| Intervertebral disc degeneration(IVDD)is the primary cause of spinal degenerative diseases such as intervertebral disc herniation,spinal canal stenosis and lumbar spine instability.With aging of our population,the incidence of IVDD-related diseases is increasing,which not only seriously impairs patients’ work and life quality,but also brings heavy economic and spiritual burden to the society and family.Currently,the most effective treatment for disc degeneration is the surgical decompression of neural canal.Various conservative and surgical treatment strategies for IVDD cannot be regarded as a perfect plan to cure IVDD-related diseases.In order to break through the current bottleneck of IVDD treatment,the current therapeutic strategies focusing on early reversal of IVDD based on microenvironment conditioning,genetic intervention,cell transplantation and tissue engineering have gradually become the mainstream research hotspot.In healthy condition,the intact disc is surrounded by the annulus fibrosus and cartilaginous endplate,resulting in a closed intervertebral disc microenvironment of immune isolation.Microdamage of intervertebral disc caused by exercise can break this sealed state,further triggering a cascade of inflammatory responses,resulting in environmental disturbances in the nucleus pulposus.Therefore,the inflammatory microenvironment is an important reason for the imbalance of intervertebral disc matrix anabolism and degradation.In the early pathological stage of IVDD,the imbalanced environment in nucleus pulposus leads to abnormal metabolism of nucleus pulposus cells,which further promotes degradation of proteoglycan and collagen of extracellular matrix(ECM),and dehydration,leading to gradual transformation of nucleus pulposus into fibrous connective tissue.With the progression of studies on the correlation between regeneration and immunity,inducing macrophages to exert immune modulating effects has becoming the core idea in regenerative medicine.As important immune-modulatory cells,macrophages play multiple regulatory roles in immune response.Under the stimulation of different cytokines,macrophages can transform to different phenotype,which is defined as macrophage polarization.Macrophage polarization can be classified into M1 pro-inflammatory type and M2 antiinflammatory type,according to their different immune-modulatory functions.The latter one can be further subdivided into M2 a,which plays immunoregulation,and M2 c,which involves in tissue remodeling.According to current research progress,the infiltration of M1-type macrophages is positively correlated with the progression of intervertebral disc degeneration.However,considering the positive role of M2-polarized macrophages in other tissue repair,whether M2 c macrophages play the same positive role in promoting the regeneration of intervertebral disc degeneration has not been determined.The preliminary study of our study found that the co-culture of nucleus pulposus cells with M2 c macrophages in vitro could promote the expression and secretion of extracellular matrix in nucleus pulposus cells.Based on this basis,the first hypothesis was proposed in our study: M2 c macrophages are assumed to promote intervertebral disc degeneration and repair by regulating the matrix metabolism of nucleus pulposus cells.The inter-cellular exchange of information is an important biological activity to maintain the physical metabolic function of organs and maintain the stability of internal environment.As an important carrier for exchange between cells,exosomes derive from cellular multivesicular body and have double phospholipid membrane with 30-100 nm in diameter.They contain a variety of proteins and non-coding RNAs,such as mi RNA and lnc RNA.When stimulated by different microenvironments,cells can be active to interstitially released exosomes which are subsequently internalized by other recipient cell through endocytosis.A variety of biological molecules contained in exosomes act as mediators to transmit regulatory information to recipient cells,interfere transcription and activate related signaling pathways,so as to affect the proliferation,migration,differentiation,secretion,apoptosis and other biological functions of recipient cells.Therefore,exosomes can mimic the microenvironmental conditioning function of original cells to a certain extent.Many studies have found that exosomes derived from macrophages play complex and diverse roles in pathophysiological processes such as inflammatory regulation,tissue repair and tumorigenesis.Based on the above theories and research,the second hypothesis is proposed in our study: M2 c macrophages can potentially regulate the metabolism and vitality of nucleus pulposus cells by secreting exosomes to deliver non-coding RNA or other active substances into nucleus pulposus cells.In order to prove these two hypotheses mentioned above,we firstly established the coculturing system of M2 c macrophage and nucleus pulposus cells,and observed the alteration of proliferation,migration and matrix protein expression and secretion of nucleus pulposus cells,in order to verifying that M2 c macrophage could regulate the metabolism of nucleus pulposus extracellular matrix and promote the transition of nucleus pulposus cells to pro-regenerating phenotype.Afterwards,we extracted M2 c macrophage exosomes to verify if the exosomes meditate the phenotype influence of M2 c macrophage on nucleus pulposus cells,and determined the optimal concentration of M2 c macrophage exosomes.Then,M2 c macrophage exosomes were loaded with hyaluronic acid hydrogel and implanted into intervertebral disc degeneration model of rat coccyx,to verify the effect of exosomes on improving intervertebral disc degeneration in vivo.Finally,based on proteomic analysis and bioinformatics prediction,the key protein of M2 c macrophage exosomes’ intervention on nucleus pulposus cells were identified,and the core molecular mechanism of "exosomal mi RNA-key protein-downstream pathway" was established and demonstrated.Part I: The effect of M2 c macrophage exosomes on extracellular matrix secretion of nucleus pulposus cellsObjective: After induce and identify M2 c polarization of macrophages in vitro,a coculture system was established to investigate the phenotypic effects of M2 c polarized macrophages on proliferation,migration,synthesis and secretion of matrix proteins and metalloproteinases on rat nucleus pulposus cells.Exosomes derived from M2 c macrophages were extracted and identified.M2 c macrophages regulate the phenotype of nucleus pulposus cells by secreting exosomes was demonstrated,and the optimal concentration of exosomes was determined.Methods: 1,Primary culture and polarization induction of bone marrow derived macrophages: rat bone marrow hematopoietic stem cells were extracted,and rat macrophage colony stimulating factor(M-CSF)was used to induce the differentiation of stem cells into macrophages.Polarization of macrophages towards M2 c was induced by cytokines IL-10 and TGF-β.CD11 b and CD163 were used as bio-markers of M2 c polarization.Immunofluorescence staining and flow cytometry were used to identify M2 c polarized macrophages.2,Primary cultured rat nucleus pulposus cells were extracted from rat coccyx intervertebral disc.3,The co-culture system of M2 c macrophages and nucleus pulposus cells was established by Transwell system.The proliferation of nucleus pulposus cells was detected by Ed U staining,and the migration of nucleus pulposus cells was observed by crystal violet staining.The expression of type II collagen,proteoglycan,matrix metalloproteinase(MMP13),disintegrin and metalloproteinase with thrombospondin 5(ADAMTS5)in nucleus pulposus cells was detected by immunofluorescence staining and Western Blot.4,GW4869 was used to block exosome secretion of M2 c macrophages in advance,and the proliferation,migration,matrix protein and metalloproteinase secretion of nucleus pulposus cells were detected through co-culture.5,Exosomes of M2 c macrophages were extracted by ultra-centrifugal method.Exosomes’ morphology was observed by scanning electron microscopy and NTA particle size analysis,and exosomes markers(TSG101,CD9,CD63)were identified by Western blot.6,nucleus pulposus cells were treated with M2 c macrophage exosomes at different concentrations(0μg/ml,50μg/ml,100μg/ml,150μg/ml),afterwards the proliferation,migration,matrix protein and metalloproteinase secretion of nucleus pulposus cells were tested.Results: 1,flow cytometry and immunofluorescence staining showed that CD11 b and CD163 molecules were highly expressed in macrophages under the stimulation of IL-10 and TGF-β,indicating that the polarization induction of M2 c macrophages was successful.2,after co-culture with M2 c macrophages,Ed U and crystal violet staining showed that the proliferation and migration of nucleus pulposus cells were significantly enhanced,and the expressions of type II collagen and proteoglycan were significantly up-regulated,while the expressions of MMP13 and ADAMTS5 were significantly down-regulated.3,when GW4869-pretreated M2 c macrophages were co-cultured with nucleus pulposus cells,the proliferation and migration of nucleus pulposus cells were still significantly enhanced,while there was no significant difference between GW4869 pretreated M2 c macrophages and control group(without pretreated M2 c macrophages).However,the effects of GW4869 pretreated M2 c macrophages on the expression of ECM proteins(type II collagen and proteoglycan)and metalloproteinases(MMP13 and ADAMTS5)were significantly impaired and reduced compared with M2 c cocultured group without pretreated GW4869.4,Exosomes of M2 c macrophages were extracted by ultra-centrifugation.Scanning electron microscopy showed that exosomes were round and umbrella shape,NTA particle size analysis showed that exosomes diameter was about 30-150 nm,and western blot showed significant expression of exosomes markers(TSG101,CD9,CD63).The results indicated that M2 c macrophage exosomes were successfully extracted.5,M2 c macrophage exosome did not significantly promote the proliferation and migration of nucleus pulposus cells,but it could significantly stimulate the expression of type II collagen and proteoglycan in nucleus pulposus cells,and inhibit the secretion of MMP13 and ADAMTS5,and its effect reached the maximum when the concentration of exosome was 150μg/ ml.Conclusion: M2 c macrophages can significantly promote the proliferation and migration of nucleus pulposus cells,and significantly stimulate the expression of ECM proteins(type II collagen and proteoglycan)and inhibit the expression of ECM metalloproteinases(MMP13 and ADAMTS5).M2 c macrophages macrophage exosomes had no significant effect on proliferation and migration of nucleus pulposus cells,but they still significantly improved matrix metabolism of nucleus pulposus cells,promoted the expression of type II collagen and proteoglycan,and inhibited the secretion of MMP13 and ADAMTS5.The optimal concentration of M2 c exosomes was 150μg/ml.Part II: In vivo study of M2 c macrophage exosomes combined with HA hydrogel to promote the repair of intervertebral disc degeneration in ratsObjective: By using HA hydrogel as a carrier,M2 c macrophage exosomes were locally implanted into the degenerative disc of rat coccyx intervertebral disc to evaluate its proregenerating effect.Methods: 1,M2 c macrophage exosomes(M2c-Exo)were labeled with PKH26 fluorescence and mixed into HA hydrogel,and the release of M2c-Exo from HA hydrogel was observed at different time points.2,The degeneration model of rat coccyx intervertebral disc was established by acupuncture method.The retention time of M2c-Exo in rat coccyx intervertebral disc was analyzed by PKH26-labelling and in vivo imaging.3,After construct the degeneration model of rat intervertebral disc,corresponding intervention were performed on the coccyx disc according to the pre-grouping(Sham operation group,degeneration group,HA hydrogel group,AND M2c-Exo@HA hydrogel group).Small animal MRI,Hematein Eosin(H&E)staining,ferro O solid green staining and immunohistochemical staining(type II collagen and proteoglycan)were performed respectively at 4,8 weeks post operation.MRI index,histological morphology,disc degeneration index,Col II and Aggrecan expression were analyzed.Results: 1.HA hydrogel could be used as a effective carrier for the continuous release of M2c-Exo,and the retention time of M2-Exo in rat coccyx intervertebral disc was significantly prolonged to 21 days.2,the MRI index,histological morphology,disc degeneration index,type II collagen and Aggrecan expression in M2c-Exo@HA hydrogel group were significantly higher than those of the injury control group and the HA hydrogel group at 8 weeks post operation.Conclusion: The HA hydrogel can be used as a carrier to release M2c-Exo and prolong its action in vivo.M2c-Exo@HA hydrogel complex can improve the histological structure and imaging manifestations of degenerative discs to a certain extent,and promote the expression of type II collagen and Aggrecan.Part III: M2 c macrophage exosomes improved the ECM secretion of nucleus pulposus cells through mir-124 /CILP/TGF-β axisObjective: To screen and verify the key molecule of M2c-Exo effects on nucleus pulposus:Cartilage intermediate layer protein(CILP),the downstream pathways of CILP and the its upstream mi RNAs in M2c-Exo were predicted and demonstrated,and the molecular mechanism of M2-Exo to improve the ECM metabolism of nucleus pulposus cells was clarified.Methods: 1,4D label-free proteomics(LFP)technique was used to filter out the differentially expressed proteins in nucleus pulposus with the treatment of M2c-Exo.After analyze their functions and signal pathway enrichment of differentially expressed proteins,the key protein CILP for M2c-Exo effects was screened out,then verified by Western Blot.2,by using Targetscan database,mi RNA in M2c-Exo which targets CILP were predicted.Through reverse transcription-polymerase chain reaction(RT-PCR)were used to demonstrate mi RNAs was delivered into the nucleus pulposus cells by M2c-Exo.Luciferase Assay were used to demonstrate that mi RNA could target the 3’-end nucleic acid sequence of CILP m RNA.3,The rescue experiments were used to prove that M2c-Exo inhibited the expression of CILP in nucleus pulposus cells through mi RNA and promoted the expression of matrix proteins(type II collagen and Aggrecan).4,Western Blot was used to demonstrate that M2c-Exo can activate TGF-β/smad3 pathway and improve ECM metabolism through regulating CILP protein.Results: 1,4D LFP proteomics analysis showed that 74 proteins were up-regulated and24 were down-regulated in M2c-Exo treated nucleus pulposus cells.Gene Ontology enrichment and KEGG analysis showed that differential proteins were associated with inflammatory regulation,oxidative stress,integrin adhesion,matrix metabolism,NF-κB pathway,proteasome pathway and HIF-1α pathway.2,4D LFP proteomics and Western Blot analysis indicated that the expression of CILP protein in nucleus pulposa cells was most significantly down-regulated by M2c-Exo.3,Targetscan database analysis indicated that mi R-124 targeted-regulated CILP;RT-PCR results showed that Mir-124 was delivered into the nucleus pulposus cells by M2 cExo.Quantitative analysis of luciferase reporter gene showed that mi R-124 could bind to the 3’sequence of CILP m RNA.4,Proteoglycan expression of nucleus pulposus cells was downregulated under the action of Mir-124 inhibitor,while ADAMTS5 expression was up-regulated,and this dys-regulation was relieved by overexpressing CILP.5,After treating with M2c-Exo,the phosphorylation of Smad3 in nucleus pulposus cells was enhanced;when the inhibition of CILP expression was relieved by transfection with mi R-124 inhibitor,overexpressed CILP could significantly inhibited the phosphorylation of Smad induced by TGF-β;as a result,the ECM secretion of nucleus pulposus cells was inhibited and the metalloproteinase secretion was enhanced.Therefore,intervention with M2 c macrophage exsome could reverse the inhibition of Smad3 phosphorylation and ECM protein expression induced by mi R-124 inhibitor.Conclusion: CILP is the key molecule mediating the effects of M2 c macrophage exosomes on nucleus pulposus cells.M2c-Exo regulates nucleus pulposus ECM metabolism and promotes intervertebral disc regeneration by delivering mi R-124,inhibiting CILP expression and activating TGF-β pathway in nucleus pulposus cells.SummaryIn this study,we demonstrated the positive regulation of M2 c macrophage-derived exosomes on nucleus pulposus cell matrix metabolism.A hyaluronic acid complex loaded with M2 c macrophage exosomes was constructed using tissue engineering techniques,and it was found that the complex significantly improved the degeneration of rat coccyx intervertebral disc in vivo.Then,through high-throughput proteomics and bioinformatics prediction,we found that cartilage mesenchymal protein CILP was a key molecule for the regulation of M2 c macrophage exosomes on nucleus pulposus cell.Through bioinformatics prediction and functional verification of mi RNA,it was found that mi R-124 in M2 c macrophages could target the CILP m RNA 3’ terminal sequence and induce the degradation of CILP m RNA,thus inhibiting the expression of CILP in nucleus pulposus cell.Finally,in present study,salvage test was conducted to verify that under the stimulation of M2 c macrophages exosomes,TGF-β/SMAD-3 pathway was indirectly activated by the inhibition of CILP protein,thus promoting the synthesis and secretion of type II collagen and proteoglycan in nucleus pulposus cells.Therefore,the mechanism of metabolic modulation in nuclear pulposus and intervertebral disc regeneration based on M2 c macrophage exosome/Mir-124/CILP/TGF-β axis was established in our study,providing a theoretical and practical basis for the application of exosome derived from immunomodulatory cells combined with tissue engineering strategy for modulating the micro-environment of intervertebral disc and pro-regeneration after degeneration. |
PDF Full Text Request