Epoxyeicosanoids Prevent Intervertebral Disc Degeneration In Vitro And In Vivo

Objective1.To study the effect of EETs on rat nucleus pulposus cells induced by TNF-in vitro and its molecular mechanism;2.The construction and evaluation of rat model of intervertebral disc degeneration is simple,efficient and reliable,find the relationship of rat tail disc degeneration time accurate degree3.Using the model of rat intervertebral disc degeneration to study the effect of EETs on the intervertebral disc degeneration,so as to understand that EETs can effectively prevent and restrain the early degeneration of intervertebral disc in rats.Methods1.In vitro,take weeks to 12-14 weeks male SD(Sprague Dawley)rats were separated and cultured.Rat nucleus pulposus cells;Using CCK-8 method to detect different concentrations of effects of 14,15-EET on the proliferation of nucleus pulposus cells;using western bloting Western blot,real-time quantitative RT-PCR method to detect measurement of 14,15-EET on TNF alpha induced degeneration of nucleus pulposus cells in type Ⅰ collagen,type Ⅱ collagen,proteoglycan and expression levels of MMPs influence aggregation,using western bloting method for the detection of 14,15-EET on and the activation of NF kappa BMAPKs signaling pathway;2.40 male SD rats aged 12-14 weeks were divided into 10 groups,rats in each group.Each group was divided into 4 groups.Each group was divided into groups,and the Co6-Co7,Co7-Co8 intervertebral discs were punctured by 21-G needle.Center for puncture from the skin of about 5 mm nucleus,the tip rotates 360 DEG,and wait for about 30 seconds.The X-ray and MRI imaging of the rat tail vertebrae were performed in each rat after 3 hours,48 hours,3 days,7 days,10 days and 14 day.The rate of change of intervertebral disc height index(Dffl%)and intervertebral disc height(RDHC)was calculated.To score the degeneration of the rat tail intervertebral disc(Ⅰ-Ⅳ),I is the normal intervertebral disc,Ⅳ is the serious degeneration,find the intervertebral disc degeneration degree rule.;3.In vivo,12 week old male SD rats,tail intervertebral disc degeneration model was established in SD rats by needle puncture of the 21G.Treatment with 14,15-EET rats for 2 weeks,after 4 weeks after the corresponding intervertebral disc were detected.The effect of nucleus pulposus and annulus structure changes by 7T small animal MR scanning method for the detection of 14,15-eet;using histomorphometric analysis of intervertebral disc tissue HE staining and alcian blue staining to assess effect of 14,15-eet on the role of nucleus pulposus cells in vivo.ResultsThe in vitro and in vivo experiments,we observe that eets inhibit intervertebral disc degeneration,the role and promote the extracellular matrix proteoglycans and type Ⅱcollagen accumulation and reduced the level of type Ⅰ collagen and matrix metalloproteinase related.EETs inhibited TNF-induced(NF-K B)signaling pathway in P-65 phosphorylation and inhibition of the MAPKs signaling pathway of extracellular regulated protein kinase(Extracellular Regulated Protein Kinase,ERK)and the phosphorylation of c-Jun N-terminal kinase(c-Jun N-terminal,Kinase,JNK),and the phosphorylation of P38 and AKT.Phosphorylation of the inhibitory effect was not significant.At the same time,we constructed a simple,reliable and efficient model of rat tail intervertebral disc degeneration.By using this model,we found that EETs prevented the intervertebral disc degeneration by inhibiting the loss of extracellular matrix.Conclusion1.Our results confirmed that EETs inhibited the intervertebral disc degeneration by acting on a variety of pathways.2.A simple,reliable and efficient animal model is a simple,reliable and efficient model for the degeneration of the rat tail intervertebral disc.3.Exogenous or endogenous EETs levels may be a new strategy for the treatment of degenerative disc disease,such as cervical spondylosis and lumbar disc herniation.