Acid-sensing ion channel ۱a regulates the survival of nucleus pulposus cells in the acidic environment of degenerated intervertebral discs

Objective(s): Activation of acid-sensing ion channel ۱a (ASIC۱a) is responsible for tissue injury caused by acidosis in nervous systems. But its physiological and pathological roles in nucleus pulposus cells (NPCs) are unclear. The aim of this study is to investigate whether ASIC۱a regulates the survival of NPCs in the acidic environment of degenerated discs. Materials and Methods: NPCs were isolated and cultured followed by immunofluorescent staining and Western-blot analysis for ASIC۱a. Intracellular calcium ([Ca۲+]i) was determined by Ca۲+-imaging using Fura-۲-AM. Cell necrosis, apoptosis, and senescence following acid exposure were determined using lactate dehydrogenase (LDH) release assay, annexin V-fluorescein isothiocyanate/propidium iodide dual-staining and cell cycle analysis, respectively, followed by Western-blot analysis for apoptosis-related proteins (Bax, Bcl-۲, and caspase-۳) and senescence-related proteins (p۵۳, p۲۱, and p۱۶). Effects of treatment with psalmotoxin-۱ (PcTX۱, blocker of ASIC۱a) on [Ca۲+]i and cell survival were investigated. Results:ASIC۱a was detected in healthy NPCs, and its expression was significantly higher in degenerated cells. When NPCs were treated with PcTX۱, acid-induced increases in [Ca۲+]i were significantly inhibited. PcTX۱ treatment also resulted in decreased LDH release, cell apoptosis and cell cycle arrest in acid condition. Acid exposure decreased the expression of Bcl-۲ and increased the expression of Bax, cleaved caspase-۳ and senescence-related proteins (p۵۳, p۲۱, and p۱۶), which was inhibited by PcTX۱. Conclusion: The present findings suggest that further understanding of ASIC۱a functionality may provide not only a novel insight into intervertebral disc biology but also a novel therapeutic target for intervertebral disc degeneration.

Keywords:

Acidic , Acid-sensing ion channel ۱a , Apoptosis , Intervertebral disc , degeneration , Nucleus pulposus cells , Senescence