Effect And Mechanism Of Exosomes Derived From Bone Marrow Mesenchymal Stem Cells On Degenerative Endplate Chondrocytes

Low back pain(LBP)is one of the most serious medical and social problems in the world,with more than 80 percent of adults experiencing low back pain in their lifetime.Disc herniation or stenosis caused by disc degeneration is an important cause of low back pain.At present,the main methods for treating intervertebral disc degeneration include conservative treatment and surgical intervention.However,conservative treatment and surgical intervention can only alleviate symptoms,but cannot fundamentally slow down or stop the progression of disc degeneration.Therefore,it is urgent to find a new treatment method to restore the function of intervertebral disc and delay the degeneration of intervertebral disc.Mature intervertebral discs are mainly composed of tissues lacking blood vessels,and the nutrient supply of the peripheral capillaries is very limited.Most of the nutrients need to be obtained through the free diffusion of the cartilage endplate.With the aggravation of intervertebral disc degeneration,cracks,inflammation and calcification occur in the cartilage endplate,the number of chondrocytes in the endplate decreases,and the supply of nutrient channels is interrupted,which further aggravates intervertebral disc degeneration and forms a vicious cycle.Modic change is a common form of endplate degeneration in clinical practice.Some studies have shown that inflammatory cytokines such as IL-1β and TNF-α are activated in Modic change.In recent years,Mesenchymal stem cells(MSCs)have attracted much attention in the treatment of intervertebral disc degeneration due to their strong proliferation,differentiation potential and immunomodulatory functions.However,MSCs are prone to aging,tumor formation and difficult to survive in harsh environments,which greatly limit their application.Recent studies have shown that MSCs can produce a variety of functional effects on target cells through paracrine,in which Exosomes play an important role.At present,encouraging achievements have been made in the therapeutic effects of Exosomes derived from mesenchymal stem cells(MSC-exos)on intervertebral discs.Several studies have reported that MSC-EXOS can reduce the damage of nucleus pulposus cells in different ways.However,there are few studies on degenerated endplate chondrocytes by MSCEXOS.Only one study reported Exosomes derived from bone marrow mesenchymal stem cells,BMSC-Exos)effect on degenerative endplate chondrocytes under oxidative stress model induced by tert-butylhydrogen peroxide(TBHP).Endplate degeneration has been shown to be associated with inflammatory factors such as IL-1β.In this study,we used IL-1β to stimulate inflammatory models of human endplate chondrocytes.To preliminarily investigate the protective effect of exosomes derived from human marrow mesenchymal stem cells(hMMSCs)on chondrocytes of the endplate of intervertebral disc in an inflammatory environment simulated by IL-1β and the downstream mechanism of the effect.Methods1.Isolation,identification and amplification of primary hMMSCs: Primary hMMSCs were obtained by using the properties of plastic adhesion of BMSCs.The main surface molecular markers of MSCs were identified by flow cytometry.HMMSCs were cultured and amplified by special serum-free medium.2.Extraction,purification and identification of Exosomes Derived from human marrow mesenchymal stem cells(HMMSCS-Exos): The supernatant of BMSCs from the third to fifth generations was collected,and exosomes were isolated and purified by size exclusion chromatography.The number and particle size of extracellular vesicles were detected by Zeta View,the morphology of exosomes was detected by transmission electron microscopy,and surface molecular markers were identified by Western blot.3.Separation and culture of endplate chondrocytes: The cartilage endplate removed during lumbar open surgery was collected,and the endplate chondrocytes were separated by digestion with trypsin and type II collagenase digestion,and identified by toluidine blue staining and safranin O staining.Endplate chondrocytes were cultured and subcultured in DMEM-F12 complete medium.4.CCK8 assay was used to detect the effect of Exosomes at different concentrations(0ug-50ug/ m L)on proliferation of endplate chondrocytes under the intervention of IL-1β(10ng/ m L),and Exosomes at appropriate concentrations were searched for subsequent intervention experiments.5.According to the intervention measures,chondrocytes of endplate were divided into three groups :(1)control group,(2)IL-1β intervention group,and(3)IL-1β+ exosome intervention group.Apoptotic endplate chondrocytes were detected by Hoechst fluorescence staining and apoptosis rate of endplate chondrocytes was detected by flow cytometry under three interventions.Western blot(WB)was used to detect the changes of apoptosis proteins Bax,Bad and Caspase3.The m RNA expression levels of inflammatory factors Cox2,IL-6 and TNF-α were detected by RT-q PCR.6.Under the premise of exosome intervention,AKT inhibitor MK-2206 was added to explore whether exosome inhibits the apoptosis of endplate chondrocytes in the IL-1βenvironment through AKT-mTOR signaling pathway.According to the intervention measures,chondrocytes of endplate were divided into four groups :(1)control group(2)IL-1β intervention group(3)IL-1β+ exosome intervention group(4)IL-1β+ exosome +mk-2206 intervention group.Western blot was used to detect the changes of AKT-mTOR signaling pathway proteins P-Akt,P-mTOR,Beclin1,LC3 II and LC3 I under the four intervention conditions.Flow cytometry was used to detect the apoptosis rate of endplate chondrocytes under the four different intervention conditions.Results:1.The obtained adherent bone marrow cells were mainly in the shape of a long spindle under the microscope,which could aggregate into clumps or whirlpools.The main molecular markers of MSCs,CD73 and CD90,were identified by flow cytometry,but CD34 was not expressed.2.The average diameter of the extracted vesicles was 123.3nm by nano-particle size tracking(NTA)technique.The characteristic internal depression conformation of exosomes was observed under transmission electron microscope.3.Under the microscope,the adherent cells obtained by trypsin and type II collagenase digestion were mainly triangular,and some were spindle shaped.,which could be dyed blue and purple by toluidine blue.4.CCK8 results showed that the average optical density(OD)value increased with the increase of exosome concentration,and the maximum OD value was obtained when the exosome concentration was 30ug/ m L.5.The number of apoptotic cells and apoptosis rate of endplate chondrocytes in IL-1β group were significantly higher than those in control group.The number of apoptotic cells and apoptosis rate of endplate cartilage in L-1β+ exosome group were lower than that in IL-1β group.Compared with the control group,the expression levels of apoptosis proteins Bax,Bad and Caspase3 and m RNA expression levels of inflammatory factors COX2,IL-6 and TNF-α in IL-1β group were significantly up-regulated.The expression levels of apoptotic proteins Bax,Bad and Caspase3 and the m RNA expression levels of inflammatory factors COX2,IL-6 and TNF-α were significantly down-regulated in L-1β+ exosome group compared with IL-1β group.6.The expression levels of P-Akt and P-MTOR proteins in IL-1β group decreased,while the expression levels of autophagy related protein Beclin1 and LC3II/LC3 I ratio increased.Compared with THE IL-1β group,the expression of P-Akt and P-MTOR in IL-β + exosome group increased,while the expression of Beclin1 and LC3II/LC3 I ratio decreased.Compared with IL-β + exosome + MK-2206 group,the expression levels of P-Akt and P-MTOR decreased,the expression levels of Beclin1 and LC3II/LC3 I increased,and the apoptosis rate of endplate chondrocytes increased.Conclusion:Exosomes derived from bone marrow mesenchymal stem cells promote the proliferation of endplate chondrocytes and protect endplate chondrocytes by alleviating ILIL-1β induced apoptosis and inflammation of endplate chondrocytes.BMSC-Exos can activate the Akt-MTOR pathway to reduce IL-β induced autophagy and apoptosis of endplate chondrocytes.