Summary

The serum protein profiles of early-stage knee osteoarthritis model mice were investigated using liquid chromatography-mass spectrometry, which revealed the differentially expressed serum proteins and mainly activated pathways.

Abstract

Introduction

Recently, new criteria for detecting early-stage knee osteoarthritis (EKOA) without evaluating radiographic abnormalities have been proposed, and various methods for early diagnosis of knee OA have been explored. The diagnosis of EKOA requires the identification of biomarkers that reflect changes in joint structure. However, no single clinically applicable biomarker characteristic of EKOA has yet been identified. This study aims to characterize the serum protein profiles of early-stage osteoarthritis model mice using liquid chromatography-mass spectrometry (LC-MS/MS) and to investigate the potential biomarkers.

Method

Serum and knee samples were collected from five control mice and 15 osteoarthritis model mice that underwent destabilization of the medial meniscus (DMM). Osteoarthritic knee samples were collected 4, 8, and 12 weeks post-DMM. Knee osteoarthritis severity was scored using the OARSI scoring system. All serum samples were analyzed via LC-MS/MS after removing highly abundant proteins using a ProteoMiner kit (Bio-Rad). The average OARSI scores were analyzed using the Steel–Dwass test. The proteomics data was analyzed using transformed normalized abundances in the t-test. Differentially expressed proteins were defined as those with a >2.0- or <0.5-fold change in expression. The list of proteins was imported into KeyMolnet, and the “Interrelation search” algorithm was used to generate the network of molecular interactions between the related proteins. The top canonical pathways associated with the generated network were extracted from the KeyMolnet knowledge base. IBM SPSS Statistical Software (version 25.0; IBM Corp., Armonk, NY, USA) was used for all analyses. Statistical significance was set at p < 0.05.

Results

The average OARSI scores of the medial tibial plateau and medial femoral condyle at eight weeks post-DMM (3.40 ± 1.02 points, p = 0.03; 2.60 ± 1.71 points, p = 0.03) and 12 weeks post-DMM (8.30 ± 3.12 points, p = 0.03; 4.80 ± 0.75 points, p = 0.03) were significantly higher than the corresponding values in the control group. Compared to those in the control model, 15, 35, and 56 proteins showed different expression levels at 4, 8, and 12 weeks post-DMM, respectively. Differentially expressed proteins four weeks post-DMM included adenylate kinase, type IV intermediate filament protein, nestin, and insulin-like growth factor-binding proteins. The pathways activated four weeks post-DMM differed from those activated at 8 and 12 weeks post-DMM. They included the intermediate filament signaling pathway, the p160 SRC signaling pathway, and the microtubule-associated protein signaling pathway.

Conclusion

Serum protein expression and activated pathways in the osteoarthritis model differed from those in the control model. Based on the progression of OA severity, four weeks post-DMM was considered to correspond to the state of EKOA. Adenylate kinase, type IV intermediate filament protein, nestin, and insulin-like growth factor-binding proteins, which are differentially expressed four weeks postoperatively, may be involved in the pathogenesis of EKOA and serve as potential biomarkers of EKOA. Moreover, activated pathways in four weeks postoperatively, including the intermediate filament signaling pathway, the p160 SRC signaling pathway, and the microtubule-associated protein signaling pathway, may contribute to understanding the EKOA mechanism.