The Protective Effect Of Quercetin On Intervertebral Disc Degeneration Based On Oxidative Stress

Objective :(1)To establish a reliable and robust cell senescence model of nucleus pulposus cells(NPCs)using D-galactose(D-Gal),and the mechanism of oxidative stress induced by D-Gal was investigated.(2)To observe the effects of quercetin(QUE)on D-Gal induced NPCs senescence,senescence related secretion phenotype(SASP)and extracellular matrix(ECM).To investigate the molecular mechanism of QUE regulating autophagy to reduce D-Gal-induced degeneration of NPCs through p38 MAPK signaling pathway.(3)A model of intervertebral disc degeneration induced by needle puncture was established to explore the protective effect of QUE on intervertebral disc degeneration.Methods:(1)CCK-8 assay and cell cycle were performed to evaluate the effect of D-Gal on proliferation activity of NPCs.The effects of D-Gal on senescence and SASP of NPCs were detected by senescence related markers.The ECM of senescent NPCs were detected by RT-q PCR,immunofluorescence and Safranin-O staining.The oxidative stress mechanism of NPCs induced by D-Gal was determined by detecting ROS,SOD and MDA.(2)CCK-8 assay and cell cycle were performed to investigate the effect of QUE on the viability of NPCs and the cells were inculcated with D-Gal.Effects of QUE on senescence and SASP induced by D-Gal in NPCs were evaluated by SA-b-Gal staing,SA-b-Gal activity and RT-q PCR.The Transewell and co-culture system were performed to evaluate the effects of conditioned media on the migration and polarization of monocytes.Effect of QUE on MAPK signaling pathway was evaluated by Western blot.(3)The activation effect of QUE on autophagy of NPCs was detected by Western blot,immunofluorescence and electron microscopy.Pretreatment with autophagy inhibitor 3-MA to explore whether QUE could protect the NPCs by improving autophagy.p38 MAPK was inhibited by SB203580 to explore QUE's effect on p38 MAPK-m TOR signal pathways in D-Gal-induced NPCs.(4)The protective effect of QUE on disc degeneration induced by needle puncture was evaluated by X-ray and MRI.The pathological changes of the intervertebral disc were evaluated by HE staining.The content and distribution of extracecellar matrix were stained by Alcian blue and Safranin O stained.The apotosis,senescence and autophagy related protein were evaluated by Immunohistochemistry.Results:The results of CCK-8 and cell cycle showed that the proliferation activity of NPCs was decreaed in 50 g/L D-Gal group.SA-?-galactosidase activity and senescence related proteins p53,p21 and p16 were significantly increased with the increase of D-Gal concentration.RT-q PCR results suggested that the SASP was increased in the senescent NPCs,especially CCL2 and MMP13.The results of RT-q PCR,immunofluorescence and Safranin O stained showed that the collagen II and aggrecan of senescent NPCs were significantly decreased.We further demonstrated that NPCs senescence was caused by D-Gal-induced oxidative stress,with increases in reactive oxygen species(ROS),the protein level of advanced glycation end products(AGEs)and the lipid peroxidation product,malondialdehyde(MDA),and decreased superoxide dismutase(SOD)activity.(2)CCK-8 results showed that 25 u M QUE had no toxicity of NPCs,and reversed the inhibitory effect of D-Gal on the activity of NPCs,Cell cycle results showed that QUE inhibited G1 cycle arrest induced by D-Gal.Cell senescence and SASP were significantly decreased after incubation with QUE compared with D-Gal group.The results of co-culture system were showed that QUE inhibited the migration and of mononuclear macrophages and reduced the M1 type macrophages.Western blot results showed that QUE could protective NPCs from oxidative stress injury by regulating JNK,ERK and p38 signaling pathways.(3)Western blot and immunofluorescence results showed that QUE could activate autophagy and autophagy flow.The expression of Beclin-1 and LC3 II/I was increased,while the expression of p62 was decreased.Pretreatment with 3-MA significantly reduced the effect of QUE on autophagy activation of NPCs.Inhibition of autophagy decreased the protective effect of QUE on ECM of NPCs and promoted the expression of senescence related proteins and SASP.QUE decreased the phosphorylation of p38 MAPK and m TOR induced by D-Gal.Inhibition of p38 MAPK phosphorylation could activate autophagy and promote autophagy flow in NPCs.(4)The animal model of disc degeneration was successfully established by needle puncture,and the height index of the disc was significantly reduced and Pfirrmann grade was significantly increased.QUE delayed the degeneration of the disc induced by needle puncture,increased the disc height index and decreased Pfirrmann classification.The results of HE staining showed that the area of NP and number of NPCs were decreased,the structure of NP and AF wasdisordered,but QUE delayed the progress of intervertebral disc degeneration and improved the modified score.The results of Alcian blue,Safranin O and ACAN immunohistochemical staining indicated that a large amount of proteoglycan was lost and abnormal distribution in the damaged NP in the puncture model group,while the loss and abnormal distribution of proteoglycan were reduced in the QUE group.Immunohistochemical staining indicated that the expression of apoptosis related protein Bax was more obvious in the puncture model group,while the expression of autophagy related protein Beclin-1 was decreased compared with the QUE treatment group.Conclusion :(1)50 g/L D-Gal inhibited NPCs viability,blocked cell cycle,increased senescence related protein and SASP,and promoted ECM catabolism.(2)QUE improves D-galactose-induced senescence and SASP of NPCs by regulating MAPK signaling pathway.(3)QUE alleviates oxidative stress-induced NPCs degeneration through p38 MAPK mediated autophagy.(4)QUE alleviates the intervertebral disc degeneration induced by needle puncture from imaging and histopathology.All the data indicates that QUE is a potential therapeutic strategy in the prevent and treatment of IVDD.