Study On The Role And Mechanism Of Platelet-rich Plasma-activated Exosomes In Regulating Macrophages Inflammasome And Nucleus Pulposus Cells Apoptosis In Intervertebral Disc Degeneration

Background and objectiveIntervertebral disc degeneration(IDD)is a common multi-factor disease in orthopedics,which can cause a variety of spinal related diseases,among which the lumbar and leg pain caused by IDD affects more than 40%of the world population.Up to 15%of patients require hospitalization to relieve their clinical symptoms.At present,the effective treatment for this disease is still dependent on surgical discectomy,but surgical resection will bring serious side effects and intractable complications.Therefore,seeking new treatment methods for intervertebral disc degeneration has become a new research direction to solve this social phenomenon.Recent studies have shown that platelet-rich plasma(PRP)or Exosomes(Exosomes)have been widely used in clinical studies of orthopedic diseases,but the role and mechanism of PRP-derived exosomes in the treatment of IDD remains unclear.The purpose of this study was to investigate the effects of PRPderived exosomes on intervertebral disc degeneration and its mechanism through cell and animal experiments.Methods1.Effect of PRP-derived exosomes on macrophages differentiation① Isolated and cultured PRP-derived exosomes from SD rats and macrophages from SD rats;② Macrophages were stimulated by co-culture of lipopolysaccharide(LPS)with 0,5,40μg/ml platelet-rich plasma or PRP-derived exosomes 0,5,40μg/ml.Cytokine changes were detected by qRT-PCR and expression of pathway proteins was detected by western blot.2.Regulation of PRP-derived exosomes on NLRP3 inflammasomeMacrophages were co-stimulated by LPS,platelet-rich plasma,and PRP-derived exosomes for 24 h and cultured in the presence of ①with different activators of NLRP3 inflammasome(ATP,Nigericin,MSU);②MG-132(10μM)or 3-MA(5 mM);③3-MA(5 mM)、BafA1(100 nM)or CQ(10μM)BafA1(100 nM)or CQ(10 μM).3.Regulation of PRP-derived exosomes on nucleus pulposus cells and its effect on IDD in vivo① The effects of different concentrations of platelet-rich plasma and PRPderived exosomes on nucleus pulposus were detected by CCK8 assay and protein assay under IL-1β stimulation.②MRI and HE staining were used to observe the improvement of disc degeneration in rat mode of IDD after treatment with PRPderived exosomes or platelet-rich plasma.③ Intervertebral disc recovery after treatment with platelet-rich plasma or PRP-derived exosomes was verified in a rat model.ResultsIn this study,①we found that PRP-derived exosomes regulate NF-κB and MAPK pathways to inhibit the polarization of macrophage M1,and regulate STAT6 phosphorylation to affect the polarization of macrophage M2.② In terms of mechanism,PRP-derived exosomes promote NLRP3 autophagy degradation by increasing NLRP3 ubiquitination,thereby reducing IL-1β and caspase-1 production.③PRP-derived exosomes also inhibited IL-1β-mediated apoptosis of nucleus pulposus cells.Finally,in vivo results also confirmed that PRP-derived exosomes can reduce the expression of inflammatory factors,thereby moderating the progression of IDD.ConclusionIn conclusion,we demonstrate that PRP-derived exosomes alleviate IDD inflammation by regulating ubiquitination and autophagy degradation of NLRP3 inflammasome,which provides a new idea for treating IDD with PRP-derived exosomes.