Cytosolic Escape Of Mitochondrial DNA Triggers CGAS-STING-NLRP3 Axis-Dependent Nucleus Pulposus Cell Pyroptosis

Objective: To explore the role of cGAS-STING-NLRP3 axis-dependent nucleus pulposus cell pyroptosis in oxidative stress-induced intervertebral disc degeneration.To explore the molecular regulatory mechanism of mitochondrial DNA cytosolic escape in cGAS-STING-NLRP3 axis activation.To evaluate accurate repair strategies targeted to inhibit cGAS-STING-NLRP3 axis activation to alleviate intervertebral disc degenerationMethods:The molecular map of nucleus pulposus cell dysfunction during intervertebral disc degeneration was analyzed by transcriptome sequencing.The expression levels of cGAS,STING and NLRP3 proteins were evaluated by western blotting,immunofluorescence and immunohistochemical staining.TBHP treatment induced oxidative stress of nucleus pulposus cells.The activation level of cGAS-STING axis was detected by western blotting,immunofluorescence staining,immunoprecipitation and proximity ligation assay.Western blotting was used to evaluate the expression level of pyroptosis-associated molecules.TEM and PI/Hoechst staining were used to observe the morphological changes and the proportion of cell death.Caspase-1 activity detection kit and ELISA were used to detect the enzyme activity of caspase-1 and IL-1β secretion.RT-q PCR,agarose gel electrophoresis and immunofluorescence staining were used to analyze mitochondrial DNA in cytoplasm.Opening of mitochondrial permeability transition pores was detected by using mitochondrial permeability transition pore opening detection kit and mitochondrial membrane potential detection kit.Small molecular inhibitors were used to inhibit opening of mitochondrial permeability transition pores and STING activation,and evaluate their protective effects on needle-puncture-induced intervertebral disc degeneration model.Results:Clinical sample-based transcriptome sequencing analysis showed that oxidative stress,inflammatory response and pyroptosis were significantly enriched in degenerated nucleus pulposus tissues,and cytosolic DNA sensing pathway and NLRP3 inflammasome assembly were also significantly activated.The expression levels of cGAS,STING and NLRP3 proteins were positively correlated with the degeneration of nucleus pulposus tissues.The cGAS-STING axis was drastically activated in oxidative stress-stimulated nucleus pulposus cells.Oxidative stress could also induce NLRP3-dependent nucleus pulposus cell pyroptosis.Inhibiting STING activation and knock downing STING expression could reduce NLRP3-dependent nucleus pulposus cell pyroptosis.Oxidative stress triggered opening of mitochondrial permeability transition pores and cytosolic accumulation of mitochondrial DNA.Inhibiting opening of mitochondrial permeability transition pores improved the mitochondrial homeostasis,reduced cGAS-STING axis activation and alleviated NLRP3 inflammasome-dependent nucleus pulposus cell pyroptosis.In the needle puncture-induced intervertebral disc degeneration model,inhibiting opening of mitochondrial permeability transition pores and STING activation could reduce cGAS-STING-NLRP3 axis activation,and alleviated the inflammatory degeneration of intervertebral disc.Conclusion: Cytosolic escape of mitochondrial DNA triggered oxidative stress-induced cGAS-STING-NLRP3 axis-dependent nucleus pulposus cell pyroptosis.Mitochondrial instability-triggered mitochondrial permeability transition pore opening was an important pathway for cytosolic escape of mitochondrial DNA.Pharmacological inhibitors targeting opening of mitochondrial permeability transition pores and STING activation could delay the process of intervertebral disc degeneration.