| Background and objectiveEpidemiological surveys show that more than 80% of people would experience neck and shoulder pain or low back pain in their lives.Neck and back pain has a huge impact on people’s lives and work.It is generally believed that neck and back pain is mainly developed from intervertebral disc degeneration.However,due to incomplete research on the mechanism of intervertebral disc degeneration,clinical treatment has been passive and the applications of non-surgical biotherapy are very slow.Patients,who having to undergo surgical treatment,face the risk of corresponding postoperative complications,especially recurrence and adjacent segment degeneration.Therefore,in-depth study of the mechanism of intervertebral disc degeneration is of great significance for early initiative interventions and reduction of operation rate.Current studies have demonstrated that intervertebral disc degeneration is a multi-factorial process,including genetic susceptibility,infection,biomechanical load abnormalities,aging,nutrient transport disorders in the endplate,and smoking.Studies have shown that degeneration of the nucleus pulposus is the primary cause of intervertebral disc degeneration,and the metabolic imbalance of extracellular matrix,the occurrence and development of inflammation and apoptosis are the three main pathophysiological changes of nucleus pulposus degeneration.Based on previous studies,in vitro experiments have attempted to repair damaged structures and relieve pain through anti-cytokine and inhibition of related signaling pathways.However,due to the complexity of internal environment,routes of administration and lack of specific targets,etc.The clinical applications have been stalled.Therefore,it is very important to find more specific targets and improve the mechanism network.Through protein combined transcriptome analysis of clinical samples with different grades of degeneration,the research group found that the expression of ossification related genes in the degeneration tissues were significantly higher than that in normal tissues,and the difference of ossification related genes FGF23 was particularly significant.Therefore,this project will further explore the role and mechanism of FGF23 in intervertebral disc degeneration,improve the mechanism network of intervertebral disc degeneration,and provide relevant experimental basis and theoretical basis for the creation of new biological therapy strategies.Part I: High-throughput sequencing found that FGF23 was significantly upregulated in degenerative nucleus pulposus cellsMethods:(1)Normal or mildly degenerative nucleus pulposus tissues were collected clinically and primary nucleus pulposus cells were extracted.(2)Primary nucleus pulposus cells were cultured in vitro and could be subcultured when the cells were nearly 80%fused(3)Total protein was extracted and divided into normal control group and degenerative group for proteomic analysis.(4)FGF23 protein expression was detected by immunohistochemistry.(5)Immunofluorescence and Western blot were performed to detect the expression of FGF23 protein in different concentrations of IL-1β.(6)ELISA was used to detect the content of FGF23 protein secreted into DMEM by various nucleus pulposus cells.(7)CCK8 kit was used to explore the experimental concentration of FGF23 neutralizing antibody.Results:(1)The primary nucleus pulposus cells were cultured and subcultured to p2-p4.(2)The proteomics analysis showed that MAPK pathway was significantly activated in the degenerative group,and the expression of pathway related protein FGF23 was the most significant difference.(3)The immunohistochemistry suggested that FGF23 expression was up-regulated during intervertebral disc degeneration.(4)The analysis of immunofluorescence detection and Western blot showed that the level of FGF23 protein in cells increased significantly under the intervention of IL-1β,and reached the peak at the IL-1β concentration of 25 ng/ml.(5)ELISA showed that the level of extracellular FGF23 protein increased to the peak when the IL-1β concentration is 25 ng/ml.(6)CCK8experiment test indicated that FGF23 recombinant protein at a concentration of 100ug/ m L and FGF23 neutralizing antibody at a concentration of 50ug/ m L significantly decreased the activity of nucleus pulposus cells.Conclusion: P2-P4 of nucleus pulposus cells with good morphology were obtained through the culture of human primary nucleus pulposus cells.The results of high-throughput omics analysis had been proved by the experiments at the tissue and cellular-level.The content of secretory protein FGF23 increased significantly in intervertebral disc degeneration,suggesting that FGF23 may play an important role in intervertebral disc degeneration.The appropriate intervention concentration of FGF23 recombinant protein and neutralizing protein was explored.Part II: Effects of FGF23 on nucleus pulposus cells degenerationMethods:(1)The apoptosis rate of nucleus pulposus cells was detected by flow cytometry after different concentrations of FGF23 recombinant protein intervention in nucleus pulposus cells for 24 hours.Total protein of nucleus pulposus cells was extracted and apoptosis-related protein expression was detected by Western Blot.(2)After transfection with interfering plasmid si FGF3 for 48 hours,the apoptosis rate of nucleus pulposus cells was detected by flow cytometry.Total protein of nucleus pulposus cells was extracted and apoptosis-related protein expression in different groups was detected by Western Blot.(3)FGF23 neutralizing antibody stimulated degenerated nucleus pulposus cells,and flow cytometry was used to detect the apoptotic ratio of nucleus pulposus cells.Total protein of nucleus pulposus cells was extracted and apoptosis-related protein expression in different groups was detected by Western Blot.(4)Expression changes of genes and proteins related to extracellular matrix metabolism in each group were detected by real-time RT-PCR and Western Blot after intervention of FGF23 recombinant protein with different concentrations.(5)The expression of genes and proteins related to extracellular matrix metabolism in different groups were detected by real-time RT-PCR and Western Blot at 48 h after transfection with the interfering plasmid si FGF3.(6)FGF23neutralizing antibody stimulated degenerated nucleus pulposus cells,and real-time RT-PCR and Western Blot were used to detect the expression changes of genes and proteins related to extracellular matrix metabolism in different groups.Results:(1)With the increase of FGF23 concentration,the proportion of apoptotic nucleus pulposus cells detected by flow cytometry increased gradually;Western Blot showed that the expression of pro-apoptotic proteins as Cleaved Caspase3 and BAX were significantly increased,while the expression of anti-apoptotic protein Bcl2 was significantly down-regulated.(2)Transfection of si FGF23 significantly alleviated the pro-apoptotic effect of IL-1β on nucleus pulposus cells.Significantly reduced IL-1βsignificantly up-regulated the expression of pro-apoptotic protein Cleaved Caspase3 BAX and down-regulated the expression of anti-apoptotic protein Bcl2.(3)FGF23 neutralizing antibody showed a similar effect to si FGF23,and had an anti-apoptotic effect on degenerated nucleus pulposus cells,with a certain concentration correlation.(4)The expression of genes related to extracellular matrix degradation metabolism was significantly down-regulated only when the high concentration of FGF23 recombinant protein interfered with the degeneration of nucleus pulposus.(5)Both si FGF23 and FGF23 neutralizing antibodies promoted the anabolism of nucleus pulposus extracellular matrix,but had no effect on degradation metabolism.Conclusion: Using FGF23 recombinant protein,si FGF23 and FGF23 neutralizing antibody,it was confirmed that FGF23 promoted apoptosis of nucleus pulposus cells and inhibited the anabolism of nucleus pulposus extracellular matrix,but had no significant effect on degradation metabolism in normal and degenerative nucleus pulposus cells.Part III: The mechanism of FGF23 promoting nucleus pulposus degeneration mediated by stc2-pi3 k /Akt pathwayMethods:(1)The degenerative nucleus pulposus induced by IL-1β was transfected with si FGF23,total RNA and proteins were collected,and downstream targets of FGF23 were screened by transcriptomic sequencing.(2)FGF23 recombinant protein and si FGF23 were used to treat normal and degenerative nucleus pulposus cells respectively,and the expression changes of STC2 and PI3K/Akt pathway related proteins were detected by immunofluorescence,Western Blot and real-time RT-PCR.(3)STC2 overexpression plasmid was constructed and transfected into nucleus pulposus cells for 48 hours,then FGF23 recombinant protein was added.After 24 hours,apoptosis of nucleus pulposus cells was detected by flow cytometry,and expression of apoptosis-related proteins and PI3K/Akt pathway related proteins was detected by Western Blot.(4)STC2 overexpression plasmid was constructed and transfected into nucleus pulposus cells for 48 hours,then FGF23 recombinant protein was added.After 24 hours,Real-time RT-PCR and Western Blot were used to detect genes related to extracellular matrix metabolism.(5)The PI3K/Akt pathway inhibitor,called LY294002,treated the degenerative nucleus pulposus cells transfected with si FGF23,and the apoptosis-related proteins and genes and proteins related to extracellular matrix metabolism were detected by flow cytometry,real-time RT-PCR and Western Blot.Results:(1)Transcriptome sequencing indicated that the STC2 and PI3K/Akt pathways were significantly different in the interference group,comparing with the control group.(2)FGF23 had negative regulation effect both on STC2 and PI3K/Akt pathways,which was consistent with transcriptome sequencing results.(3)FGF23 promotes apoptosis of nucleus pulposus cells through STC2,and STC2 mediates the regulation of FGF23 on PI3K/Akt pathway.(4)STC2 can alleviate the inhibition of FGF23 on anabolism related genes.(5)PI3K/Akt pathway plays an important role in the regulation of FGF23 on nucleus pulposus cell apoptosis and extracellular matrix metabolism.Conclusion: Firstly,transcriptome sequencing was used to screen downstream targets dependent on FGF23 to regulate intervertebral disc degeneration,and thorough verification was carried out.Subsequently,the effects of STC2 and PI3K/Akt pathways on the regulation of FGF23 on nucleus pulposus cells apoptosis and extracellular matrix metabolism were explored through the response experiments.Finally,the internal mechanism of regulation of FGF23 on intervertebral disc degeneration through STC2-PI3K/Akt pathway was clarified.SummaryIn this study,it was found for the first time that ossification related gene FGF23 was expressed and secreted in intervertebral disc nucleus pulposus cells,and the expression level was positively correlated with the degree of intervertebral disc degeneration.Further studies revealed that,mediated by the STC2-PI3K/Akt signaling pathway,FGF23 could induce apoptosis of nucleus pulposus cells,inhibit the synthesis of extracellular matrix in nucleus pulposus,and promote the progression of intervertebral disc degeneration.The study further improved the mechanism network of intervertebral disc degeneration,and provided ideas and theoretical basis for the exploration of related molecular biological therapy targets. |
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