| Background and ObjectiveIntervertebral disc degeneration(IDD)is the main cause of cervical spondylosis,lumbar disc herniation and other spinal degenerative diseases.For spinal degenerative diseases,conservative and surgical treatment are mainly used.Although conservative treatment can alleviate the symptoms of early IDD to a certain extent,it can not reverse the biological function of the degenerative disc.In the late treatment of IDD,there are many problems,such as the inability to restore the normal disc height and load-bearing capacity,the deterioration of existing disc degeneration,and even the acceleration of adjacent vertebral degeneration.Therefore,in the early stage of degeneration,it is of great significance to explore active and effective ways to prevent and control IDD.The disc is composed of three parts: the endplate of the upper and lower cartilages,the nucleus pulposus in the central part and the fibrous ring in the periphery.The nucleus pulposus mainly bears the pressure of the spine,while the fibrous ring mainly bears the axial load of the spine and various stresses of the body movement.IDD first occurs in the nucleus pulposus.The decrease of the number and function of nucleus pulposus cells is the early pathological basis of degeneration.At present,a series of research achievements have been made on the degeneration mechanism and repair regeneration of nucleus pulposus,but it is still difficult to effectively apply to improve IDD.The main reason is that there is a lack of another component of the disc,the repair after the degeneration/rupture of the fibrous ring.Maintaining the integrity of the structure and function of the fibrous ring is very important for maintaining the shape and position of the nucleus pulposus and the physiological pressure in the disc under load.It can be seen that in the early stage of IDD,it can effectively promote the regeneration and repair of the annulus fibrosus,and then strengthen the annulus fibrosus to prevent the degeneration of the nucleus pulposus from further protruding,which is expected to provide an efficient prevention and treatment means for IDD.IDD is the result of many factors,such as biomechanics,heredity,nutritional deprivation,inflammation and so on.Autophagy,as an important programmed cell death mode,plays an important role in regulating cell proliferation,differentiation,aging and death.It has been proved to be closely related to a variety of degenerative diseases.Autophagy has also been reported to exist in IDD,which may play a "double-edged sword" role in the process of degeneration,and it is gradually becoming a research hotspot in the field of IDD.Material and Method(1)Construction of annulus fibrocyte culture system: isolation and culture of rat annulus fibrocytes,and construction of a controllable pressure hypoxia cell culture system.(2)The role of Wnt/?-catenin signaling pathway in regulating the autophagy of annulus fibrosus cells: after inhibiting or over expressing the key molecules of Wnt/?-catenin signaling pathway,the autophagy level of annulus fibrosus cells and the related indexes of chondrogenesis and osteogenesis were detected.(3)The role of autophagy in regulating Wnt/?-catenin signaling pathway: after inhibiting or over expressing the key molecules of autophagy pathway,detect the expression of the key molecules of Wnt/?-catenin signaling pathway in annulus fibrosus cells,and detect the related indexes of chondrogenesis and osteogenesis.(4)Construct the model of tail vertebra controlled compression in rats.(5)Detection of Wnt/?-catenin signaling pathway and autophagy pathway: under different pressure intensity,the expression of Wnt/?-catenin signaling pathway and autophagy pathway key molecules were detected from gene and protein levels.(6)Detection of chondrogenesis and osteogenesis: under different pressure intensity,detection of relevant indicators of chondrogenesis and osteogenesis.(7)Wnt/?-catenin signaling pathway regulates autophagy-KLPP,an inhibitor and activator of catenin signaling pathway,was used to detect autophagy level,chondrogenic and osteogenic properties in the caudal intervertebral disc of rats under different pressure intensities,and to analyze its internal correlation.(8)Autophagy regulates Wnt/?-catenin signaling pathway: KLPP loaded with autophagy inhibitor and activator was injected into the intervertebral disc of the tail vertebra of rats respectively to detect Wnt/?-catenin signaling pathway activity,chondrogenic and osteogenic properties,and analyze its internal correlation.(9)SPSS 21.0 statistical software was used to deal with the above data.Mean ± SD(Mean ± SD)was used to express the experimental data.The differences between the two groups were statistically significant(P < 0.05).Result(1)Western blot and RT-PCR showed that the expression levels of LC3 B and Beclin1 were increased,and the positive rate of MDC was also increased.(2)LDH and CCK-8 were detected at 0h,6h,12 h and 24 h after the intervention of pressure hypoxia.The results showed that 3-MA,an autophagy inhibitor,could enhance LDH release activity(reflecting cell death),and 3-MA could simultaneously reduce CCK-8 level(reflecting cell activity),indicating that autophagy played a role in protecting fibrocyte.(3)After 0 and 2 weeks of tail vertebrae compression,the expression of Wnt3 a and ?-catenin,the key molecules of Wnt/?-catenin signaling pathway,and the expression of type II collagen and proteoglycan were down regulated by immunohistochemistry,suggesting that the activation of Wnt/?-catenin signaling pathway may be negatively correlated with IDD.(4)Western blot and RT-PCR showed that Wnt3 a and ?-catenin,the key molecules of Wnt/ ?-catenin signaling pathway,were up-regulated gradually.(5)LDH and CCK-8 detection showed that IWP-2,an Wnt/?-catenin inhibitor,inhibited the cell death,and Li Cl,an Wnt/?-catenin activator,increased the cell death,suggesting that the activation of Wnt/?-catenin signaling pathway may be positively correlated with IDD.(6)Calcein-AM/PI staining and quantitative display: the new peptide self-assembly gel material KLPP has better biocompatibility than traditional KLD-12 self-assembly gel material,has little toxicity to chondrocytes and promotes cell proliferation.Compared with KLD-12,the new type of KLPP can promote chondrocytes to migrate from its interior to its surface,with appropriate porosity,and can be used as a good drug delivery carrier.ConclusionUnder the condition of pressure hypoxia,Wnt/?-catenin signaling pathway was negatively correlated with the autophagy of fibrocyte.Under the condition of pressure hypoxia,Wnt/?-catenin signaling pathway and autophagy are closely related to the chondrogenic and osteogenic activities of annulus fibrocyte cells.Under the condition of pressure and hypoxia,we can regulate Wnt/?-catenin signal pathway to regulate autophagy level,and finally promote the soft ossification and ossification of fibrous ring. 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