Role Of Carbohydrate Sulfotransferase 3 In Intervertebral Disc Degeneration And Underlying Mechanism

Background and ObjectiveNeck and back pain are common clinical symptoms,which seriously affects patients'daily work.Some patients even lose the ability to take care of themselves due to neck and back pain.There are 800 million people suffering from neck and back pain all over the world.The high cost of treatment has brought heavy burden to the medical system of many countries,and even caused social conflicts.Therefore,neck and back pain are health problems that need to be solved urgently.A number of studies have shown that intervertebral disc degeneration is closely related to neck and back pain.The intervertebral disc is the connective tissue between adjacent vertebral bodies,containing sufficient water and playing essential role in maintaining the stability and mobility of the spine.During intervertebral disc degeneration,the content of water reduced,followed by lose of elasticity of the intervertebral disc,then,the intervertebral disc is prone to herniation or collapse under excessive biological stress,leading to clinical symptoms.Chondroitin sulfate is the most important water-retaining component in the intervertebral disc,which was attributed to the negatively charged sulfate ions arising from chondroitin sulfate sulfation.Thus,the chondroitin sulfate sulfation is important for maintaining water content as well as the normal structure and function of the intervertebral disc.Carbohydrate Sulfotransferase 3(CHST3)acts as the critical enzyme catalyzing chondroitin sulfate sulfation,multi-center clinical studies have shown that single nucleotide polymorphism in the CHST3 gene closely related to intervertebral disc degeneration,but the internal regulatory mechanism remains to be clarified.Thus,this study intends to investigate the interaction and regulation mechanism between CHST3and intervertebral disc degeneration.Part I:Differential expression of CHST3 during intervertebral disc degeneration and the underlying mechanismMethods:(1)Human intervertebral disc tissues with various grades of degeneration were collected,the expression of CHST3 in human tissues was detected through Real-time RT-PCR and Western Blot analysis;(2)Normal and degenerated intervertebral discs of mice were collected,the expression of CHST3 in mice tissues were detected through immunohistochemistry to further clarify the influence of intervertebral disc degeneration on CHST3 expression;(3)Pro-inflammatory factors,IL-1?and TNF?,were used to stimulate nucleus pulposus cells to assemble the inflammation during intervertebral disc degeneration,and induce degeneration of cells,the expression of CHST3 was detected to evaluate the effects of inflammation on CHST3,and Luciferase assay was conducted to explored the underlying mechanism that inflammation regulating CHST3 expression relies on;Results:The expression of CHST3 in human and mouse degenerated intervertebral disc tissues significantly decreased.Furthermore,with pro-inflammatory factors stimulation,both m RNA and protein levels of CHST3 were significantly reduced in nucleus pulposus cells.Luciferase assay showed that pro-inflammatory factors inhibit promoter activity of CHST3;In addition,The NF-?B and JAK/STAT pathways,inflammation related pathway,are significantly activated in the nucleus pulposus cells stimulated with pro-inflammatory factors.The downstream effectors of NF-?B and JAK/STAT pathways,NF-?B1 and STAT3,could bind to the promoter sequence of CHST3 and inhibit promoter activity,which indicates that NF-?B and JAK/STAT pathway directly regulate the expression of CHST3 under inflammatory environment.Conclusion:Inflammation reduces the expression of CHST3 through NF-?B and JAK/STAT pathways during intervertebral disc degenerationPart II:The effect of CHST3 on intervertebral disc degenerationMethods:(1)CHST3 interfering plasmid si CHST3 and overexpressing plasmid oe CHST3 were transfected into normal and degenerated nucleus pulposus cells,which were induced by pro-inflammatory factors,respectively.In order to specify the role of CHST3in nucleus pulposus cells,we detected the apoptotic cells through flow cytometry,in addition,the expression of apoptosis related protein(Cleaved Caspase 3 and BAX)were detected by Western Blot,the expression of extracellular matrix metabolism related genes,including degradation related genes MMP-1,-3,-9,ADAMTS-4,-5 and synthesis related gene Aggrecan,COL2A1,were detected by Real-time RT-PCR.(2)CHST3^-/-mice were constructed using CRISPR-Cas9 gene editing technology,followed by genotype identification;(3)Data about mice body length and weight were collected for analyzing the development of CHST3^-/-mice,the intervertebral discs of mice were assessed by MRI scanning and histopathological staining for degeneration,the expression of extracellular matrix metabolism related genes(Aggrecan,MMP-3 and ADAMTS-4)were detected by immunohistochemistry;(4)The adeno-associated virus carrying oe CHST3 was used to evaluate the potential therapeutic value of CHST3 for intervertebral disc degeneration through intradiscal injection on puncture-induced rat model of intervertebral disc degeneration.Results:With CHST3 interference,we found increased apoptosis and expression of extracellular matrix degradation related genes as well as decreased expression of extracellular matrix synthesis related genes in normal nucleus pulposus.CHST3overexpression showed the opposite results in degenerated nucleus pulposus cells,indicating that CHST3 exerts critical effect on maintaining normal nucleus pulposus cell activity and function,and delaying the degeneration of nucleus pulposus cells;In order to further explore the effect of CHST3 on intervertebral disc degeneration,we designed and synthesized CHST3 sg RNA according to the CHST3 gene sequence.Then,Cas9 RNA and CHST3 sg RNA were transferred into zygotes of wild-type mice by microinjection,the zygotes were cultured in vitro until 2-cell embryos,and transplanted into pseudo-pregnant female mice of the ICR strain to obtain F0 generation mice,and then further expanded reproduction to obtain wild type(WT),CHST3^+/-and CHST3^-/-F2 mice.Western Blot of tissue showed significantly reduced expression of CHST3,which indicates reliable knockout of CHST3 in CHST3^-/-mice.The off-target analysis results showed that none of the first 20 potential off-target sites of CHST3 sg RNA manifesting off-target;The body length data told that CHST3^-/-mice show a significantly shorter than WT and CHST3^+/-mice at 4 weeks after birth.In addition,MRI scanning and histopathological staining showed intervertebral disc degeneration manifestation in4-week-old CHST3^-/-mice,and immunohistochemistry showed reduced expression of extracellular matrix synthesis related gene Aggrecan and increased expression of extracellular matrix degradation related genes MMP-3 and ADAMTS-4 in the intervertebral disc tissues of CHST3^-/-mice,which indicates that CHST3 knockout induce spontaneous intervertebral disc degeneration in mice;what's more,compared with degenerated intervertebral disc induced by puncture,intradiscal injection of adeno-associated virus carrying CHST3 overexpressing plasmid significantly alleviated the signs of degeneration as showed by MRI and real-time RT PCR indicating higher expression of genes related to extracellular matrix synthesis and lower expression of genes related to extracellular matrix degradation,indicating that CHST3 overexpression has potential therapeutic value for intervertebral disc degeneration.Conclusion:CHST3 is a protective gene that maintains normal intervertebral disc structure and function and delays the process of intervertebral disc degeneration,which indicates potential significance as a target for intervertebral disc degeneration biological therapyPart III:CHST3 regulates intervertebral disc degeneration through autophagyMethods:(1)The expression of autophagy markers,LC3 and ATG7,in the intervertebral disc tissues of CHST3^-/-and WT mice were detected by immunohistochemistry;(2)CS with different sulfation patterns(including CS-A,CS-C,CS)were adopted to stimulates nucleus pulposus cells,and the autophagy activity in nucleus pulposus cells under different condition were detected through transmission electron microscope,the expression of LC3 and ATG7 were detected by immunofluorescence and Western Blot;(3)Autophagy inhibitors(chloroquine)were used to treat the degenerated nucleus pulposus cells overexpressing CHST3,and nucleus pulposus cells apoptosis were detected through flow cytometry.The expression of extracellular matrix metabolism related genes in nucleus pulposus cells were detected through Real-time RT-PCR.Results:The expression of LC3 in the intervertebral disc tissues of CHST3^-/-mice was significantly lower than that of WT mice,indicating that CHST3 positively regulates autophagy activity;Furthermore,CS with CHST3 mediated sulfation,CS-C,significantly increases the number of autophagosomes,autolysosomes as well as the expression of LC3 and ATG7,which was not seen in CS-A,CHST11?12?13-mediated sulfation of CS,indicating that CS with different sulfation pattern exert various effect on autophagic activity,and CHST3 may regulate autophagy by affecting the CS sulfation;In addition,autophagy inhibitor significantly weakened the protective effect of CHST3 on nucleus pulposus,as manifested by increased apoptosis,increased expression of extracellular matrix degradation related genes,and decreased expression of extracellular matrix synthesis related genes,indicating that autophagy plays an important role in CHST3 regulating degeneration of nucleus pulposus cells and even intervertebral disc.Conclusion:CHST3 affects the autophagy activity in nucleus pulposus cells by regulating CS sulfation,and ultimately retards the process of intervertebral disc degeneration.SummaryIn this study,we have found reduced expression of CHST3 during intervertebral disc degeneration on human and mice tissues.Then,through cell and molecular experiments,we identified that CHST3 is regulated by inflammation and NF-?B and JAK/STAT pathways in degenerated intervertebral discs.In addition,we have found the essential role of CHST3 in maintaining the activity and function of nucleus pulposus cells based on cell experiments,what's more,by using CHST3^-/-mice and puncture-induced rat models of intervertebral disc degeneration,we further confirmed that CHST3 shows protective effect on intervertebral discs and potential therapeutic value for intervertebral disc degeneration biological treatment;Finally,we found that CHST3 affects the autophagy activity in nucleus pulposus cells by regulating chondroitin sulfate sulfation,and ultimately retards the process of intervertebral disc degeneration,which was regarded as the downstream mechanism of CHST3 regulating intervertebral disc degeneration.Through this study,we preliminarily clarified the interaction and underlying mechanism between CHST3 and intervertebral disc degeneration.