The Anti-effect Of Lysyl Oxidase (LOX) On Nucleus Pulposus Degeneration And Relevant Mechanisms

The degeneration of the nucleus pulposus(NP)is the main factor leading to intervertebral disc degeneration.In the degenerated NP,the extracellular matrix(ECM)synthesis decreases,and NP cells undergo apoptosis and senescence,which ultimately leads to the loss of the structure and function of NP.Lysyl oxidase(LOX)can maintain the stability and strength of ECM by catalyzing the covalent binding of collagen and elastin.Studies have shown that LOX promoted the synthesis of ECM in chondrocytes,which played a therapeutic effect on the degeneration of bone and articular cartilage.However,the effect of LOX on degenerative NP has yet to be verified.Therefore,this study focuses on exploring the effect of LOX on degenerative NP firstly,then investigate the potential regulated mechanism.The main research contents and results are as follows:(1)The anti-apoptotic effect of LOX on NP cells and its mechanism TNF-α-induced cell apoptosis model was constructed in vitro to detect the effects of LOX on TNF-α-induced NP cell viability,apoptosis and extracellular matrix synthesis.Potential molecular mechanism was then explored.Results showed that the expression of LOX in TNF-α-induced NP cells was significantly down-regulated;the addition of exogenous LOX significantly increased the viability and the ECM synthesis of TNF-α-induced NP cells;LOX also inhibited the apoptosis of NP cells.The results of in vivo and in vitro studies showed that LOX significantly down-regulated Fas/Fas L signaling pathway,while LOX also transported into the nucleus of NP cells,and significantly down-regulated the phosphorylation of p53.(2)The anti-senescence effect of LOX on substrate stiffness induced NP cells A mechanical model for inducing NP cell senescence in vitro was established.This study used a stiffness-controllable polyvinyl alcohol(PVA)hydrogel simulating the stiffness of normal(5 k Pa)and severely degenerated(20 k Pa)NP tissue.Primary rat nucleus pulposus cells were cultured on hydrogels.The effect of LOX on NP cell proliferation,SA-β-Gal staining,cell cycle,telomerase activity,NP cell surface markers,Young’s modulus and cytoskeleton arrangement were explored,and the potential related mechanisms were then investigated.Results showed that increased substrate stiffness down-regulated NP cell proliferation,telomerase activity and increased the positive SA-β-Gal staining.Increased substrate stiffness also arrested the cell cycle in the G1 phase of most NP cells.Additionally,the expression of NP cell marker was decreased,Young’s modulus of NP cells was significantly increased,and the morphology of NP cells was long spindle-shaped with a large number of lamellipodia.LOX significantly increased NP cell proliferation,telomerase activity and decreased SA-β-Gal positive NP cells.LOX also maintained the expression of NP cell marker,decreased cellular Young’s modulus.The morphology of NP cells was polygonal with filopodia.The mechanism research results showed that LOX improved the ECM microenvironment by increasing ECM synthesis.Increased ECM induced integrin-β1 significantly up-regulated,and ultimately leading to a significant up-regulation of cyclin-dependent kinases(CDKs)cyclin D1,which faciliated NP cell cycle transition from G1 phase to S phase.(3)The anti-degeneration effect of LOX on NP Based on the results of the above two parts,this part evaluated the effect of LOX on NP degeneration in vivo.Using a poly(lactic-co-glycolic acid)(PLGA)nanoparticles to wrap LOX,then particles were mixed with agarose hydrogel(PLGA-LOX-Agarose,PLA)for making a LOX sustained-releasing system,PLGA nanoparticles without wrapping LOX agarose hydrogel was used as a control group(PLGA-Agarose,PA).The characterization results of the material showed that the size of PLGA nanoparticles was between 100-200 nm,and the encapsulation efficiency and drug loading efficiency of LOX are 82.59% and 1.02%,respectively.The cumulative release curve of PLA in PBS showed that the cumulative release rate reached 87.93% on the 30 th day.NP cells were mixed with hydrogels and cultured for 14 days or 42 days.Compared with the PA group,PLA significantly down-regulated NP cell apoptosis.With long-term culture(42days),expression of NP cell surface molecular markers CD24 and brachyury were stably expressed in PLGA hydrogel.The deposition of proteoglycan and type II collagen was also significantly higher than that in the PA group.A lumbar disc degeneration of rat was established.The PA and PLA hydrogels cultured in vitro for 14 days were then implanted respectively into the degenerated lumbar intervertebral discs.After 4 weeks,histological results showed that compared with the control and PA group,the PLA group could partially restore the structure of NP.PLA group can better maintain the expression of NP cell surface molecular markers CD24 and brachyury compared with the PA group.In addition,PLA can also promote the deposition of NP proteoglycans and type II collagen.In summary,the results of this study show that LOX can inhibit TNF-α-induced NP apoptosis and promote ECM synthesis of NP cells by significantly down-regulating Fas/Fas L and p53 phosphorylation.In addition,LOX plays an anti-senescence effect on substrate stiffness induced NP cells improving ECM synthesis and up-regulating integrin-β1-cyclin D1.In vivo results show that LOX can play an anti-degeneration effect on NP tissue.The results of this article will contribute to further understand the regulatory role of LOX in cellular physiological functions,as well as provide a theoretical and experimental foundation for clinical treatment of intervertebral disc degeneration.