Publication: Role of p2x4 and n-methyl-d-aspartate- 2b receptors on pain modulation in thalamus of complete freund’s adjuvant-induced chronic polyarthritis rat upon corm-2 and ifenprodil treatments
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Date
2025-06
Authors
Khir, Nurul Ajilah Mohamed
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Abstract
Rheumatoid arthritis (RA) is a chronic inflammatory disease and pain being a major debilitating symptom reported by RA patients. This symptom frequently persists even after inflammation subsides, suggesting altered mechanisms beyond peripheral inflammation. CORM-2 and Ifenprodil were selected for their targeted modulation of central nociceptive pathways, specifically through inhibition of P2X4 and NMDAR- 2B receptors implicated in chronic RA pain. This study aimed to investigate effects of CORM-2 (P2X4R antagonist) and Ifenprodil (non-competitive NMDAR-2B antagonist) treatments on nociceptive and apoptotic mechanisms in thalamus of complete Freund’s adjuvant (CFA)-induced chronic polyarthritis rat. Eighty Sprague- Dawley male rats were randomly divided into five groups (n=16) consisting of non- arthritic control (N), arthritic control (A), arthritic groups treated with CORM-2 (A+CORM-2), Ifenprodil (A+Ifenprodil), and Diclofenac (A+Diclofenac) (positive control). The arthritic rats received intrathecal injection of either CORM-2 (20μg), Ifenprodil (0.5μg/μL), or Diclofenac (6μg) for seven days (day-16 to -22 post-arthritic induction). Meanwhile, A and N control groups were administered with normal saline (0.9%). Ankle joints’ diameter and pain behaviour responses (spontaneous behaviour activities, von-Frey and hot-plate tests) were recorded on day-0 (baseline), day-15 (pre-treatment), and day-23 (post-treatment). The rats were sacrificed on day-24 and thalamus was collected for ELISA, RT-qPCR, immunohistochemistry, and
histological examination by Nissl staining. Arthritic rats demonstrated significant increase in bilateral ankle joints’ diameter with reduced spontaneous behaviour activities, and persistent development of tactile allodynia and thermal hyperalgesia. Treatments with CORM-2 and Ifenprodil significantly improved spontaneous activities and attenuated tactile allodynia and thermal hyperalgesia development as comparable to Diclofenac. Ifenprodil significantly reduced ankle joints’ diameter in arthritic rats, while no notable change was observed with CORM-2 treatment. Significant mRNA upregulation and increased proteins expression of NMDAR-2B, P2X4R, and BDNF were observed in bilateral thalamic VPL regions of arthritic rats which were strongly attenuated by CORM-2 and Ifenprodil. A marked increase in hypertrophied microglia expression supported by the mRNA upregulation were observed in arthritic groups and these parameters were significantly inhibited by both treatments. Significant increase in pro-apoptotic markers level (protein kinase B; PKB, caspase-3 and caspase-8) with no significant change observed in anti-apoptotic Bcl-2 was identified in arthritic groups and these proteins level were significantly reversed by CORM-2 and Ifenprodil treatments. Reduced Nissl-positive neuron cells were detected in thalamic VPL of arthritic groups and the cells were significantly increased in CORM-2- and Ifenprodil-treated groups. In conclusion, CORM-2 and Ifenprodil exerted their anti-nociceptive, anti-inflammatory, and anti-apoptotic effects on CFA- induced chronic polyarthritis rats which are comparable to Diclofenac. The antagonists could be explored in future as adjunct therapies aimed at specifically managing chronic pain that persists despite adequate control of inflammation, which is a major unmet need in RA management. This study also provides insights into the role of P2X4R and NMDAR-2B in modulating central nociceptive mechanisms during pathogenesis of RA pain.