The Roles And Mechanisms Of ASICs In The Progression Of Intervertebral Disc Degeneration

Background Stem cell approaches have become an attractive therapeutic option for intervertebral disc degeneration（IVDD）.Nucleus pulposus mesenchymal stem cells（NP-MSCs）participate in the regeneration and homeostasis of the intervertebral disc（IVD）,but the molecular mechanisms governing these processes remain to be elucidated.Acid-sensing ion channels（ASICs）,a membrane H⁺-gated subgroup of the degenerin/epithelial Na⁺channel（DEG/ENa C）family,have emerged as key receptors for extracellular protons in central and peripheral neurons.Some researches have illustrated the connection between acidic p H and ASIC function during IVD degeneration.However,the functional status of ASICs in NP-MSCs,a critical cell population required for IVD maintenance and regeneration,remains unclear.Objectives To investigate the expression of ASICs in the tissues of patients with intervertebral disc degeneration,to uncover the key subunits that may play important roles in the progression of IVDD;To confirm whether ASICs are involved in regulating the proliferation and senescence of NP-MSCs;To elucidate the role and mechanisms of ASICs in the IVDD process so as to provide potential therapeutic strategies for the treatment of IVDD.Methods Western blot was performed to detect the expression of ASIC isoforms in primary IVD isolates with variant degrees of degeneration and the expression of a series of proteins such as p53,p16,Rb1 and p27 which are involved in cell cycle arrest and senescence programming;NP-MSCs were cultured under p H 6.6 conditions that mimic the degenerated intervertebral disc and CCK-8 experiment was used to detect cell proliferation ability;Flow cytometry was conducted to detect cell cycle changes during the degeneration process;Senescence-associatedβ-galactosidase（SA-β-Gal）and enzyme-linked immunosorbent assay（ELISA）were used to detect the senescence phenotype.Calcium imaging detected the flow of Ca²⁺in acidic environment,and the influence of ASICs inhibitors were evaluated on the above experiments.Results ASIC1 and ASIC3,but not ASIC2 and ASIC4 are upregulated in human IVDs according to the degree of clinical degeneration;Subjecting IVD-derived NP-MSCs to p H 6.6 culture conditions to mimic pathological IVD changes resulted in decreased cell proliferation due to cell cycle arrest and induction of senescence.Key molecules involved in senescence including p53,p21,p27,p16 and Rb1 were all increased.Calcium imaging results show the instantaneous influx of Ca²⁺in acidic environment.ASICs inhibitors can alleviate the senescence phenotype of NP-MSCs under acidic conditions to varying extents.Conclusion ASIC1 and ASIC3 are highly expressed in degenerative tissues and are positively correlated with the degree of degeneration;ASIC1 and ASIC3 may promote NP-MSCs cell senescence and intervertebral disc degeneration by activating p53-p21/p27 and p16-Rb1 signaling pathways.