Research On The Role Of ERK5Signaling Pathway In The Nucleus Pulposus Cells Degeneration

Objective：To explore the functions of ERK5signaling pathway in the nucleus pulposus cells，whether it might provide a therapeutic approach for the development of molecular therapies fordisc degeneration.Methods：1、nucleus pulposus cells were isolated from Human normal intervertebral disc,immunohistochemistry and immunofluorescence assay were used to detect the expression ofERK5and P-ERK5in Nucleus pulposus cell, the cytotoxicity of different concentration ofBIX02188was using XTT method.2、nucleus pulposus cells were inhibited by differentconcentration of BIX02188for two hours, then actived by H2O2in five minutes, western blotassay were employed to detect the P-ERK5.3、nucleus pulposus cells were exposed to twodifferent group:5μM BIX0218and blank control group, Forty-eight hours late, RT-PCR assaywere employed to detect the gene mRNA expression of Type Ⅱ collagen,glycosaminoglycans, SOX9.4、nucleus pulposus cells were exposed to two different group:5μM BIX0218and blank control group, and conditioned media analyzed for accumulation ofnitric oxide(NO), IL-6, matrix metalloproteinase-3(MMP-3), and tissue inhibitor of matrixmetalloproteinase-1(TIMP-1) after48hours.Ruslts：1、ERK5and P-ERK5were detected in nucleus pulposus cells nucleus and cytoplasm,there is no cytotoxicity of BIX02188under the concentration of10μM.2、the expression ofP-ERK5in nucleus pulposus cells could be actived by H2O2at the concentration of200μM, theexpression of P-ERK5actived by H2O2can be inhibited by BIX02188at the concentrationbetween3μM to10μM.3、48hours later inhibited by BIX02188, the mRNA expression ofType Ⅱ collagen, glycosaminoglycans, SOX9in nucleus pulposus cells were significantdecreased in contrast to blank control group (n=5,P<0.05);4、48hours later inhibited byBIX02188，the expression of NO、IL-6、MMP-3were significant elevated in contrast to blankcontrol group (n=5,P<0.05)，however there is no significant changing in TIMP-1. Conclution：1、ERK5and P-ERK5were detected in nucleus pulposus cells nucleus andcytoplasm；2、Inhibition of ERK5in nucleus pulposus cells significant decreased the geneexpression of Type Ⅱ collagen, glycosaminoglycans, SOX9and significant elevated theexpression of NO、IL-6、MMP-3, however there is no significant changing in TIMP-1.