The Deubiquitinase USP11 Ameliorates Intervertebral Disc Degeneration By Regulating Oxidative Stress-induced Ferroptosis Via Deubiquitinating And Stabilizing Sirt3

More and more studies have reported that intervertebral disc degeneration（IVDD）is the main incentive and independent risk factor for low back pain（LBP）.Therefore,it is of great practical significance to study the exact pathogenesis of intervertebral disc degeneration and develop targeted molecular drugs for the intervention and treatment of IVDD.Iron death is a novel form of programmed cell death（PCD）characterized by glutathione（GSH）depletion and the inactivation of core glutathione peroxidase 4（GPX4）regulated by the antioxidant system（the glutathione system）.The close relationship between oxidative stress and iron death has been studied in a variety of diseases,but the interaction between oxidative stress and iron death in IVDD has not been explored.In this study,we first collected human intervertebral disc tissues,successfully extracted HNP cells,and established a mouse model of IVDD to carry out relevant studies.The results proved that Sirt3 expression gradually decreased with the increase of IVDD grade,and Sirt3 expression was statistically negatively correlated with the degree of IVDD.And iron death is involved in the pathological mechanism of intervertebral disc degeneration.Next,we found that after Sirt3 gene knockout in mice(Sirt3^-/-),oxidative stress-mediated iron death was significantly elevated,resulting in IVDD and poor pain-related behavioral scores.So far,this study has proved that oxidative stress-mediated iron death plays a key role in the pathological process of IVDD,and Sirt3 is a key factor to inhibit oxidative stress-mediated iron death,as well as an effective target to alleviate pain response in IVDD,providing theoretical support for further exploration of intervention methods in IVDD.Immunoprecipitation combined mass spectrometry（IP/MS）and co-immunoprecipitation（co-IP）showed that USP11 and Sirt3could bind to each other,and the binding regions were located in the DUSP domain of USP11and the SIR2 domain of Sirt3,respectively.Secondly,we demonstrated that Sirt3 is degraded through the ubiquitin-proteasome system,and USP11 can stabilize Sirt3 expression.It was then verified that USP11 could stabilize Sirt3 expression by deubiquitinating K48 sites.In vitro overexpression of USP11 significantly improves oxidative stress-mediated iron death,thereby alleviating IVDD by increasing Sirt3 content.Finally,by building a USP11 whole-gene knockout mouse(USP11^-/-)mediated IVDD model and achieving disc-specific Sirt3overexpression through AAV-Sirt3,it was demonstrated that USP11 knockout can lead to severe IVDD and pain behavior,while disc-specific overexpression of Sirt3 can reverse the above phenomenon.It was demonstrated that USP11 alleviates disc degeneration and pain behavior by stabilizing Sirt3 expression.In summary,this study emphasizes the importance of USP11 and Sirt3 in the pathological process of IVDD by regulating oxidative stress-mediated iron death.Enhancing the combination of USP11 and Sirt3 or enhancing the expression of SIRT3 to inhibit oxidative stress-mediated iron death is a promising target for intervention and treatment of IVDD.