axis in Post-Traumatic Osteoarthritis

Summary

Here, we present a protocol to systematically outline the establishment and evaluation methods of osteoarthritis rat models, through comparison, providing scientific evidence for the differing pathogenesis between acute mechanical injury-induced and chronic metabolic/environmental factor-induced osteoarthritis. The ACLT model is the main focus, emphasizing changes in the EP4-Ca²⁺-mitochondrial dysfunction signaling pathway.

Abstract

Post-traumatic osteoarthritis (PTOA) is a degenerative joint disease triggered by trauma or intense mechanical stress, leading to joint cartilage degeneration and functional impairment. Prostaglandin E2 (PGE₂) contributes significantly to cartilage degradation following mechanical injury by activating its receptor, Prostaglandin E receptor 4 (EP4), on chondrocyte membranes. The homeostasis of articular cartilage primarily relies on the dynamic balance between cartilage degradation and repair, a process finely regulated by chondrocytes. The Ca²⁺/Calmodulin-dependent Protein Kinase II (CAMKII) signaling pathway has been shown to play a critical role in mediating chondrocyte function restoration. In this study, we observed increased expression of EP4, Inositol 1,4,5-trisphosphate receptor (IP3R), and phosphorylated CaMKII in interleukin-1 beta (IL-1β) stimulated chondrocytes, suggesting a possible link between EP4 signaling and calcium dysregulation. However, direct evidence confirming the involvement of the EP4-Ca²⁺/CaMKII axis in PTOA is still lacking. Further investigations using genetic or pharmacological interventions are needed to clarify this potential mechanism. These findings provide a preliminary basis for exploring calcium homeostasis and EP4 signaling as targets for PTOA treatment.

Introduction

Although physical activity is widely promoted for its health benefits, it remains a major cause of lower limb musculoskeletal injuries, which can lead to various adverse outcomes¹. Among these, PTOA has become an increasing burden for adolescents and young adults who experience joint injuries at a young age². Alarmingly, approximately 48-52% of individuals who sustain sports-related joint injuries develop PTOA within 11-17 years^3,4. The knee joint, one of the most frequently injured joints in youth sports and recreational activities, is particularly vulnerable in....

Protocol

All experimental procedures involving animals were reviewed and approved by the Animal Ethics Committee of Guangdong Provincial Hospital of Chinese Medicine (ethical approval number: 2024014-2). The study was conducted in accordance with the regulations on the ethical use of laboratory animals stipulated by the State Council of the People's Republic of China.

1. Animals

1. Use Sprague-Dawley (SD) outbred SPF rats to reduce potential confounding effects associated with the estrous cycle. Select a total of nine male SD rats, aged 4 weeks and weighing between 90 g and 100 g, for this experiment.
   NOTE: These....

Results

ACLT-induced PTOA exhibits premature cartilage destruction and behavioral deficits relative to FD-induced chronic KOA (Figure 2 and Figure 3)
Joint space narrowing and cartilage degeneration are the primary outcomes of OA. Previous studies have identified overweight and environmental factors (e.g., humid climate) as potential contributors to OA onset^{12,13<.......}

Discussion

Compared to chronic OA induced by environmental and dietary factors, ACLT-induced joint trauma leads to a more rapid progression of the disease. Our results, based on behavioral assessments including joint swelling, hindlimb grip strength, and gait analysis, revealed significant differences between the control group, the PTOA group, and the positive control group induced by environmental and dietary factors. After 12 weeks of intervention, excessive joint swelling, accompanied by a significant reduction in hindlimb grip .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.2% Collagenase II	Sigma-Aldrich, USA	CAS No.: 9001-12-1	Primary chondrocyte isolation and culture
0.2% Triton X-100	Beyotime, China	ST795	Immunofluorescence
0.25% trypsin-EDTA	Wuhan Boster Biological Technology Co., Ltd.	AR1071	Decalcification
0.25% Trypsin-EDTA	Gibco, USA	25200072	Primary chondrocyte isolation and culture
4% Paraformaldehyde Fixative Solution	Biyuntian, China	P0099-3L	Tissue fixation
Anti-CaMKII Antibody	Huabio, China	ET1608-47	Western blot (1:1000)
Anti-Collagen II Antibody	Abcam, UK	ab34712	Western blot (1:1000)
Anti-COX2 Antibody	Abcam, UK	ab179800	Western blot (1:1000)
Anti-Cyt-C Antibody	Selleck, China	F2534	Western blot (1:1000)
Anti-IP3R Antibody (B-2)	Santa Cruz, USA	sc-377518	Western blot (1:1000)
Anti-MMP9 Antibody	Abcam, UK	ab76003	Western blot (1:1000)
Anti-p-CaMKII Antibody	Selleck, China	F0342	Western blot (1:1000)
Anti-β-actin Antibody	Cell Signaling Technology (CST), USA	8H10D10	Western blot (1:2000)
BSA Blocking Solution	Sigma Aldrich, USA	N/A	5% BSA for Western blot blocking
Centrifuge (5424 R)	Eppendorf, Germany	N/A	755 × g centrifugation speed
Chloroform	Sigma Aldrich, USA	N/A	Solvent for PA
Climate-controlled chamber (RXZ-158A)	Domestic manufacturer, China	N/A	Humid environment model
Color Reverse Transcription Kit	Invitrogen, Suzhou, China	N/A	cDNA synthesis
CTAn software	Bruker	versions 1.9 and 4.1	For quantitative morphometric analysis
CTVol software	Bruker	version 2.0	For qualitative and three-dimensional visualization
DAPI Staining Solution	Beyotime, China	N/A	Nuclear staining (5 µg/mL)
Digital Grip Strength Meter	Puxin Instrument	N/A	Measuring hindlimb grip strength
DMEM/F12 Medium	Corning, USA	N/A	Cell culture medium
EDTA (Ethylenediaminetetraacetic acid)	Aladdin	N/A	Decalcification of bone tissues
ELISA Kit for PGE2	Elabscience, Wuhan, China	PGE2E-EL-0034	ELISA detection
Enhanced Chemiluminescence Kit	Millipore, USA	WBKLS0500	Western blot detection
Ethanol	Guangdong Guangshi Reagent Technology Co., Ltd.	CAS: 64-17-5	Tissue dehydration
Fetal Bovine Serum (FBS)	Corning, USA	N/A	10% concentration for culture
FITC-labeled Goat Anti-Rabbit IgG	Beyotime, China	P0179 kit	Immunofluorescence (1:500)
GraphPad Prism	GraphPad Software, USA	Version 8.0.2	Data analysis
Hematoxylin and Eosin (H&E) Staining Kit	Servicebio, Wuhan	G1005	Histology
IL-1β	PeproTech, USA	400-01B	10 ng/mL for PTOA cell model
ImageJ	NIH, USA	Version 1.48v	Image analysis
Image-Pro Plus software	Media Cybernetics	Version 6.0	Quantitative gait analysis from scanned footprints
Isoflurane	Shanghai Yaji Biological	YS1412C	Anesthesia for animal surgery
Leica Embedding Center	Leica Microsystems	EG1160	Paraffin embedding
Leica Microtome	Leica Microsystems	RM2235	Sectioning paraffin-embedded tissues
Leica Water Bath	Leica Microsystems	HI1210	Floating paraffin sections
Light microscope (ECLIPSE Ti2-E)	Nikon, Japan	N/A	Histological observation
Micro-CT Scanner (ZKKS-MCT-Sharp)	Domestic manufacturer, China	N/A	Bone imaging
Mouse-HRP Secondary Antibody	Cell Signaling Technology (CST), USA	7076	1:2000 dilution
NIS-Elements Imaging Software	Nikon	N/A	Capturing and analyzing microscope images
NRecon software	Bruker, Germany	Version 1.6	CT reconstruction
Palmitic Acid (PA)	Sigma Aldrich, USA	P0500	500 µM stock for FD cell model
Paraffin Wax for Sectioning, Pathology Grade, Melting Point 52-54 °C	Guangdong Guangshi Reagent Technology Co., Ltd.	Y258797-500g	Used for paraffin embedding during tissue processing
Paraffin Wax for Sectioning, Pathology Grade, Melting Point 56-58 °C	Guangdong Guangshi Reagent Technology Co., Ltd.	Y258799-500g	Used for final hard wax embedding in tissue sectioning
PBS (Phosphate Buffered Saline)	Gibco / Thermo Fisher	C10010500BT	Cell washing and tissue rinsing
Penicillin-Streptomycin	Corning, USA	N/A	1% for cell culture
PVDF Membrane (0.45 µm)	Millipore, USA	IPVH00010	Western blot membrane
Rabbit-HRP Secondary Antibody	Cell Signaling Technology (CST), USA	7074	1:2000 dilution
Red Ink	HERO	HERO 204	Footprint tracking in gait analysis
RIPA Lysis Buffer	Keygen, China	N/A	Protein extraction
Safranin O–Fast Green Staining Kit	Servicebio, Wuhan	G1053	Histological staining
Spiral Micrometer (Vernier Caliper)	Huangshan, China	N/A	Joint width measurement
Subchondral Bone Analysis Software (CTAn)	Bruker, Germany	Versions 1.9 and 4.1	Morphometric analysis
SYBR Green PCR Master Mix	AG Scientific, China	N/A	qPCR analysis
Western blot reagents (RIPA buffer, SDS-PAGE gel components, loading buffer, TBST, etc.)	Beyotime Biotechnology (China)	N/A (used as a group entry)	All routine reagents used for Western blotting procedures
Xylene	Guangdong Guangshi Reagent Technology Co., Ltd.	CAS: 1330-20-7	Tissue clearing