The Mechanisms Of SIRT3-regulated Mitochondrial Homeostasis In AGEs-induced Intervertebral Disc Degeneration

Part 1.The effect and mechanisms of targeting mitochondrial oxidative stress on AGEs-induced apoptosis of nucleus pulposus cellsObjective: To explore the mechanisms of mitochondrial pathway in the process of AGEsinduced apoptosis of NP cells,and to clarify the therapeutic effect of targeting mitochondrial oxidative stress.Methods: The level of AGEs in NP tissue was detected by immunohistochemistry,and the correlation between AGEs and the IDD grades was analyzed.The primary NP cells were cultured by AGEs stimulation or Mito TEMPO/Sk Q1 pretreatment in vitro,and the proliferation activity of NP cells was detected by Ed U staining,Annexin V-APC/7-AAD staining and TUNEL staining for apoptosis,Western staining for cleaved-caspase3;Mitochondrial membrane permeability was detected by Calcein-AM staining,JC-1 staining for mitochondrial membrane potential,Western staining for Bcl-2,Bax and cytochrome C,DCFH-DA probe loading for ROS.Results: The levels of AGEs in NP tissues were positively correlated with the IDD grades;The results of in vitro studies showed that AGEs could significantly inhibit the proliferation and induce the apoptosis of NP cells.Mitochondrial membrane permeability was increased by AGEs stimulation,which promotes the release of cytochrome c into the cytoplasm;The pretreatment of mitochondrial-targeted antioxidants Mito TEMPO/Sk Q1 could attenuate AGEs-induced oxidative stress and mitochondrial membrane permeability,following by decreased NP cell apoptosis.Conclusion: Mitochondrial dysfunction is critical for AGEs-induced NP cell apoptosis,targeting mitochondrial oxidative stress could significantly improve the mitochondrial function of NP cells,and then inhibit AGEs-induced NP cell apoptosis.Part 2.The effect and mechanisms of SIRT3 regulating mitochondrial redox homeostasis on AGEs-induced apoptosis of nucleus pulposus cellsObjective: To clarify the effect of AGEs on the mitochondrial antioxidant system of NP cells,and to further explore the effect and mechanism of SIRT3 regulating mitochondrial redox homeostasis on AGEs-induced apoptosis of NP cells.Methods: The levels of mitochondrial oxidoreductase in NP cells were examined using western blotting.The levels of SIRT3 protein in human NP tissues and NP cells were examined using western blotting;the SIRT3 function loss-and gain-function experiments were constructed by SIRT3 interference RNA transfection and overexpression lentivirus infection,and the ROS levels in mitochondria were detected by Mito SOX staining and colocated with Mito Tracker staining;the apoptosis of NP cells was detected by Annexin VAPC/7-AAD staining.Results: The in vitro experiments confirmed that AGEs could significantly reduce the protein levels of mitochondrial oxidoreductase in NP cells;the protein level of SIRT3 in NP tissues was negatively correlated with the IDD grades,and negatively correlated with the level of AGEs deposition;AGEs could significantly reduce the SIRT3 protein level and its deacetylase activity;the loss-and gain-function experiments of SIRT3 confirmed that SIRT3 is critical for maintaining mitochondrial redox homeostasis and alleviating the apoptosis of NP cells.Conclusion: The decline of mitochondrial antioxidant system is important for AGEs-induced oxidative stress in NP cells,which is closely related to the SIRT3 dysfunction.Targeted recovery of SIRT3 protein function can not only effectively restore the mitochondrial redox homeostasis of NP cells,but also significantly alleviate AGEs-induced apoptosis of NP cells.Part 3.The mechanisms of NMN regulating NP cell apoptosis via SIRT3 and mitochondrial pathway and its effect on IDDObjective: To clarify the effect and regulatory mechanism of NMN on SIRT3 function and redox homeostasis of NP cells,and to further clarify its therapeutic effect on IDD.Methods: The levels of AMPK-PGC-1? pathway and mitochondrial oxidoreductase in NP cells were examined using western blotting.NP cells were co-cultured with a-769662,compound C,RAGE neutralizing antibody,NMN and AGEs,or SIRT3 interference RNA was pretransfected,then using western blotting for detecting the levels of AMPK-PGC-1? pathway,mitochondrial oxidoreductase and SIRT3 in NP cells,the ROS levels in NP cells were detected by Mito SOX staining,and Annexin V-APC/7-AAD staining for apoptosis;the degeneration model induced by AGEs deposition was established by the injection of SD rats' tail intervertebral disc,and observed by MRI,hematoxylin eosin and saffron green staining;western blotting and immunofluorescence staining for detecting SIRT3 and cleaved caspase-3 protein levels,TUNEL staining for detecting apoptosis.Results: The in vitro experiments confirmed that AGEs could significantly inhibit the activation of AMPK-PGC-1? pathway in NP cells;NMN could significantly activate the AMPK-PGC-1? pathway in NP cells,increase the protein levels of mitochondrial oxidoreductase,and then restore the redox homeostasis,alleviate the apoptosis of NP cells induced by AGEs.SIRT3 loss-function experiment confirmed the necessity of SIRT3 in this process;NMN injection could significantly alleviate NP cell apoptosis and IDD in AGEsinduced IDD model.Conclusion: NMN could activate the AMPK-PGC-1? pathway,increase the protein levels of SIRT3 and mitochondrial oxidoreductase in NP cells,restore the mitochondrial redox homeostasis of NP cells,and play an important role in alleviating AGEs-induced NP cell apoptosis and IDD progression.