The Role And Mechanism Of Mechanical Sensitive Protein YAP1 In The Degeneration Of Nucleus Pulposus Cells Induced By Tension Stimulation

Objective: To establish a model of nucleus pulposus cell degeneration loaded by continuous cyclic tension stimulation,to investigate the mechanism of the regulatory role of mechanosensitive protein YAP1 in inducing nucleus pulposus cell degeneration under continuous cyclic tension conditions,and to analyze potential targets for intervertebral disc degeneration therapy.Methods: Sprague-Dawley rat caudal disc nucleus pulposus tissue was extracted,digested with conventional collagenase and cultured;the P2 nucleus pulposus cells were stimulated by continuous cyclic distraction using the Flexcell-6000T^TM Cell Strain Loading System and divided into normal control group（NC）and cyclic mechanical tension group（CMT）,the tensile strength of the CMT group was 10% and the duration was 0h,12 h,24h The expression of YAP1 and Piezo1 was verified by RT-q PCR and Western Blot;the animal model of caudal disc degeneration was induced by CINS,and the phenotype was verified by immunohistochemistry and fluorescence staining;the key molecule YAP1 was selected for follow-up experiments,and the nucleoplasm separation assay and immunofluorescence were used to verify the expression of YAP1 in and out of the nucleus;the nucleus pulposus overexpression and knockdown model was constructed and the protein expression was verified by Western Blot.Finally,the upstream molecules of the Hippo signaling pathway were validated,Piezo1 was identified as the study target and modulated using the corresponding agonist,and the levels of YAP1 and related proteins were observed.Results: In the CINS-induced rat caudal intervertebral disc degeneration model,the cell strain loading system with a stimulation frequency of 0.5 Hz,tensile strength of 10%,and loading time of 24 h could simulate the induced degeneration model;the expression of mechanosensitive protein YAP1 gradually decreased with the prolonged loading time,and the p-YAP fraction increased;overexpression of YAP1 promoted the synthesis and secretion of the myeloid matrix,which could In the process of degeneration induced by cyclic mechanical tension,mechanosensitive signals activated the opening and expression of Piezo1 channels,and Piezo1 did not regulate the active expression of YAP1 and the entry and exit of YAP1 into and out of the nucleus through the Hippo signaling pathway.Conclusion: Sustained cyclic traction（0.5 Hz,10%）induces a nucleus pulposus degenerative phenotype in vitro,and increasing YAP1 functional activity mitigates nucleus pulposus degeneration progression,and Piezo1 may become a new target for the treatment of disc degeneration.