Tnf-? Induces Apoptosis Of Human Nucleus Pulposus Cells Via Activating The TRIM14/NF-?B Signalling Pathway

Object:Cervical spondylosis is a common chronic degenerative disease of the cervical spine in the elderly.It shows clinical symptoms caused by nerve root compression,which seriously affects the normal life of most patients.However,so far,the pathogenesis of cervical spondylosis is not completely clear.This study intends to determine the degree of apoptosis of nucleus pulposus cells and the occurrence and development of cervical spondylosis by detecting the apoptosis of nucleus pulposus tissue in patients with cervical spondylosis,and to detect the expression of nucleus pulposus cell apoptosis-related genes stimulated by TNF-? in order to To clarify the correlation between the expression of T Trim14,NF?B p65 and PPM1 A and apoptosis of nucleus pulposus cells in the course of chronic degenerative diseases,and to clarify the regulatory mechanism.Methods:1.The nucleus pulposus tissue of patients with cervical spondylosis and non-cervical spondylosis were collected for tissue section staining.HE staining was detected to clarify the changes of the nucleus pulposus tissue structure in patients with cervical spondylosis,and the Tunnel staining was used to clarify the withering of nucleus pulposus tissue in patients with cervical spondylosis,which may be correlation with the process of cervical spondylosis.At the same time,the nucleus pulposus cells in the human nucleus pulposus tissue were isolated and extracted.Under the induction of TNF-? inflammatory factor,the activity an apoptosis of the nucleus pulposus cells were detected to determine the effect of inflammatory factors on the apoptosis of the nucleus pulposus cells.2.To clarify in human nucleus pulposus cells,TNF-? inflammatory factor affects the apoptosis of nucleus pulposus cells by affecting the expression changes of which factors.Human nucleus pulposus cells were extracted for the m RNA and protein under different concentrations of TNF-? inflammatory factor treatment, and the expression of Trim14 and NF?B P65 induced by TNF-? inflammatory factor was detected by q RT-PCT and Western Blot.3.To clarify the effect of TNF-? inflammatory factor on human nucleus pulposus cells apoptosis,we constructed Trim14 interference plasmid and overexpression plasmid to detect apoptosis related genes by apoptosis flow cytometry and q RT-PCR detection.The effect on NF-?B P65 was also detected to clarify the regulatory mechanism of Trim14 on NF-?B P65.4.In order to clarify the relationship between Trim14 and PPM1 A protein,we added the Trim14 interference plasmid in human nucleus pulposus cells to interfere with the expression of Trim14.Moreover,we also detect the expression levels of PPM1A and PPM1 B to clarify the regulatory effect of Trim14 on phosphatase, and then detect PPM1 A ubiquitin by co-IP to determine the effect of TNF-? inflammatory factor on PPM1 A activity through Trim14.5.We take the nucleus pulposus tissue of patients with cervical spondylosis and patients with non-cervical spondylosis and extract their m RNA to detect the expression of TNF-?,Trim14,PPM1 A,NF?B P65 to clarify the changes in the expression of these factors during the pathogenesis of cervical spondylosis.Results:1.Tissue section staining of nucleus pulposus tissue in patients with cervical spondylosis and non-cervical spondylosis revealed that apoptotic bodies increased significantly in the nucleus pulposus tissue of patients with cervical spondylosis. Extracting nucleus pulposus cells,induced by different concentrations of TNF-? inflammatory factors,significantly inhibited the activity of nucleus pulposus cells and promoted their apoptosis.It indicated that in the nucleus pulposus tissue,the apoptosis of nucleus pulposus cells increased significantly,which may be caused by the increased content of TNF-? inflammatory factor.2.Human nucleus pulposus cells treated with different concentrations of TNF-? can significantly promote the expression level and phosphorylation level of NF-?B p65,and also promote the expression of Trim14.3.Using the Trim14 interference plasmid to interfere with the expression of Trim14 in human nucleus pulposus cells,it was found that it significantly inhibited the apoptosis of nucleus pulposus cells induced by TNF-? inflammatory factor.The expression of Bcl2 was significantly increased,and the expression of Bax protein was significantly reduced.Reduced the expression level and phosphorylation level of NF-?B p65.After using the overexpression plasmid to increase the expression of Trim14,it was found that it significantly increased the apoptosis of nucleus pulposus cells induced by TNF-? inflammatory factor.The expression of Bcl2 was significantly reduced,and the expression of Bax protein was significantly increased.NF-?B p65 expression level and phosphorylation level.It showed that Trim14 was significantly increased under the action of TNF-? inflammatory factor,thereby accelerating the apoptosis of nucleus pulposus cells by promoting the increase of NF-?B p65 expression and phosphorylation level.4.Using the Trim14 interference plasmid to interfere with the expression of Trim14 in human nucleus pulposus cells,it was found that under the effect of TNF-?,the expression of PPM1 A can be significantly increased.At the same time,we used co-IP detection to find that Trim14 can promote the ubiquitination level of PPM1A,thereby inhibiting the removal of PPM1 A Phosphorylation ability increases the phosphorylation level of NF-?B,thereby activating the NF-?B signaling pathway to increase the expression of apoptotic genes and promote the occurrence of apoptosis in human nucleus pulposus cells.5.In the nucleus pulposus tissue of patients with severe cervical spondylosis,the expression of TNF-?,Trim14 and NF-?B increased significantly,while the expression of PPM1 A decreased significantly.Conclusion:In summary,in our study,we found that the apoptosis of cervical medulla cells in cervical spondylosis patients was significantly increased,which may be related to the increased expression of inflammatory factor TNF-? activating the Trim14 / NF-?B p65 regulatory pathway,which further clarified the pathogenesis of cervical spondylosis and also The clinical treatment of the occurrence of cervical spondylosis provides new ideas.