Background & Aims

Chronic pain and pathological joint changes associated with Osteoarthritis (OA) are major health concerns globally. Currently available treatments are symptomatic and often associated with significant side effects. Thus, the mediators and mechanisms that monitor pain and inflammation in OA are of great interest in the search for new treatment modalities. Our previous findings have provided evidence that proteasome-mediated proteolysis critically regulates pathological pain by selectively degrading cellular proteins1,2. However, the underlying mechanisms remain obscure. The project aims to characterise the proteomic profile of chronic pain by identifying key regulatory biomarkers of pain. Further, the role of the proteasome system in the regulation of molecular mechanisms of pain will be specified.

Methods

The study is based on 24 female Lewis rats. OA was induced in 12 rats by monosodium iodoacetate (MIA) injection into the left knee joint, while 12 control rats received saline. At the onset of OA all animals were randomly divided into 4 groups: 2 OA and 2 control groups each containing 6 rats. The control and OA groups were injected s.c either with vehicle or MG132 (1 mg/kg) once daily for 21 days. Joint stiffness and pain were recorded. All animals sacrificed and the dorsal spinal cord L2-4 were dissected and stored at minus 80oC until analysis.
Quantitative mass spectrometry (MS). Samples were analysed on a NanoLC™ 425 System coupled to a Triple TOF™ 6600 mass spectrometer. For data acquisition, the mass spectrometer was set to scan full spectra for 250 ms, followed by up to 100 MS/MS scans with 30 ms accumulation time. Candidate ions were isolated, and MS/MS spectrum was collected. A specific ion-library was created and searched using the ProteinPilot™ software for data analysis.

Results

A total of 1583 proteins were quantified with 54 up- and 28 down-regulated in OA when compared with normal controls. The bioinformatic analysis of the 82 DEPs proteins identified biological processes/pathways associated with vesicle-mediated transport, anion and protein bindings, as well as positive regulation of immune response. The comparison within the OA groups, treated with or without MG132, identified 21 DEPs with 10 up- and 11 down-regulated proteins. After overlapping both the OA- and MG132 treatment-related proteomic files, we identified 3 novel potential biomarkers Haptoglobin (Hp), Microtubule-associated proteins-1 (Map 1) and a Complement component C9. All three biomarkers expression were increased in OA compared to controls. The higher levels of Hp, Map 1 and C9 were reversed after MG132-treatment.

Conclusions

Our finding showed a comprehensive proteomic landscape of the spinal cord in OA and control rats. Further bioinformatic analysis identified specific biomarkers and biological processes associated with pain. In consistency with previous observations, our findings confirmed the role of Map 1, a neuron-specific cytoskeletal protein associated with the inflammatory origin of knee pain3. In addition, the role of complement cascade in immune response was also identified. A robust effect with reversal of pain behavior and decreased Map 1, C9 and Hp expression suggest that proteasome is one of the potential intracellular pathways critical for the development of both pain and inflammation in OA. The proteasome system may represent a promising system/mechanism for treatment modality in OA. Thus, novel safe proteasome inhibitors with limited adverse effects would be beneficial to attenuate the development of arthritis and hence reduce the pain.

References

1.Ahmed et al., Pain. 2012; 153 (1): 18-26.
2.Guimarães et al., Pain. 2021 Jun 1;162(6):1722-1731.
3.Eymard et al., Jt. Bone Spine 2016, 83, 389–393.

Presenting Author

Aisha S. Ahmed

Poster Authors

Aisha Ahmed

PhD

Karolinska Institutet

Lead Author

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Junyu Chen

Department of Orthopaedic Surgery, West China Hosptial, Sichuan, P.R. China

Lead Author

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Junyu Chen

Orthopedic Research Institution, West China Hospital, Sichuan University, P.R. China

Lead Author

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Vera M. Mendes

CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal

Lead Author

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Bruno Manadas Manadas

CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal

Lead Author

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Hugo Leite-Almeida

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga,

Lead Author

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Topics

  • Models: Chronic Pain - Inflammatory