Lycorine Suppresses Endplate-chondrocyte Degeneration And Prevents Intervertebral Disc Degeneration By Inhibiting NF-?B Signalling Pathway

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Evaluation of the catabolic and anabolic protease expression in the cartilaginous endplate(CEP)of patients with intervertebral disc(IVD)degeneration.Objective:To compare the catabolic and anabolic protease expression in CEP between disc degeneration patients and lumbar fracture patients.Methods:Samples of degenerated IVD tissue(n=25)were obtained from patients with intervertebral disc degeneration.Samples of non-degenerated IVD tissue(n=7)were obtained from patients with lumbar fracture.Preoperative lumbar MRI,Safranin O-fast green staining and immunohistochemistry(IHC)staining of CEP tissues were conducted to evaluate the severity of CEP degeneration.Quantitative real-time PCR(qPCR)was conducted to investigate the mRNA expression levels matrix metalloproteinase(MMP)-3,MMP-13,a disintegrin and metalloproteinase with thrombospondin motifs(ADAMTS)-4,ADAMTS-5,Sox-9,aggrecan and Col-2a1.Results:IHC results showed the increased expression of catabolic proteases,such as ADAMTS-4,ADAMTS-5,MMP-3 and MMP-13 in CEP from degenerated-IVD.The Pfirrmann scores were positively correlated with the expression level of ADAMTS-4,ADAMTS-5,MMP-3 and MMP-13 and negatively correlated with the expression level of synthetic molecules,such as Sox-9,aggrecan and Col-2al in CEP.The age of the patients was also an independent risk factor of CEP degenerationConclusion:The expression of catabolic proteases was increased in the CEP of patients with degenerated IVD.Part ? The protective effect of lycorine(LY)on CEP-derived chondrocytes in vitroObjective:To examine the effect of LY on CEP-derived chondrocytes and its underlying mechanism.Methods:CEP and nucleus pulposus(NP)cells were separated from the intervertebral disc of rats.The cytotoxic effects of LY were determined by Cell Counting Kit-8(CCK-8)assay,flow cytometry analysis of apoptosis and cell cycle.qPCR,Western Blot,Alcian blue and DMMB colorimetric assay were conducted to investigate the expression levels of MMP-3,MMP-13,ADAMTS-4 and ADAMTS-5 after treated with IL-1? and LY.The effects of IL-1(3 and LY on signaling pathway were invested by plasmid transfection,Western Blot,cell immunofluorescence and luciferase reporter-gene assays.Results:No cytotoxic effect of LY was found when applied at concentration<0.5?M.No obvious cell apoptosis or cell cycle arrest were observed after treatment with 0.4?M of lycorine.The mRNA and protein levels of ADAMTS-4,ADAMTS-5,MMP-3 and MMP-13 were significantly upregulated by IL-1?,whilst LY suppressed their expression in a dose-dependent manner.LY treatment reduced the IL-1?-induced phosphorylation of IkB-? and increased the level of IkB-?,without changing the phosphorylation of ERK,P38,JNK,TAK1 or IKK-a/(3.In cell immunofluorescence,IL-1? activated P65 nuclear translocation while LY retained P65 in the cytoplasm The luciferase-reporter activity stimulated by IL-1? was also reduced by LY in a dose-dependent manner.Conclusion:LY suppressed IL-1?-induced expression of catabolic proteases by inhibiting NF-kB signalling.Part ? The protective effect of LY on IVD degeneration in vivo Objective:To further evaluate the protective effect of LY by systemic administration on rat-tail CEP and IVD degeneration model.Methods:High Performance Liquid Chromatography(HPLC)quantification of LY in rat plasma was conducted after received 5mg/kg of LY by oral or intraperitoneal(i.p.)administration.Rat-tail CEP and IVD degeneration models were established and divided into four groups:Sham group;PBS group;low-dose LY group and high-dose LY group.After 2 months of i.p.administration of LY at the dose of 5mg/kg,hearts,livers,lungs and kidneys were collected for H&E staining.Rat-tail sections were stained with H&E,Picrosirius red and Safranin O-fast green staining.The thickness and the ratio of cartilage tissue area of CEP were measured.Immunostaining of ADAMTS-4,ADAMTS-5,MMP-3,MMP-13 and immunofluorescence staining for MIF in rat-tail sections were also conducted.CEP and NP cells were treated with IL-1?(10 ng/ml)with or without LY(0.4 ?M)and then qPCR and Western Blotting were performed for evaluating the levels of TNF-a,IL-6,IL-1? and MIF.Results:The mean plasma concentration of LY reached the effective drug dose after both oral and i.p.administration.LY prevented CEP degeneration in the rat-tail CEP and IVD degeneration models.Immunostaining postive cell rates of ADAMTS-4,ADAMTS-5,MMP-3 and MMP-13 in CEP were higher in the PBS group and decreased in LY groups.LY also effectively prevented IVD degeneration in the rat-tail models.Histological score was significantly higher in PBS group and decreased in LY groups.Western Blotting and qPCR detected MIF expression in CEP cell but not in NP cell in vitro.MIF immunofluorescence staining of the CEP was higher in the PBS group,but decreased in LY groups.Conclusion:LY might prevente CEP degeneration and IVD degeneration in the rat-tail model.LY might prevent IVD degeneration by inhibiting MIF release from the endplate to the NP tissue.