Function And Mechanism Of Nucleus Pulposus Derived Exosomal MiR-196b-5p In Cartilage Endplate Cell Apoptosis

Low back pain is a highly prevalent clinical problem and intervertebral disc degeneration(IVDD)is now accepted as the major pathophysiological mechanism responsible for this condition.After nearly 50 years of in-depth research,IVDD is considered to be a complex and multifactorial integrated disease model.IVDD is a pathological process which consist of inflammatory response,cell loss and degradation of extracellular matrix under the stimulation of major pathogenic factors,such as oxidative stress,DNA damage,pro-inflammatory factors,nutritional deficiency and abnormal mechanical load,but its pathogenesis has not been fully defined.Intervertebral disc is composed of annulus fibrosus,nucleus pulposus and cartilage endplate.Based on anatomy,it is believed that the main function of cartilage endplate is to provide nutrition for nucleus pulposus.However,the communication between nucleus pulposus and cartilaginous endplate has not been studied.As a new carrier of information communication between cells,exosome can be secreted by almost all living cells.Exosomes contain nucleic acids,proteins,lipids and other biological molecules and maintain stable activity,which can be absorbed by recipient cells to play a variety of biological functions.Therefore,in this study,a series of in vivo and in vitro experiments were conducted to explore the role and mechanism of nucleus pulposus derived exosomal miRNA in the apoptosis of cartilage endplate cells,and to provide a new theoretical basis for revealing the mechanism of IVDD.Part 1.Establishment of human nucleus pulposus cell strain,cartilage endplate cell strain and nucleus pulposus degeneration modelObjective: To establish stable nucleus pulposus cell strain,cartilage endplate cell strain and nucleus pulposus degeneration model,and provide high quality cell strain for subsequent experiments.Method:1)Human nucleus pulposus cells and cartilage endplate cells were isolated and cultured.2)Identify nucleus pulposus cells by immunofluorescence and cellular morphology.3)Identify cartilage endplate cells by immunofluorescence and alcian blue staining.4)The model of nucleus pulposus degeneration induced by TNFα was established and identified by immunofluorescence staining and q RT-PCR.5)CCK-8 detected cell activity and cell growth curve.Result:1)The morphology and growth characteristics of nucleus pulposus cells and cartilage endplate cells are similar.Cell are spindle-shaped,completely adherent after 3-5days and reach more than 80% in 10-12 days.In subculture,cell adherent after 24 hours.The logarithmic period of cell growth was 3-7 days,and the cell density reached more than 80% after 10 days.2)Nucleus pulposus and cartilage endplate have strong function of synthesizing extracellular matrix.100ng/ml TNFα had little effect on the activity and growth state of nucleus pulposus,but inhibited the synthesis of extracellular matrix.Conclusion: Human nucleus pulposus cell strain,cartilage endplate cell strain and nucleus pulposus cell degeneration model was successfully established.Part 2.Isolation and identification of nucleus pulposus derived exosomes and screening and analysis of differentially expressed miRNAs.Objective: Nucleus pulposus exosomes were isolated and identified,and differentially expressed miRNAs were screened by sequencing to provide research directions for subsequent experiments.Method:1)Exosomes were isolated by density gradient hyper-centrifugation,and identified by transmission electron microscopy,nanoparticle tracking analysis and Western Blot.2)High throughput Small RNA was used to screen differentially expressed miRNAs in exosomes of normal and degenerated nucleus pulposus.Differentially expressed miRNAs were conducted GO analysis and KEGG analysis by DAVID.Target gene of miR-196b-5p were predicted by public databases miRanda,Pic Tar and Target Scan.Result:1)Nucleus pulposus exosomes were double concave-like disks with an average particle size of about 153.6 nm.The membrane proteins CD9,CD63 and TSG101 were positive,while the nuclear protein Calnexin was negative.2)The differentially expressed miRNAs in the exosomes of degenerated nucleus pulposus included 10 up-regulated and 6 down-regulated miRNAs.The expression level of miR-196b-5p was the largest and the difference was the most significant,and its predicted target gene was HOXC8.3)Bioinformatics analysis suggested that the main biological processes involved in the degenerated nucleus pulposus exosomes were cell differentiation,phosphorylation and apoptosis,and signal transduction might be through the MAPK pathway.Conclusion: It is confirmed that the objects of this study are exosomal miR-196b-5p,target gene HOXC8,cell apoptosis and MAPK pathway.Part 3.Function and mechanism of nucleus pulposus derived exosomal miR-196b-5p in cartilage endplate cell apoptosis.Objective: To explore the function and mechanism of nucleus pulposus derived exosomal miR-196b-5p in cartilage endplate cell apoptosis in vitro.Method:1)Exosomes were labeled with PKH67 and uptake of exosomes by cartilage endplate cells was observed.2)After exosome incubation,proliferation,invasion and apoptosis of cartilage endplate cells were tested.3)The effects extracellular matrix of cartilage endplate under exosomes were detected by immunofluorescence and q RT-PCR.4)The target gene HOXC8 of miR-196b-5p was verified by dual luciferase reporter gene assay.The changes of miR-196b-5p after exosome intake by cartilage endplate cells were verified by q RT-PCR.5)Western Blot was used to detect the changes of target genes and to verify the cell apoptosis induced by p38 MAPK signaling pathway.Result:1)Nucleus pulposus derived exosomes can be ingested by cartilage endplate.2)Degenerated nucleus pulposus exosomes had no significant effect on proliferation of cartilage endplate cells,but could inhibit cell invasion and promote cell apoptosis.3)Degenerated nucleus pulposus derived exosomal miR-196b-5p inhibits HOXC8 expression in cartilaginous endplate cells.4)Inhibition of HOXC8 expression in cartilage endplate can activate the p38 MAPK signaling pathway and promote cell apoptosis.Conclusion: The degenerated nucleus pulposus derived exosomal miR-196b-5p promoted the apoptosis of cartilage endplate by inhibiting the expression of HOXC8 and thereby activating the p38 MAPK signaling pathway in vitro.Part 4.The role of nucleus pulposus derived exosomes in the rat model of intervertebral disc degeneration by acupuncture.Objective: To explore the function and mechanism of nucleus pulposus derived exosomal miR-196b-5p in cartilage endplate cell apoptosis in vivo.Method:1)To establish the acupuncture model of degeneration of caudal vertebral disc in rats.2)Degree of intervertebral disc degeneration and histological changes were determined by X-ray,MRI and biopsies staining.3)The expression of miR-196b-5p,HOXC8,p38 MAPK signaling pathways and apoptosis related factors in cartilage endplate degeneration were tested by immunohistochemistry and q RT-PCR.Result:1)The degeneration of caudal vertebral disc in rats can be induced by acupuncture injury.2)Histological changes of the cartilage endplate degeneration include structural disorder or disappearance,blurred boundaries,reduced functional cells,and immune cell aggregation.3)miR-196b-5p,p38,Bax and Caspase-3 were up-regulated in the degenerative cartilage endplate tissues,while HOXC8 and Bcl-2 were down-regulated.Conclusion: The degenerated nucleus pulposus derived exosomal miR-196b-5p promoted the apoptosis of cartilage endplate by inhibiting the expression of HOXC8 and thereby activating the p38 MAPK signaling pathway in vivo.