| Background and objectiveNowadays,Low back pain?LBP?is one of the most common chronic diseases in clinic,seriously affecting the daily life of patients,causing physiological and psychological damage to patients,and bringing huge economic burden to society and patients.For the specific cause of low back pain,the most important is the intervertebral disc degeneration?IDD?.At present,patients show a trend of youth,and patients will further increase with the advent of an aging society.No effective non-surgical treatment in clinical practice attribute to the mechanism of IDD is far from fully revealed.But the operation still exists some disadvantages.It only carried out the resection of the pathological part without solving the pathogeny of degenerated disc.Furthermore,it increased the risk of degeneration of adjacent segments,because it changed the original anatomy and biomechanics of some segments of the spine.Therefore,it is of great significance to explore the specific mechanism of IDD.The normal intervertebral disc includes nucleus pulposus,annulus fibrosus and cartilage endplate.The peripheral annulus fibrosus tightly surrounds the central nucleus pulposus and attached with cartilage endplate.It's the largest area without vascular and nerve,and the structure of which is similar to a"sandwich".The intervertebral disc suffers from terrible environment consist of low sugar,low oxygen,low p H value,high permeability and load fluctuation.IDD is a complicated progress including aggravated inflammatory reaction,reduced water,decreased height,and moved nucleus pulposus,leads to low back pain by compressing nerve.Because MRI sensitive to the water of tissue,the decreased signal intensity and height of the intervertebral disc can be found in T2weighted MRI.The Pfirrmann classification based on MRI,which used to distinguish the severity of degeneration in clinical.At present,the research shows that the degeneration of NP is the most important in the degeneration mechanism of intervertebral disc.The main pathophysiological changes can be divided into three parts:the inflammation,the metabolic imbalance of extracellular matrix and apoptosis,in which the development of inflammation and the metabolic imbalance of extracellular matrix are dominant.The classical inflammatory factors IL-1?and TNF-?can obviously induce the degeneration of intervertebral disc.However,inflammatory factors take place in many tissues,they are basically lack of tissue specificity,so the therapy which based on inflammation still need to overcome many problems before applied to a clinical setting.Besides,the metabolic imbalance of extracellular matrix mainly includes the decreased synthesis and the increased decomposition end.40 k Da chitinase 3-like protein 1?CHI3L1?was first discovered and described as a highly conserved glycoprotein secreted by synovial cells in 1990.CHI3L1 gene consists of10 exons,which are located on chromosome 1q31-q32.The product,chi3l1/YKL-40,consists of 383 amino acids.With the development of study,researchers found that CHI3L1 can be secreted not only by synovial cells,but also by many other cells,including chondrocytes,osteosarcoma cells,smooth muscle cells,etc.Its function is usually related to inflammatory effect and tissue remodeling.But there is still a great controversy on whether the role of CHI3L1 in inflammatory response is to promote inflammation or not.Previous studies have shown that the expression level of CHI3L1 in NP tissue is significantly increased in disc degeneration,but the mechanism of which has not been studied.In our previous study,we found that the expression of CHI3L1 was significantly increased in the degenerated nucleus pulposus by high through-put label-free proteomics.Therefore,based on the above basis,the purpose of this study is to conduct in-depth study on the role and mechanism of CHI3L1 in disc degeneration,so as to provide theoretical basis for the future mechanism treatment of disc degeneration.Part?Expression level of CHI3L1 in nucleus pulposus cells of intervertebral discMethods:?1?The normal and degenerative disc tissues were collected from human disc to extract nucleus pulposus cells according to Pfirrmann grade.?2?The NP cells were cultured in the DMEM containing 10%FBS and incubated at 37?in 5%CO2.The medium was changed every 3 days.The cell generation produced until 80%of the cells fused.?3?The expression of related genes in the degenerated nucleus pulposus cells was detected by real-time quantitative PCR.?4?Immunohistochemistry was used to analyze the intervertebral discs of mice in different stages and states.?5?The total RNA and protein of nucleus pulposus cells were collected after 72 hours of intervention with IL-1??25ug/ml?and TNF-??50ug/ml?respectively.The levels of CHI3L1 in different groups were detected by real-time quantitative PCR and Western blot.?6?The protein level of CHI3L1in the nucleus pulposus cells treated with different concentrations of inflammatory factors was determined by enzyme-linked immunosorbent assay.Results:?1?Human nuclear cells were successfully extracted and cultured in vitro.?2?In degenerated nucleus pulposus cells,the levels of CHI3L1 and ADAMTS4 were significantly increased,while the levels of ACAN and CHSY1 were significantly decreased.?3?It was found that CHI3L1 was mainly expressed in nucleus pulposus by immunohistochemistry.?4?By real-time quantitative PCR and Western blot,it was found that the expression level of CHI3L1 in the model group was significantly higher than that in the control group.?5?The results of ELISA assay showed dose dependent elevation of secreted CHI3L1 level in NP cell supernatant.Conclusions:We have obtained the ideal nucleus pulposus cells by the experiment.CHI3L1 expression is NP specific in IVD tissue.Furthermore,the elevated secreted CHI3L1 level induced by an inflammation,which indicated its expression is inflammation sensitive in NP cells.Part?The effect of CHI3L1 on IDDMethods:?1?Human primary nucleus pulposus cells were cultured in vitro.?2?Verify the efficiency of the designed CHI3L1 si RNAs.?3?After 48 hours of treatment with plasmid or si RNA,the experimental group:normal nucleus pulposus cells were treated with CHI3L1 overexpression plasmid or si CHI3L1;the control group was untreated normal nucleus pulposus cells.?4?In addition,two kinds of inflammatory factors were used to establish the degeneration model of nucleus pulposus cells in vitro,the experimental group:the nucleus pulposus cells in the degeneration model in vitro were treated with CHI3L1 overexpression plasmid or si CHI3L1 for 48h;the control group was the nucleus pulposus cells in the untreated degeneration model in vitro.?5?The total RNA of each group was collected by Trizol method,and then the RNA expression of different genes was detected by Real-time PCR.?6?The expression of MMP of different group were detected by Transwell analysis.Groups:Normal nucleus pulposus cell,nucleus pulposus cell model of degeneration,nucleus pulposus cell model of degeneration with overexpression CHI3L1 and nucleus pulposus cell model of degeneration with si CHI3L1.Results:?1?Four human nuclear cell lines were successfully cultured.?2?The designed si CHI3L1 significantly inhibit the level of chi3l1 in nucleus pulposus cells.?3?The expression of MMP1,MMP3,MMP13,ADAMTS4 and ADAMTS5 in the overexpression group of CHI3L1 was significantly higher than that in the control group,but the expression in the transfection group of si CHI3L1 was opposite.?4?In the normal nucleus pulposus cell group,CHI3L1 and si CHI3L1 had no significant effect on the regulation of ACAN,CHSY1 and COL2.However,in the degeneration model group of nucleus pulposus cells in vitro,it was found that overexpression of CHI3L1 could significantly promote the expression of these genes,while transfection of si CHI3L1 could significantly inhibit the expression of these genes.?5?Overexpression of CHI3L1 inhibited the secretion of MMP,while si CHI3L1 promoted the secretion of MMP.Conclusions:4 normal human nuclear cell lines were obtained.Through Real-time PCR and Transwell analysis,it was found that CHI3L1 could significantly inhibit the expression of inflammation related genes during the process of degeneration of nucleus pulposus cells in vitro.At the beginning,CHI3L1 had no obvious regulation on the expression of matrix related genes.However,CHI3L1 could promote the expression of matrix related genes with the presence of certain inflammation.Part?The mechanism of CHI3L1 protecting disc degeneration by regulating Akt3 signalingMethods:?1?High through-put RNA sequencing was used to find the downstream target genes of CHI3L1 during the process of disc degeneration.?2?Bioinformatics were used to analyze the mechanism of CHI3L1 protecting the degeneration of nucleus pulposus cells.?3?The potential downstream target genes of IDD analyzed by high through-put RNA sequencing were screened by q PCR.?4?The downstream target gene of CHI3L1 was confirmed by rescue experiment in vitro.?5?By regulating the level of CHI3L1 protein in nucleus pulposus with recombinant protein or polyclonal blocking antibody,the regulation of chi3l1 on downstream target genes and its effect on nucleus pulposus were clarified.Results:?1?It was found that there was a significant difference in transcriptome level gene expression between the two groups by using high through-put RNA sequencing technology.?2?After using bioinformatics to analyze the sequencing results,we found the potential downstream target genes of CHI3L1.?3?It was found that AKT3 might be the downstream target gene of CHI3L1 in disc degeneration by q PCR.?4?The results of rescue experiment indicate that AKT3 is the downstream target gene of CHI3L1 during the process of disc degeneration.?5?After using recombinant protein to regulate CHI3L1,AKT3,p-AKT3 and the downstream targets of AKT3 were activated by recombinant protein regulation,MMPs and ADAMTS4 were inhibited,and expression of ACAN and CHSY1 was promoted.The reverse results were found by polyclonal antibody.The results of Annexin V-FITC/PI double staining flow cytometry showed that the apoptotic cell were significantly increased by polyclonal antibody regulation,and reduced by recombinant protein regulation.Conclusions:CHI3L1 inhibit inflammation and apoptosis,and promote ECM synthesis during the degeneration of IVD.It's confirmed that CHI3L1 can play the above roles by regulating the downstream Akt3 signaling.SummaryIn this study,we first investigated the role of the inflammation related gene CHI3L1in the nucleus pulposus cells.We found the specific expression of CHI3L1 in the nucleus pulposus cells by immunohistochemistry.By regulating the level of CHI3L1,we found that CHI3L1 can inhibit the expression of inflammation related genes and promote the expression of matrix synthesis related genes.AKT3 is the potential downstream target of CHI3L1 in IDD process,which was confirmed by rescue experiment.It is proved that CHI3L1 can inhibit inflammation,promote extracellular matrix synthesis and inhibit apoptosis by directly regulating the downstream target AKT3,so as to protect intervertebral disc degeneration. |
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