| Background and objectiveLow back pain(LBP)and chronic neck pain,as the common chronic high incidence diseases in clinic at present,has seriously caused serious psychological and physical damage to patients,affecting the daily life of patients,and bringing huge economic burden to society.Intervertebral disc degeneration(IDD)is one of the common causes of low back pain and chronic neck pain.However,in the current clinic,the patients present a significant trend of youth,which may be related to the increase of inappropriate using time of electronic products and the long-term and intensive work of young people.With the intensification of social aging,it can be predicted that the number of IDD patients will further increase in the future.However,the limited understanding of the specific pathogenesis of IDD leading the lack of effective molecular targeted and precise non-surgical treatment plans.Only surgical treatment can solve the symptoms of nerve compression caused by IDD.Surgical treatment cannot fundamentally solve the degenerative progress of the intervertebral disc,and only alleviates the corresponding symptoms of nerve compression.Besides,it changes the original anatomical structure of the corresponding segments of the spine,resulting in changes in the overall biomechanics of the spine,and increases the stress of the adjacent segment of the intervertebral disc.Thus,the incidence of adjacent segment disease(ASD)is significantly improved.Therefore,the exploration of the specific mechanism of IDD is of great significance for the treatment of intervertebral disc degeneration.The normal structure of intervertebral disc includes three parts: nucleus pulposus,annulus fibrosus and cartilage endplate.The central nucleus pulposus is tightly wrapped by the peripheral annulus fibrosus,and the cartilage endplate is attached to the upper and lower parts,forming an anatomical structure similar to "sandwich".Intervertebral disc as the largest non-vascular nerve area of the whole body,all of them exist in the harsh environment of low oxygen,low sugar,low PH value,high load fluctuation and high permeability.In the overall progression of IDD,IVD continuously loses water and the intervertebral height gradually decreases.Magnetic resonance imaging(MRI)examination is sensitive to the imaging of water in the organism,the change of spinal cord signal intensity and the reduction of intervertebral height can be found in the T2-weighted imaging of the patient’s MRI examination,so,the commonly used Pfirrmann grading standard for intervertebral disc degeneration in clinic also depends on the imaging performance of the patient’s MRI examination.Current research indicates that the degenerations of NP is important in the mechanism of intervertebral disc degeneration.The pathophysiology changes that occur in NP can be divided into five phenotypes: oxidative stress,autophagy,apoptosis and proliferation,pyroptosis,and the development of inflammation and metabolism disorders of the extracellular matrix(ECM).Among them,the development and occurrence of inflammation and the disturbance of ECM metabolism occupy the dominant position.Metabolic disturbance of the ECM is mainly manifested as increased degradation metabolism and decreased synthesis metabolism due to inflammation.Studies have shown that IL-1β and TNF-α,as classic inflammatory factors,can significantly induce the occurrence and progression of intervertebral disc degeneration.However,they exist in various tissues and organs of mammals and lack the tissue specificity.Therefore,there are still many problems and challenges in the clinical translation of inflammation-based drug therapy based on this.Currently,IDD research focuses on upstream genes participating in the indirectly regulation of nucleus pulposus ECM metabolism and components through downstream targets.ECM has many important biological functions,which highlighting the importance of studying the mechanism of nucleus pulposus ECM components in the degeneration of nucleus pulposus cells and the balance of ECM metabolism.The Pfirrmann grading standard is used in the clinical diagnosis and treatment of intervertebral disc degenerative diseases.The herniation part of Pfirrmann IV and V grade intervertebral disc always occurred ossification,which seriously compresses the spinal nerve roots.Besides,a large number of studies have shown that intervertebral disc degeneration is closely related to tissue ossification,so ossification related genes also play an important role in intervertebral disc degeneration.The integrin binding sialoprotein(IBSP)gene was first discovered in 1992 and located on chromosome 4q21-q23,and contains six small exons(51-159 bp)and one large exon(～2.6 kb).It is a member of the Small Integrin-Binding Ligand N-linked Glycoproteins(SIBLING)family and primarily expressed in bone tissues.IBSP is a secreted glycoprotein that has 317 amino acids and plays important physiological roles in promoting cell adhesion,HA nucleation,and collagen binding.IBSP in the SIBLING family can specifically bind and activate matrix metalloproteinases(MMP)with angiogenic ability.Elevated MMP levels caused by IBSP in the intervertebral disc may induce inflammation and angiogenesis,implying its potential role in disease progression.The role of IBSP in the progression of IDD has not been described in the current research.Only the literature indicates that IBSP can be expressed in both articular cartilage,annulus fibrosus and nucleus pulposus cells.In the previous study,our group carried out protein and RNA sequencing of normal and degenerated nucleus pulposus tissues of intervertebral disc,and found that the expression of the ossification related gene IBSP in the degenerated group was significantly higher than that in the control group,which indicated that may plays a vital role on IDD.Therefore,based on the above basis,the topic aims of this was conducting in-depth research on the role of ossification related gene IBSP in intervertebral disc degeneration and its specific mechanism,and is intended to provide relevant theoretical basis for the mechanism treatment of intervertebral disc degeneration in the future.Part Ⅰ Expression level of IBSP in nucleus pulposus cells of intervertebral discMethods:(1)Collecting normal and degenerative intervertebral disc tissue according to Pfirrmann grading standards and extracting nucleus pulposus cells.(2)Culturing the nucleus pulposus cells in vitro using DMEM containing 10% fetal bovine serum under a37℃,5% CO2 environment,changing the cell medium every 2-3 days and dividing the cells after 80% fusion.(3)Exploring the key differential genes in the process of nucleus pulposus degeneration of intervertebral disc through integrated transcriptome and proteome analysis.(4)Detecting the expression of relevant target genes in degenerative nucleus pulposus cells using real-time quantitative PCR.(5)Examining the expression of target genes in mice intervertebral disc at different time points and under different treatments through immunohistochemical analysis.(6)Constructing an in vitro degenerative nucleus pulposus cell model by treating the cells with 25ug/ml IL-1β or50ug/ml TNF-α for 72 hours,collecting total protein and RNA from the degenerative model group and the blank control group,and detecting IBSP levels using protein blotting and real-time quantitative PCR.(7)Analyzing the protein level of IBSP in intervertebral disc nucleus pulposus cells treated with different concentrations of inflammatory factors using ELISA.Results:(1)Human nuclear cells were successfully extracted and cultured in vitro.(2)The integrated transcriptome and proteome analysis found that there was significant differential expression of IBSP after nucleus pulposus degeneration.(3)Compared to the control group,the expression levels of IBSP and MMP3 in degenerative nucleus pulposus cells significantly increased,while the matrix synthesis-related genes expression levels significantly decreased.(4)Immunohistochemistry result also showed high expression of IBSP in degenerative nucleus pulposus tissues.(5)Real-time quantitative PCR and Western blot experiments found that the expression levels of IBSP in the degenerative model groups constructed by the intervention of two different classic inflammatory factors after 72 hours were significantly higher than the expression levels of them in the blank control group.(6)The ELISA experiment analysis found that with the increase of inflammation factor concentration,the level of IBSP in the nucleus increased until it reached saturation.Conclusions: Excellent morphology of human-sourced nucleus pulposus cells with a screw shape was obtained through culturing primary cells.Furthermore,the protein or RNA level of IBSP was significantly higher in degenerated nucleus pulposus cells compared to the control group.Part Ⅱ The effect of IBSP on IDDMethods:(1)Cultivate the collected human primary nucleus pulposus cells in vitro until the P2 generation for subsequent experiments.(2)The efficiency of the designed IBSP si RNAs was verified.(3)IBSP overexpression intervention and si RNA si IBSP transfection were performed on normal nucleus pulposus cells,and these were treated for48 hours to form the experimental group,while the control group consisted of untreated normal nucleus pulposus cells.(4)In addition,two classical in vitro intervertebral disc nucleus pulposus degeneration models were established using two different inflammatory factors.Besides,IBSP overexpression intervention and si RNA si IBSP transfection were performed on the nucleus pulposus cells in the degeneration models,and these were treated for 48 hours to form the experimental group,while the control group consisted of untreated nucleus pulposus cells in the in vitro degeneration model.(5)Total RNA was collected from each group of nucleus pulposus cells using the Trizol method,and the c DNA was reverse-transcribed and the expression level of the target gene was detected using real-time PCR assay.(6)The differences in MMP expression among the normal nucleus pulposus cell group,the blank group of the in vitro degeneration model,the IBSP overexpression intervention group of the in vitro degeneration model,and the si IBSP transfection group of the in vitro degeneration model were detected using the Transwell assay.Results:(1)Six lines of nucleus pulposus cells were successfully established in vitro.(2)The si IBSP transfection significantly inhibited the level of IBSP in nucleus pulposus cells.(3)In vitro intervertebral disc nucleus pulposus degeneration models,the levels of MMP1,MMP3,MMP9,ADAMTS4,and ADAMTS5 in the nucleus pulposus expressed by over-expression of IBSP were significantly higher than those in the control group,while the opposite results were observed in the si IBSP transfection group.Nevertheless,in the normal nucleus pulposus cells,si IBSP transfection only significantly decreased the expression of MMP1 and ADAMTS4.(4)In vitro intervertebral disc nucleus pulposus degeneration models,the levels of ACAN,COL2,and CHSY1 in the nucleus pulposus expressed by over-expression of IBSP were significantly lower than those in the control group,while the opposite results were observed in the si IBSP transfection group.However,in the normal nucleus pulposus cells,si IBSP transfection only significantly improved the expression of COL2.(5)Accordding the results of Transwell analysis,over-expression of IBSP significantly promoted the secretion of MMPs by nucleus pulposus cells,while si IBSP significantly suppressed the secretion of MMPs by nucleus pulposus cells.(6)The apoptosis of NP cells was obviously improved by the IBSP over-expression,while significantly decreased by the si IBSP treatment.Conclusions: 6 normal human nucleus pulposus cell lines were obtained.Through real-time PCR and Transwell technology,it was found that regardless of in normal nucleus pulposus cells or in vitro intervertebral disc nucleus pulposus degeneration models,over-expression of IBSP significantly promotes the expression of inflammation-related genes,increases the apoptosis,and suppresses the expression of matrix synthesis-related genes,while si IBSP leads to the opposite results.In normal nucleus pulposus cells,the effect of si IBSP on phenotypes was lower than in degeneration models,which owing to the low secretion of IBSP protein in normal NP cells.Part Ⅲ The mechanism of IBSP promoting disc degeneration by regulating Integrin αvβ3/SRC/AKT3 signalingMethods:(1)Utilizing high-throughput sequencing technology to explore downstream target genes of IBSP in the progression of intervertebral disc degeneration.(2)Determining the mechanism by which IBSP promotes nucleus pulposus cells degeneration through bioinformatics analysis.(3)Verifying the potential downstream target genes of IBSP in IDD through qPCR analysis following bioinformatics analysis of transcriptome sequencing.(4)Further validating the selected downstream target genes of IBSP through in vitro rescue experiments.(5)Regulating the level of extracellular IBSP protein in nucleus pulposus tissue cells through the use of monoclonal antibodies and recombinant protein to clarify the regulation of downstream target genes by IBSP and its effect on nucleus pulposus tissue.(6)Further determining the mechanism of IBSP in intervertebral disc degeneration through animal model studies using needle puncture models with different treatments in vivo.Results:(1)High-throughput transcriptomics sequencing reveals significant differences in gene expression levels of nucleus pulposus cells in degeneration model treated with si IBSP compared to the control group.(2)Bioinformatics analysis of the sequencing results reveals potential downstream target genes.(3)The results of qPCR analysis shows that AKT3 may be a downstream target gene of IBSP in intervertebral disc degeneration.(4)The Rescue experiment,by regulating the levels of downstream key molecules,suggests that Integrin αvβ3/SRC/AKT3 is a downstream target of IBSP in intervertebral disc degeneration.(5)Regulating recombinant protein significantly inhibits AKT3 and p-AKT3 expression,activates inflammatory related MMPs and ADAMTS4 levels,and reduces matrix synthesis related ACAN and CHSY1 expression.The antibody regulation provided the significantly opposite results.In addition,flow cytometry with Annexin V-FITC/PI double staining reveals a significant increase in nucleus pulposus cell apoptosis after recombinant protein regulation,and a significant decrease after antibody regulation.(6)The IHC results confirmed the expression of key molecules in different treatment.Conclusions: IBSP has been found to promote inflammation,inhibit ECM synthesis,and induce apoptosis in IDD progress.The study has also confirmed that these effects of IBSP are mediated through regulation of the Integrin αvβ3/SRC/AKT3 axis as downstream targets.Part Ⅳ Muscone regulates the AKT signaling pathway to alleviate the intervertebral disc degeneration induced by IBSP.Methods:(1)qPCR analysis to screen the traditional Chinese medicines that could alleviate the intervertebral disc degeneration induced by IBSP.(2)Further explore the role of selected traditional Chinese medicine on the degeneration of nucleus pulposus cells induced by IBSP.(3)High-throughput sequencing and bioinformatics analysis were used to explore the potential downstream targets of the selected traditional Chinese medicine in IDD and verify them using qPCR.(4)In vitro rescue experiments were performed to further validation of the downstream target genes selected from the bioinformatics analysis.Results:(1)The qPCR experiments revealed that muscone can significantly alleviate intervertebral disc degeneration induced by IBSP.(2)The experiments of phenotypes further proved that muscone can regulates various phenotypes of degenerative nucleus pulposus cells induced by IBSP.(3)Bioinformatics analysis results suggested that signaling pathway may be the downstream target of muscone.(4)The Rescue experiment demonstrates that muscone participates in the IDD progression by regulating the AKT signaling pathway.Conclusions: In IBSP-induced intervertebral disc degeneration,muscone indeed relieves the intervertebral disc degeneration by regulating the AKT signaling pathway,thus playing a protective role on the intervertebral disc.SummaryIn this study,we first investigated the role of ossification-related gene IBSP in nucleus pulposus cells.Molecular experiments have shown that the expression level of IBSP is increased in degenerative nucleus pulposus cells.By regulating the level of IBSP,it was found that IBSP promotes the expression of inflammation related genes and inhibits the expression of matrix synthesis related genes in nucleus pulposus cells.By the transcriptome sequencing and bioinformatics analysis of multiple groups of degenerated nucleus pulposus cells with different levels of IBSP,the study identified for the first time that the Integrin αvβ3/SRC/AKT3 axis is a potential downstream target of IBSP in the progression of intervertebral disc degeneration(IDD).This was confirmed by Rescue experiments.In the study of using traditional Chinese medicine to relieve IBSP-induced IDD,it was found for the first time that muscone can effectively relieve IBSP-induced IDD through the AKT signaling pathway.This study revealed that in intervertebral disc degeneration,IBSP promotes inflammation,inhibits extracellular matrix synthesis of NP cell,and improves NP cell apoptosis through its downstream Integrin αvβ3/SRC/AKT3 axis,thereby promoting intervertebral disk degeneration,which can be effectively alleviated by muscone through the AKT signaling pathway. |
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