Exosomes Derived From Cartilage Endplate Stem Cells Inhibit Intervertebral Disc Degeneration

1.1 Background and objective:Intervertebral disc degeneration(IVDD)is an important cause of low back pain,mainly manifested by the up-regulation of pro-inflammatory cytokines and matrix metalloproteinase(MMP)expression,the decrease of functional nucleus pulposus cells(NPCs)and the changes of anatomical structure.The mechanism of the occurrence of IVDD mainly includes cartilage endplate(CEP)providing nutrients to the inner layer of the annulus fibrosus(AF)and NPCs are reduced,and CEP regulates IVD’s anabolic weakening and catabolism enhancement,which in turn leads to NPCs apoptosis and IVDD occurrence.Studying the role of cartilage endplate in regulating intervertebral disc degeneration and related mechanisms provide us with an important direction for treating IVDD.CEP is hyaline cartilage wich is located on the upper and lower sides of the intervertebral disc(IVD).In previous clinical statistical studies and analysis,the results indicated that the recurrence rate of low back pain of the IVDD patients accompanied with cartilage endplateitis was significantly higher than that of IVDD patients accompanied without cartilage endplateitis after lumbar fusion.The results may indicate that CEP may play an important role in regulating IVDD progross,but the specific detailed mechanism is still not very clear.Studies have shown that in human and mouse CEP tissues,there are progenitor cells that can differentiate into bone cells,adipocytes,and chondrocytes.These progenitor cells are defined as cartilage endplate stem cells(CESCs).It plays a very important role in maintaining the structural integrity and normal function of CEPs.Nucleus pulposus(NP)and annulus fibrosus(AF)are the main structural components of IVD.NPCs secrete extracellular matrix components to help maintain the biomechanical function of IVD.The reduction of functional NPCs can lead to IVDD.Therefore,regeneration of NPCs may be a potential strategy for the treatment of IVDD.Although studies have shown that CESCs can promote the regeneration of NPCs and regulate IVD homeostasis by transforming into nucleus pulposus cell-like cells,we do not know whether CESCs exosomes(CESC-Exos)can regulate the migration of CESCs into the nucleus pulposus and transform into NPCs through an autocrine method.Gata binding protein 4(GATA4)is a zinc finger transcription factor that binds to DNA and has been shown to regulate a variety of biological processes,including proliferation,differentiation,and angiogenesis.In the development of bone formation,GATA4 plays an important role in promoting osteogenic differentiation,which is mainly achieved by promoting the expression of key proteins such as runt-related transcription factor 2(Runx2),bone morphogenetic protein(BMP),and transforming growth factor(TGF-β).However,so far,no one has studied whether CESCs-Exos can regulate the expression of GATA4 in CESCs,and evaluated the role of GATA4 in promoting the migration and differentiation of CESCs to NPCs.Exosomes(Exos)are extracellular vesicles with a diameter of 30 to 150 nm.Exosomes exist in various body fluids,including plasma,semen,saliva,urine,amniotic fluid,synovial fluid,and breast milk.Exosomes can transport proteins and small RNAs into the intracellular environment,and play an important role in signal transduction related to proliferation,differentiation,autophagy,and other cell activities.In the pathological changes of hepatic ischemia-reperfusion injury,exosomes can promote the proliferation of normal liver cells and repair the damaged liver;it is worth noting that the exosomes derived from mesenchymal stem cells can be related to autophagy after being injected into IVD.The signal pathway inhibits the apoptosis of NPCs and improves IVDD.Autophagy is a necessary intracellular catabolic process.Under conditions of inflammation,nutrient deficiency and stress,it controls intracellular quality and maintains cell survival by degrading and recovering damaged organelles and toxic proteins.Autophagy can degrade apoptotic proteins to regulate the progression of various diseases,such as cancer,neurodegenerative diseases and osteoarthritis.In addition,exosomes from MSCs can inhibit cell apoptosis by activating the PI3K/AKT/autophagy signaling pathway.Whether CESC-Exos can inhibit NPCs apoptosis and IVDD by activating the autophagy pathway in nucleus pulposus cells has not been reported.In view of this,this article studied the effects of CESCs-Exos derived from cartilage endplate stem cells in promoting the transdifferentiation of CESCs into NPCs and inhibiting the apoptosis of NPCs,and analyzed whether the autocrine exosomes of CESCs can be activated by Hypoxic inducible factor(HIF)-1α/Wnt pathway and increase the expression of GATA4/TGF-β,promote CESC migration and transdifferentiation into NPCs,study the mechanism of CESCs-Exos inhibiting the apoptosis of NPCs,and explore the mechanism of CESCs and their secreted exosomes The role and significance in suppressing IVDD.1.2 Methods:1.2.1 Exosomes derived from CECSs activate Akt/autophagy to inhibit NPCs apoptosis and IVDD progression1.2.1.1 Verify that the CEP IVDD progression1)Collect patient clinical data,analyze the recurrence rate of low back pain in IVDD patients with cartilage endplateitis and IVDD patients without cartilage endplateitis.2)The rat animal model verifies that Tert-butyl hydroperoxide(TBHP)can induce cartilage endplateitis,and it can aggravate the progress of intervertebral disc degeneration.3)Extract and identify CESCs and their secreted exosomes,and verify that TBHP can induce degeneration of CESCs.4)Extract the exosomes produced by normal CESCs and degenerated CESCs,and perform mass spectrometry analysis,KEGG and GO data analysis.1.2.1.2 Cell function experiment and mechanism discussion in vitro1)Extract and identify NPCs cells.2)Immunofluorescence,WB and flow cytometry analysis of the difference in the inhibitory effect of exosomes produced by normal or degenerated CESCs on NPCs apoptosis.3)After NPCs are processed with normal or degenerated exosomes,immunofluorescence,electron microscopy and WB detect and analyze the autophagosome and autophagy protein expression in NPCs.4)After normal or degenerated exosomes are processed with nucleus pulposus cells,WB analyzes the activation of AKT proliferation pathway in NPCs.5)After inhibiting the AKT pathway,WB,immunofluorescence and cytological analysis are used to detect the apoptosis of NPCs.1.2.1.3 In vivo experimental research1)Construct a rat IVDD model and treat it with normal exosomes or AKT pathway inhibitor LY294002.2)Magnetic resonance imaging(MRI)and immunofluorescence staining verify that LY294002 aggravate IVDD,while normal exosomes inhibited IVDD.1.2.2 Autocrine exosomes promote the transdifferentiation of CESCs into NPCs,thereby inhibiting IVDD1.2.2.1 Scratch experiment,Transwell experiment,WB,immunofluorescence and q RT-PCR verify the role of CESCs autocrine exosomes in regulating the invasion,metastasis and differentiation of CESCs1.2.2.2 In vitro cytology experiments,scratch experiment,Transwell experiment,WB,immunofluorescence and q RT-PCR are used to analyze the hypoxia-inducible factor(HIF)-1α/Wnt pathway in promoting the expression of GATA4/TGF-β1,thereby regulating CESCs Transfer and differentiation.1.2.2.3 MRI and immunohistochemistry methods to analyze the effect of CESCs overexpressing GATA4 or not on repairing IVDD.1.3 Results1.3.1 Exosomes derived from cartilage endplate stem cells activate Akt/autophagy to inhibit nucleus pulposus cell apoptosis and IVDD1.3.1.1 Inflammation and degenerative CEP accelerate the progression of IVDDThe clinical data of the patient showed that the recurrence rate of IVDD in patients with cartilage endplateitis was 11.66%,and the recurrence rate of IVDD in patients without cartilage endplateitis was 2.5%.We used TBHP to construct an animal model of chondritis and IVDD.The results show that TBHP induces CEP inflammation and degeneration,and the degenerated cartilage endplate accelerates the progression of IVDD1.3.1.2 After obtained from CEP,CESCs is identified that it has the characteristics of stem cells and the function of secreting exosomes.The results of TEM,NTA and WB showed that rat CESCs have stem cell characteristics and can secrete exosomes.Moreover,TBHP can induce degeneration of CEP.q RT-PCR and WB results showed that with the increase of TBHP concentration and time,the gene expression levels of IL-6,TNF-α,IL-1β and MMP13 increased significantly.1.3.1.3 Compared with exosomes derived from degenerated CESCs(D-Exos),exosomes derived from degenerated CESCs(N-Exos)has the effect of activating autophagy and inhibiting the apoptosis of NPCs more effectively.Mass spectrometry and Heat Map analysis of the obtained N-Exos and D-Exos reveale that the proteins carried by N-Exos and D-Exos are significantly different.KEGG enrichment analysis,GO data analysis,WB,immunofluorescence and flow cytometry are performed on the proteins contained in N-Exos and D-Exos.The results show that compared with D-Exos,N-Exos can more effectively activate self Phagocytosis and inhibition of apoptosis.1.3.1.4 N-Exos execute the function of inhibiting nucleus pulposus(NPCs)apoptosis via activating the PI3K/AKT/autophagy pathway.After NPCs treated with N-Exos,the cell apoptosis rate and apoptotic protein are significantly reduced,and the autophagy protein ratio LC3B/A and p-AKT levels increase.After adding AKT inhibitor LY294002(20μmo L/m L)for3 days,the p-AKT pathway and autophagy pathway are inhibited,and the apoptosis of NPCs increase,indicating that N-Exos inhibite the apoptosis of NPCs by activating the PI3K/AKT/autophagy signaling pathway.1.3.1.5 N-Exos reduces rat intervertebral disc degeneration by activating PI3K/AKT/autophagy pathway.In order to further confirm that N-Exos inhibit the apoptosis of NPCs and improve IVDD by activating the AKT/autophagy pathway,we divided the rats into 5 groups: NC group,puncture groups,puncture + LY294002(20 μmol)group,puncture+N-Exos(40μg)group,puncture+N-Exos(40μg)+LY204002(20μmol)group.The results show that compared with the control group,the puncture aggravate the degeneration of the IVD.PI3K/AKT inhibitor LY294002 aggravate the progression of IVDD.Exosomes reduced the degeneration of the IVD,but LY294002 reduced the inhibitory effect of exosomes-mediated progression on IVDD.WB experiment and immunofluorescence staining results show that exosomes inhibited the expression of cleaved caspase3 and activated the p-AKT/autophagy pathway,but LY294002 attenuate these effects of N-Exos.1.3.2 CESCs promote CESCs transdifferentiation into nucleus pulposus cells and inhibit IVDD via autocrine exosomes1.3.2.1 Exosomes secreted by CESCs promote the transdifferentiation of CESCs into NPCs.CESCs were treated with CESC-Exos(20 or 40μg/m L)at 37°C for 24 hours.The treated CESCs show stronger migration and invasion capabilities.Immunofluorescence showed that the level of Collagen II(NPCs marker protein)in CESCs increased after exosomes process CESCs.Then western blotting is used to detect the levels of Collagen II,SOX9,Collagen I and Acan in CESCs.The results show that the expression of nucleus pulposus cell markers Collagen II and SOX9 increases in the treatment group CESCs.1.3.2.2 CESC-Exos promotes the invasion,migration and differentiation of CESC by activating HIF-1α.The expression of HIF-1α increased in CESCs treated with 0,20,40ug/ml exosomes.Lentivirus is used to overexpress HIF-1α to detect the role of HIF-1α in CESCs.The invasion and migrated ability has improved more obviously in the Lenti-HIF-1α-CESCs group co-treated with CESCs-Exos(40μg/m L).Immunofluorescence stainging analysis indicates that the expression of collagen I is decreased significantly in the Lenti-HIF-1α-CESCs group co-treated with CESCs-Exos(40μg/m L).Therefore,it can be concluded that CESC-Exos promotes CESCs differentiation based on activating the HIF-1αpathway.1.3.2.3.HIF-1α promotes the differentiation of CESCs into NPCs by activating the Wnt/GATA4/TGF-β pathway.KEGG enrichment analysis show that the Wnt signaling pathway is obviously activated in the process of CEP degeneration and differentiation.WB and immunofluorescence analysis find that when different concentrations of Wnt agonist 1(0,10,20 m M)are used to treated with CESCs,the expression levels of β-catenin,TCF-4,SOX9 and GATA4 in CESCs gradually increase as the concentration of Wnt agonist 1 increases.These results indicate that increasing the expression of HIF-1α can enhance the activation of the Wnt signaling pathway,thereby stimulating the expression of GATA4 and promoting the differentiation of CESC.Over-expressing GATA4 lentiviruses are used to infect CESCs.Through q RT-PCR and western blotting,it show that TGF-β1 increases most in Lenti-Gata4-CESCs group,while the expression level of EGF increase,colony stimulating factor-1(CSF-1).The expression level of platelets derived growth factor(PDGF)decreases.In the lenti-Gata4 treatment group,the expression of SOX9,collagen II,and TGF-β1 protein increases,while after co-treatment with the TGF-β inhibitor PFD,the expression of SOX9,collagen II,and TGF-β1 protein decreases,suggesting that the effect of GATA4 in promoting the differentiation of CESSC into NPCs is blocked by TGF-β inhibitors.The results of immunofluorescence analysis also show that TGF-β inhibitors blocked the effect of GATA4 in promoting the differentiation of CESCs into NPCs,while the TGF-β1 agonist SRI-011381 improves the ability of CESCs to invade,migrate and differentiate into NPCs.1.4 Conclusion:1.4.1 Cartilage endplateitis aggravates the recurrence of low back pain after lumbar fusion in IVDD patients1.4.2 There are CESCs in CEP.Compared with exosomes derived from degenerated CESCs,exosomes derived from normal CESCs can more effectively promote autophagy by activating PI3K/AKT signaling pathway and inhibit NPCs cell apoptosis and IVDD progross.These results provide further support for exosomes as a tool to prevent and treat IVDD.1.4.3 The autocrine exosomes can enhance the expression of GATA4 and TGF-β1 after activating the HIF-1α/Wnt pathway in CESCs,and promote the migration of CESCs to IVD and the transformation into NPCs,which provide a new target treatment method for IVDD.In summary,CESCs execute the vital function in inhibiting the progress of IVDD.CESCs derived exosomes can not only activate NPCs autophagy and inhibit NPCs apoptosis,but also CESCs can be regulated via autocrine exosomes.After the expression of GATA4 and TGF-β1 increases in CESCs via activating HIF-1α/Wnt signaling pathway,the migration of CESCs is enhanced and CESCs can also transform into NPCs to aleviate IVDD.