A Preliminary Study On The Expression And Role Of JAG1 In Intervertebral Disc Degeneration

Background and objectiveLow back pain is a common chronic injury that affects a wide range of people worldwide,and projections of the global burden of disease statistics between 1990 and 2019 show that low back pain is in the top three in terms of years lived with disability.Low back pain is primarily caused by intervertebral disc degeneration,and degenerative disc changes not only cause low back pain,but are also a major pathological factor in a variety of degenerative spinal diseases.As the aging of the population intensifies and there is a general shift in work and lifestyle,the incidence of low back pain is progressively on the rise,leading to significant medical pressure.Consequently,elucidating the pathogenesis of IDD has become a research focal point,carrying significant scientific value and social significance.The treatment of IDD is mainly conservative and surgical,which can only relieve the patient’s pain and cannot repair the damaged disc,and there is no effective means to prevent the early degeneration of the disc.Biological therapies are currently developing rapidly in the field of IDD diseases,and the success of many experimental animal studies has demonstrated the potential they hold for providing new ideas and approaches to the treatment of degenerative disc diseases.JAG1 is a transmembrane protein that is widely expressed throughout mammalian development and is involved in the pathophysiology of a variety of diseases.JAG1 expression was found to be elevated in osteoarthritis,and in an in vitro experiment,silencing JAG1 promoted LPS-induced proliferation of degenerating chondrocytes and inhibited apoptosis,inflammation and extracellular matrix degradation.As a class of chondrocytes,the biological similarity between articular cartilage and the nucleus pulposus has been widely recognized,and the development of IDD is highly similar to the pathophysiological process of osteoarthritis,and there are few studies related to the role of JAG1 in IDD.In this study,we found that the expression of JAG1 in degenerated IVD tissues was significantly higher than that in normal IVD tissues.Therefore,a preliminary exploration of the role and mechanism of JAG1 in intervertebral disc degeneration was conducted in order to provide relevant experimental basis and theoretical rationale for new bio-targeted therapeutic strategies.Part Ⅰ: Bioinformatics analysis identify JAG1,a key gene for intervertebral disc degenerationMethods:(1)Retrieval and download of gene expression dataset.Data preprocessing and differential gene expression analysis.(2)Based on the results of the differential analysis,the top 5 up-regulated and down-regulated genes were selected,and total RNA was extracted from different degeneration grades of nucleus pulposus tissue for validation of the bioinformatics analysis results.The target genes related to degeneration were screened.(3)Immunohistochemistry staining was conducted on nucleus pulposus tissues of different degenerative grades,and protein was extracted for WB experiment to confirm the expression of the target gene.Results:(1)The dataset included 10 IVD samples,and the samples with different degeneration grades were divided into two groups,with 5 control group samples and 5 IDD samples.By selecting the differential expressed genes based on the criteria of |log2FC|>=1and P<0.05,a total of 335 differential genes were obtained,and relevant genes were further validated.(2)The clinical PCR results showed that JAG1 was highly expressed in degenerated intervertebral discs,which was consistent with the results of bioinformatics analysis.(3)JAG1 expression was significantly increased in the high degeneration grade nucleus pulposus tissue samples.Conclusion: In this study,differential expressed genes in degenerated and nondegenerated human nucleus pulposus tissue were identified and screened through bioinformatics analysis.Based on the significant expression changes,10 genes were selected and validated for their expression in IDD nucleus pulposus and normal nucleus pulposus tissues using PCR and Western Blot experiments.JAG1 was identified as a key gene for further research,and in vitro and in vivo experiments were designed to explore its role in intervertebral disc degeneration.Part Ⅱ: Effects of JAG1 on nucleus pulposus cell degenerationMethods:(1)Normal or low degenerated disc tissues were collected in the clinic,and human primary nucleus pulposus cells were extracted and cultured in vitro to establish an in vitro nucleus pulposus cell degeneration model using IL-1β.(2)JAG1 silencing and overexpression interventions were performed to investigate the effect of JAG1 on NPC degeneration phenotype in vitro,CCK-8 assay to detect the effect of JAG1 on the proliferation activity of myeloid cells,and TUNEL assay to detect the effect of JAG1 on myeloid cell apoptosis.(3)The expression levels of inflammatory factors in different treatment groups were determined by ELISA;RNA and protein were extracted from each group of myeloid cells,and the expression differences of extracellular matrix-related genes were detected by RT-q PCR technique and Western Blot technique.(4)To detect the expression of Notch signaling-related proteins in NPC after overexpression of JAG1.(5)AdJAG1 alone or together with Notch1-specific inhibitors treated NPC to detect changes in NPC proliferation,apoptosis,inflammation and extracellular matrix anabolic and catabolic processes.Results:(1)Human myeloid cells were successfully extracted and cultured and constructed to degenerate NPC in vitro.(2)Silencing JAG1 ameliorated IL-1β-induced decrease in myeloid cell activity and apoptosis.(3)Silencing JAG1 reduced the expression levels of NPC inflammatory factors IL-6,IL-8 and TNF-α,and upregulated the expression of extracellular matrix-associated protein COL2A1 and downregulated the expression of MMP3 and ADAMTS4.(4)The levels of Notch1 and its downstream genes Hes1 and Hey1 were significantly upregulated in NPC overexpressing JAG1.(5)Overexpression of JAG1 enhanced the IL-1β-induced decrease in proliferative activity of NPC,increased apoptosis,increased release of inflammatory cytokines and decreased expression of ECM synthesisrelated proteins and increased expression of catabolic-related proteins,while Notch inhibitor could effectively reverse this process.Conclusion: In vitro experiments,JAG1 significantly affected myeloid degenerationrelated processes.j AG1 may promote IDD by regulating Notch1 signaling that activates IL-1β-induced NPC apoptosis,inflammation and ECM catabolism.Part Ⅲ: Study of JAG1 si RNA for the treatment of disc degenerationMethods:(1)Construction of a mouse tail puncture model to simulate in vivo intervertebral disc degeneration.The mice were divided into a sham operation group(Sham group),a needle puncture + negative control group(IDD+NC si RNA group),and a needle puncture + JAG1 treatment group(IDD+JAG1 si RNA group).(2)MRI was used for imaging analysis.(3)Histological analysis was performed with Haematoxylin-Eosin staining,Fast Green Safranin staining,and other methods.(4)Immunohistochemistry staining was used to detect the expression of COL2A1 and MMP3,the proteins related to nucleus pulposus degeneration.Results:(1)The mouse tail puncture model established could well simulate the pathological changes of intervertebral disc degeneration.(2)The imaging results showed that the treatment group could alleviate intervertebral disc degeneration to a certain extent.(3)HE staining and histological score results showed that JAG1 si RNA treatment could significantly delay intervertebral disc degeneration.(4)Immunohistochemical staining showed that JAG1 si RNA could affect the expression of proteins related to intervertebral disc degeneration,slowing the progression of IDD.Conclusion: The results of in vitro experiments showed that JAG1 si RNA treatment could alleviate the intervertebral disc degeneration caused by tail puncture in mice,indicating the feasibility of JAG1 as a target for disc degeneration treatment.SummaryIn this study,we found and demonstrated the high expression of JAG1 in the degenerating nucleus pulposus,and in vitro experiments demonstrated that the differential expression of JAG1 is closely related to intervertebral disc degeneration,and by inhibiting the expression of JAG1,we increased the in vitro nucleus pulposus cell activity,inhibited the apoptosis of nucleus pulposus cells,reduced the release of inflammatory factors,and also inhibited the catabolism of extracellular matrix thereby effectively delaying the degeneration of the disc,and demonstrated that JAG1 regulates the process of IDD by activating the Notch1 signaling pathway.In vivo experiments have demonstrated that JAG1 si RNA can delay disc degeneration in animals,and JAG1 may be a new effective target for the biological treatment of disc degeneration.