Research On Expressions Of HMGB1and CD163in The Cartilage End-plate And Their Relationships With The Intervertebral Disc Degeneration

Objectives:To investigate the expressions of HMGB1and CD163in the cartilage end-plate and their relationships with the intervertebral disc degeneration; and then, to reveal the mechanisms of the intervertebral disc degeneration.Methods:(1) Create acupuncture models of lumbar intervertebral disc degeneration (IDD) in the rabbits:72Japanese White Rabbits were divided into acupuncture groups and control groups at random.16epidural needle puncture was used to establish animal model of lumbar intervertebral disc degeneration; In the acupuncture groups, the discs of L2.3, L3-4, L4.5, L5.6were chosen as experimental group for surgical intervention, and in the control groups the discs of L2-3, L3-4, L4-5, L5-6were chosen as control group.(2) Harvest Samples:12Japanese White Rabbits were killed randomly at4,8,12weeks after operation per group, and4Japanese White Rabbits of each group were for lumbar X-ray and MRI examinations firstly; after that, the discs of experimental group and control group were preserved in liquid nitrogen separately.(3) Detect the changes of intervertebral discs from Imaging, HE stain, MMP-9Immunohistochemistry; and evaluate the feasibility of acupuncture models of lumbar intervertebral disc degeneration.(4) Study the co-expression of HMGB1and apoptotic cell:Hybridization in situ combined with Double-labelling immunofluorescence was used to research the co-expression of HMGB1and apoptotic cell in the cartilage end-plate of the degenerative lumbar intervertebral discs from the acupuncture models.(5) Observe the expression of HMGB1in the cartilage end-plate during the process of lumbar intervertebral disc degeneration:The methods of western blot and real-time fluorescent quantitative PCR were used to detect the expression change of HMGB-1in the cartilage end-plate of the degenerative lumbar intervertebral discs.(6) Expression of CD163mRNA in the cartilage end-plate of the degenerative intervertebral discs:Real-time quantitative PCR was used for detecting the expression of CD163mRNA in the cartilage end-plate, to explore the significance of CD163in the process of intervertebral disc degeneration.Results:(1)Compared with the control groups, the discs of experimental groups appear pathological changes of height loss, water content loss, calcification of end-plate, osteophyte of adjacent vertebrae, architecture distortion of annulus fibrosus and nucleus pulposus, cytoreduction at different levels; And MMP-9shows a high expression in the experimental groups’discs; The degree of intervertebral disc degeneration gradually increases over time.(2)HMGB1has expressed both in the rabbit intervertebral discs of control group and experimental group, the expression Locates in the cartilage endplate chondrocytes, and positive expression cells are always accompanied by uneven distributed positive particles in the matrix. HMGB1has a higher expression in every experimental group’s discs than in every control group’s discs, and the expression increases with the severity of disc degeneration; apoptotic cell has expressed both in the rabbit intervertebral discs of control group and experimental group, apoptotic cell shows an increasing tendency with the severity of disc degeneration in the experimental groups, and the expression in control groups’discs is lower than experimental groups’, but there is no obvious difference among the control groups’discs. Moreover, HMGB1could express in normal cells and apoptotic cells as well as in the stroma around the cells.(3)Compared with the control groups, HMGB1has a higher expression in cartilage endplate at4,8,12weeks after operation from Western blot, P<0.05, with statistical significance, and the expression of HMGB1shows an increasing tendency with the severity of disc degeneration. FQ-PCR reveals the high expression of HMGB1mRNA in the endplate cartilage cells, but there is no obvious variation with time.(4)The expression of CD163mRNA in the discs shows an increasing tendency with the severity of disc degeneration, which reveals that phagocytes like macrophages in the cartilage end-plate take part in the degeneration of intervertebral disc.Conclusions:(1)HMGBl may be secreted and released by the cartilage cells under degeneration, the increased expression of HMGB1may promote the endplate chondrocytes apoptosis. The expression of HMGB1in the cartilage end-plate has a positive correlation with chondrocytes apoptosis during the process of intervertebral die degeneration.(2)Phagocytes like macrophages may involve in the process of intervertebral dic degeneration, HMGB1may induce phagocytes like macrophages to enter into cartilage end-plate, which increase the apoptotic cells, release various cytokines and inflammatory cytokines, and then, initiate aseptic inflammation, promote the process of intervertebral disc degeneration.(3)HMGB1is an important initiator in the intervertebral disc degeneration process, plays an important role in the process of intervertebral disc degeneration, and related immunological mechanism remains to be further researched.