Exosome Derived From Bone Marrow Mesenchymal Stem Cells Protects Human Nucleus Pulposus Cells From Acidic PH-induced Damage

Objective: To explore the protective of exosome on nucleus pulp osus cell in acidic pH.Methods:1.NPC were divided into three groups: Group A,pH 7.1?7.3;Group B,pH 6.5?6.7 and Group C,pH 5.9?6.1.The NPCs were cultured in above-defined acidic medium,simultaneously,three different amounts of Exo were added into the media.Finally,the expression of the caspase-3,aggrecan,collagen II and MMP-13 was analyzed and compared among the different groups.2.Bone mesenchymal stem cell culture medium was replaced with exosome-free medium before the culture reached 80% confluence.After 48 h,the cultured supernatant was collected for exosome isolation.The number,size distribution and morphological of the isolated particles were analyzed using the Nanoparticle Tracking Analyzer PMX110 and transmission electron microscope.Western blot analysis was performed to detect the expression of Exo markers(TSG101,CD63).3.Finally,NPCs were cultured in the above-defined acidic medium supplemented with appropriate concentration of BMSCs-Exo.The expression of caspase-3,MMP-13,aggrecan and collagen II was detected by Western blot and q RT-PCR.Results: 1.The results of western blot analysis showed that the expression of cleaved caspase-3 and MMP-13 in Group B and C was significantly increased compared with group A(p<0.05).However,the expression of collagen II was significantly decreased in Group C compared with B(p<0.05),and the expression of aggrecan was also decreased significantly in Group B and C compared with A(p<0.05);2.The morphology of BMSC-derived Exo appeared as cup-shaped vesicles with a size of about 100 nm,as observed by transmission electron microscopy.Particle size and distribution of the isolated Exo were analyzed by the Nanoparticle Tracking Analyzer and produced a absorption peak in the region of about 125 nm.The expression of the known exosomal markers TSG101 and CD63 was confirmed by Western blot analysis.The above results identified these particles collected from MSC culture medium as BMSC-Exo.3.When BMSC-derived Exo equivalent to 20 ?M of BMSC-Exo protein was added into NPCs cultured at pH 5.9-6.1 medium,the upregulation of cleaved caspase-3 and MMP-13 as well as the downregulation of collagen II and aggrecan induced by acidic pH were significantly reversed,as shown by Western blot analysis.Conclusions:In the pathological acid environment,MSC-derived Exo promotes the expression of chondrocyte extracellular matrix,collagen II and aggrecan,and reduces matrix degradation by downregulating matrix degrading enzymes,protecting NPCs from acidic pH-induced apoptosis.This study reveals a potential promising strategy for treatment of IVD degeneration.