Gamma-Secretase Inhibitor(DAPT)Regulates The Activation Of The NF-?B Signaling Pathway By The Release Of The Activated Cytoplasmic Domain Of Notchl In Nucleus Pulposus Cells

Objective:Current studies have shown that the activation of the NF-?B signaling pathway plays an important role in intervertebral disc degeneration.However,regulating this signaling pathway remains a challenge.In this study,we investigated the junction of the Notch signaling pathway and NF-?B signalingpathway.We then regulated the NF-?B signaling pathway by inhibiting the Notch signaling pathway,delaying the degeneration of the intervertebral disc under inflammation.Methods:The hNPCs and mice disc were assigned to four groups:(1)Control group(2)Degeneration group:IL-1?(lOng/ml)and TNF-?(50ng/ml)were added into culture medium.(3)Low-DAPT group:IL-1?(10ng/ml),TNF-?(50ng/ml)and DAPT(5?mol/ml)were added into culture medium.(4)High-DAPT group:IL-1?(10ng/ml),TNF-?(50ng/ml)and DAPT(20?mol/ml)were added into culture medium.The degeneration of hNPCs was observed with or without ?-secretase inhibitor,a Notch signaling pathway inhibitor.1.Human nucleus pulposus cells(hNPCs):Western blot was conducted to observe the expression levels of Col ?,proteoglycan,Runx 2,the phosphorylation and acetylationand of p65 and the intranuclear subunit of NF-?B in hNPCs.2.RT-PCR was used to observe the expression of AGC1 and Col ?.3.The 293T cells were transfected with plasmid carried with overexpression of correlate NICD1 domains.IP was used to observe the expression of of NICD1 and p65 binding.4.Mice:The intervertebral disc of the mice was cultured for 3 and 10 days.IL-1? and TNF-? were added in the culture medium to induce the degeneration of hNPCs,which was observed with or without DAPT.The expression of Col ? in the intervertebral disc of mice was observed by imnunohistochemistry.By contrast,the expression of proteoglycan in the intervertebral disc was observed by hematoxylin and eosin staining and alcian blue staining.Results:hNPCs:DAPT of hNPCs caused a concentration-dependent increase in the IL-??-and TNF-?-induced decreased expression of Col ? and proteoglycan.IL-1?and TNF-? decreased the expression of Col ? and proteoglycan in the intervertebral disc of mice,inducing the aging of hNPCs.After adding DAPT,the expression of Col ? and proteoglycan in hNPCs increased,and the expression of transcription factors Col ? and AGC1 was enhanced.The protein and mRNA level of transcription factor Runx 2 related to Col ? and proteoglycan expression was inhibited by IL-1? and TNF-?,while DAPT rescued the inhibition.Expression of ColX illustrated the aging end-stage of hNPCs.ColX protein and mRNA level were increased by IL-1? and TNF-?,while DAPT suppressed the inhibition.Further studies showed that DAPT inhibited the IL-1?-and TNF-?-induced activation of the NF-?B signaling pathway.Western blot revealed that DAPT inhibited the phosphorylation and acetylationand of p65 and nuclear import levels of p65 and p50.Co-immunoprecipitation(Co-IP)demonstrated that the cytoplasmic domain of Notchl(NICD1)combined with p65 in hNPCs,and the ANK domain of NICD1 was its binding site with p65.Plasmids transfected 293T cells:The 293T cells were transfected by plasmids with overexpression of eorrelated NICD1 domains.Immune eoprecipitation found after deleting ANK domain that the binding phenomenon of NICD1 and p65 could not appear.Lentivirus transfected hNPCs:The hNPCs were transfected by lentivirus conteined with ANK-deletion overexoression NICD1.Immune coprecipitation showed NICD1 and p65 cannot be combined.Intervertebral disc of mice:IL-1? and TNF-? inhibited the expression of Col ?and proteoglycan in the intervertebral disc and led to the degeneration of hNPCs.After DAPT was added in the culture medium of the intervertebral disc,the expression of Col ? and proteoglycan increased in a concentration-dependent manner.Conclusion:Results showed that DAPT inhibited the IL-1?-and TNF-?-induced activation of the NF-?B signaling pathway by inhibiting the release of the cytoplasmic domain of Notch 1(NICD1)in hNPCs.Thus,the aging of hNPCs induced by inflammatory factors was delayed.1.DAPT rescued the hNPCs and intervertebral disc of mice degeneration induced by IL-1? and TNF-?.2.DAPT inhibited the IL-1?-and TNF-?-induced activation of the NF-?B signaling pathway by inhibiting the release of the NICD1 in hNPCs.3.Phosphorylation and acetylationand of p65 needed the binding,with ANK domain of NICD1 In hNPCs.