The Role And Mechanism Of YAP Regulating The TNF-α Induced Premature Senescence Of Rat Nucleus Pulposus Cell

Objective:The aim of the study is to predict the effects of YAP on TNF-α-induced premature senescence of rat nucleus pulposus cells,to explore the mechanism of Hippo signaling pathways in regulating YAP in the premature rat nucleus pulposus induced by TNF-α and to investigate the role of YAP in disc degenerationMethods:(1)Identification of primary rat nucleus pulposus cells by toluidine blue staining,HE staining and type II collagen immunofluorescence;(2)ordinary PCR,CCK8,β-galactosidase(SA-β-gal)staining and Western blot to verify the aging model of rat nucleus pulposus cells;(3)Analysis of changes in Hippo signaling pathway in NP-senescent cells induced by TNF-α using quantitative PCR,Western blot and immunofluorescence;(4)The specific drug Verteporfin was used to inhibit YAP,and to explore its regulatory mechanism in the process of TNF-α-induced senescence;(5)Western blot and β－galactosidase were used to investigate the use of lentivirus to overexpress YAP.(6)To explore the effect of YAP on the changes of rat nucleus pulposus cells by quantitative PCR,semi-quantitative PCR,immunofluorescence technology and small animal MR.Results:(1)Verify that the extracted and cultured primary cells are rat nucleus pulposus(NP)cells by three kinds of relatively specific staining related to nucleus pulposus cells;(2)40ng/ml TNF-α activates p53-p21-pRb pathway,reduce proliferation of nucleus pulposus cells,induce senile nucleus pulposus cells of SA-P-gal positive rats:(4)Hippo signaling pathway is activated in nucleus pulposus induced by TNF-α,leading to increased YAP phosphorylation However,the corresponding TNF-α induction also resulted in an increase in the production of YAP itself,and eventually the expression of YAP protein increased,and YAP entered the nucleus with increased activity.(5)After using Verteporfin,a specific YAP inhibitor,it was found that p53 in NP cells.p21.The expression of pRb increased,and SA-β-gal staining also showed that the proportion of positive cells also increased significantly compared to the control group.(6)After lentivirus overexpression of YAP,it was found that the senescence-related protein p53 in NP cells.p21.The expression of pRb decreased,and the mRNA expression of type II collagen and aggrecan increased at the mRNA level,and MMP13 decreased accordingly,and SA-β-gal staining also found that the proportion of positive cells was reduced relative to the control group,so We speculate that YAP protein has an anti-aging effect on TNF-α-induced premature NP cell senescence.(7).It was further found in vivo experiments that the injection of YAP lentivirus can alleviate the degeneration of the rat’s tail vertebra caused by acupuncture,and it may have an anti-disc degeneration effect.Conclusions:This study showed that YAP is presented as an anti-senescence protein in rat NP cells premature senescence induced by TNF-α,and it was found that overexpression of YAP may alleviate degeneration of the rat tail disc induced by acupuncture.These findings suggest that YAP may be a potential therapeutic target for delaying the progression of IDD.