| Background and ObjectiveLow back pain(LBP),experienced by 80% of people worldwide during their lifetime,is the leading cause of disability,and among these,intervertebral disc degeneration(IVDD)is the most common cause of LBP.Stress is an initiating factor in disc degeneration.Under excessive stress,the extracellular matrix(ECM)of intervertebral disc is metabolically imbalanced,which was shown as decreasing of aggrecan(AGG),collagen type II(Col II)and tissue inhibitors of metalloproteinases,and increasing of matrix metalloproteinases(MMP)-3,accompanied with increased expression of inflammatory factors as well as cyclooxygenase(COX)-2,leading to dehydration and fibrosis of the nucleus pulposus tissue,avulsion of the annulus fibrosus tissue,and calcification of the endplate cartilage.These histological changes in turn cause decreased intradiscal pressure,which in turn leads to altered biomechanical properties of the disc and a decreased ability of bearing stresses,which in turn further aggravates the metabolic imbalance of the disc ECM under excessive stress,forming a "vicious cycle",ultimately causing diseases such as discogenic low back pain and even disc herniation.The specific mechanisms by which nucleus pulposus cells(NPCs)sense external mechanical stimuli and produce biological effects are not well defined.It has been reported that integrin?5?1 expression is significantly increased under excessive stress,and its downstream p38 phosphorylation is increased in response to mechanical stimulus signals in the outside world,and additional reports have shown that excessive stress can induce the degeneration of NPCs by nuclear transcription factor kappa B(NF-?b)-related signaling pathways,so we hypothesized that excessive stress could lead to NPCs degeneration through integrin?5?1/p38/NF-?B signaling pathway.In addition,viscoelasticity is an important biomechanical property of intervertebral disc,which is a creep process in which the deformation of intervertebral disc is nonlinear with time under stress.The water content of intervertebral disc is an important structural basis.Therefore,we believe that the viscoelasticity of intervertebral disc is significantly affected by the degree of degeneration.We first explored the mechanisms by which stress causes nucleus pulposus degeneration by constructing animal models in which excessive stress causes IVDD,then validated it in vitro at the cellular level from human NPCs.The elucidation of the mechanism could help to find new therapeutic targets,thereby delaying the process of disc degeneration under stress.While in this study,creep experiments of intervertebral discs with different degeneration degrees under different mode stresses were conducted to elaborate the effects on the viscoelastic properties of intervertebral disc degeneration after the remodeling of ECM due to degeneration.The research may also contribute to a better understanding of the pathogenesis of discogenic low back pain based on the description of the "vicious cycle" process of IVDD.Part I Investigation of caudal disc degeneration and its mechanism in rats induced by excessive compressive stressMethods:(1)rat Co5?Co7 disc degeneration animal models were constructed and housed for 1 month by means of a caudal vertebral external fixation compression device(Ilizarov-type apparatus)in which the experimental group was given a compressive stress of1.3 MPa and the control group was not pressurized and housed under equivalent conditions for 1 month,and(2)disc height indexes(DHI)and its change(%DHI)before and after compression were determined by means of X-ray,while the signal intensity values of nucleus pulposus on T2 WI were measured from magnetic resonance imaging(MRI),to determine the degree of disc degeneration and the validity of the model,(3)the studied disc tissues were removed,HE staining and Safranin O-fast green staining were performed to observe their histomorphology and confirm their degree of degeneration scores(Yurube method),(4)immunohistochemistry(IHC)staining was performed to evaluate the expression of various markers associated with nucleus pulposus degeneration,including AGG,col II,MMP-3,TIMP-1 and COX-2,while the expression of integrin?5?1 was assessed meanwhile;(5)total RNA and protein were extracted from the nucleus pulposus tissue and subjected to quantitative real-time polymerase chain reaction(q RT-PCR)and Western blot(WB)analysis,to detect nucleus pulposus degeneration indicators and the expression of integrin?5?1,again,as well as the phosphorylation levels of p38 and NF-?B,which are key molecules of the signaling pathway.Results:(1)a stress-induced rat caudal disc degeneration animal model was successfully constructed,and 20 rats from control group and experimental group were enrolled respectively,and(2)X-ray examination showed that the% DHI of Co5/6,Co6/7 in control and experimental rats before and after pressure were(95.66 ± 4.76)% vs(54.76 ±8.92)%,(94.34 ± 4.57)% vs(51.36 ± 9.70)%,and the signal intensity values of the nucleus pulposus section on T2 WI were(1360.8 ± 182.4)vs(470.1 ± 279.8),(1367.0 ± 146.8)vs(527.5 ± 275.9),respectively,(3)HE staining,Safranin O– fast green staining showed that obvious degeneration occurred in the histomorphology of the intervertebral discs in the experimental group,while the Yurube scores of the control and experimental groups were(4.40 ± 0.63)and(11.55 ± 0.55),respectively,(4)IHC staining results indicated that the amount of col II,AGG and TIMP-1 in the nucleus pulposus tissue of the experimental group was significantly reduced,while the amount of COX-2 and MMP-3 was significantly increased,along with the expression of integrin.Conclusions: An animal model of excessive stress-induced disc degeneration was successfully constructed by using a caudal external fixation compression device(Ilizarovtype apparatus),and the integrin?5?1/p38/NF-?B signaling pathway plays a key role in excessive stress-induced nucleus pulposus cell degeneration.Part II Mechanisms of nucleus pulposus cell degeneration regulated by cyclic compressive stressMethods:(1)Intraoperative disc samples from patients with Pfirrmann grade?III were collected,and nucleus pulposus cells were extracted and subcultured,(2)a nucleus pulposus cell/agarose complex was created and cultured for 1 week,(3)the complex was tested for cell viability as well as IHC staining to determine nucleus pulposus cell viability and the expression of col II and AGG among them to confirm the effectiveness of the complex,and(4)the nucleus pulposus cells/agarose complexes were subjected to a cyclic compressive stress of different sizes at a frequency of 1 Hz produced by a stress loading system(flexercell,FX-5000 C,USA)and lasted for 2 h,and the grouping methods were as follows.Group A:control group;group B: moderate pressure group,0.3 MPa;group C: excessive pressure group,1.3 MPa;group D: excessive pressure + GRGDSP,1.3 MPa,the addition of integrin receptor blockers(GRGDSP)to the culture well plates 1 h before pressurization at 50 ?g/m L;Group E: excessive pressure + SB203580,1.3 MPa,the addition of p38 inhibitor(SB203580)to the culture well plates 1 h before pressurization at 5 ?M.(5)q RT-PCR and WB assays were used to determine the indicators of nucleus pulposus cell degeneration,the expression of integrin?5?1,and the phosphorylation levels of p38 and NF-?B in different groups.Results:(1)the nucleus pulposus cells/agarose complex was successfully made,and after 1 week of culture,the cell viability was well and obvious expression of col II and AGG appeared,and(2)the anabolic metabolism of nucleus pulposus cells under moderate pressure was enhanced,as indicated by col II and AGG synthesis increased;(3)however,the nucleus pulposus cells under excessive stress showed attenuated anabolism and increased catabolism,and a marked inflammatory response,as indicated by a significant increase in COX-2 and MMP-3 in group C compared with group B,whereas AGG,col II as well as TIMP-1 were significantly reduced;(4)at the same time,the expression of integrin?5?1 receptors was increased,and the phosphorylation levels of p38 and NF-?B were significantly increased;(5)after the addition of GRGDSP,the degeneration of nucleus pulposus cells in group D was significantly improved compared with group C,and the phosphorylation levels of p38 and NF-?B were significantly decreased;(6)after the addition of sb203580,the degeneration of nucleus pulposus cells in Group E was also significantly improved compared with group C,which also showed the phosphorylation of p38 and NF-?B were significantly decreased.Conclusions: Moderate stress promotes nucleus pulposus cell anabolism,while excessive stress is able to cause nucleus pulposus cell degeneration through the integrin?5?1/p38/NF-?B signaling pathway,and this effect is partially alleviated by integrin receptor blockers or p38 inhibitors.Part III Creep properties of intervertebral discs with different degrees of degeneration under various compressive stressesMethods:(1)fresh adult thoracolumbar specimens were applied to produce functional spinal unit(FSU)containing intervertebral discs and each half of the vertebral bodies above and below,and MRI was performed to determine the Pfirrmann grade of disc degeneration,(2)discs with different degrees of degeneration were subjected to compressive stress with same mode,and the creep curve(time-strain)was depicted,from which,the viscoelastic parameters were calculated using the three parameter constitutive equation as well as the lsqcurvefit function,and(3)discs with similar degrees of degeneration were subjected to cyclic compressive stress of different sizes,and the creep curve(time-strain)and viscoelastic parameters were obtained using the same way.Results:(1)FSUs required for biomechanical experiments were successfully prepared,and whose degeneration degree(Pfirrmann from I to V)was confirmed by MRI,(2)creep strain under the same mode stress significantly increased as disc degeneration progressed and the viscoelastic parameters of the discs calculated by constitutive equations changed significantly,and(3)for discs with similar degrees of degeneration,the strain of the disc increased obviously when the stress was increased,but the calculated viscoelastic parameters did not change significantly.Conclusions: Creep behavior of the intervertebral disc is clearly related to its degree of degeneration,which diminishes as it enlarges,indicating a decrease in its ability to withstand external stress,while the magnitude of stress,although related to the creep strain of the disc,does not affect its viscoelastic properties.SummaryIn this study,an animal model of disc degeneration under excessive stress was constructed by compression fixation of caudal vertebrae in rats,and the mechanism by which excessive stress causes disc degeneration was initially explored,which showed that nucleus pulposus cells under excessive stress had attenuated anabolic metabolism and enhanced catabolic metabolism,and the integrin?5?1/p38/NF-?B signaling pathway plays a key role in the process.Then,in this study,periodic stress loading with different modes was further performed on human nucleus pulposus cells extracted from mild degenerative disc specimens via a cell stress loading system.The results confirmed that excessive stress could lead to increased secretion of MMP-3 and COX-2 and decreased synthesis of col II and AGG via activation of integrin?5?1/p38/NF-?B signaling pathway in nucleus pulposus cells,resulting in attenuated anabolism,enhanced catabolism,and marked inflammatory response in nucleus pulposus cells,which in turn caused disc degeneration.Finally,the creep properties of intervertebral discs with different degrees of degeneration under various stress loading were investigated by biomechanical experiments,and the results showed that degeneration of the intervertebral discs resulted in a significant decrease in their viscoelastic properties and a significant increase in deformation under equal stress,indicating a significant decrease in their ability to bear stress.Our study provides a complete illustration of how excessive stress causes nucleus pulposus cell degeneration and its mechanism,which in turn causes alteration of the viscoelastic properties of the intervertebral disc,leading to its reduced ability to withstand external stress,which in turn further aggravates the process of disc degeneration under stress.This study contributes to a complete understanding of the "vicious cycle" of IVDD and the occurrence process of LBP,which provides insights to find new therapeutic targets,thereby delaying the process of disc degeneration under stress. |
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