| Part I Expression of TNFAIP3 in normal and degenerated intervertebral discs ObjectiveTo clarify the expression changes of TNFAIP3 and NF-κB-related signaling molecules in normal and degenerated intervertebral disc NP tissues.MethodsCollect clinical normal and degenerated NP tissues of huamn and observe the changes of TNFAIP3 expression in normal and degenerated NP tissues by immunohistochemistry;The acupuncture method was used to construct the rat IVDD model.The m RNA and protein of normal and degenerated NP tissues were extracted respectively(4 weeks,20 weeks,20 weeks of puncture degeneration,and 50 weeks).Quantitative real-time polymerase chain reaction(RT-PCR)and Western Blot were perform to detect the expression levels of TNF-α,TNFAIP3,NF-κB and TRAF6.Immunohistochemistry was used to detect the expression changes of TNF-α,TNFAIP3,NF-κB and TRAF6 molecular in the NP tissue of the 20-week and 20-week puncture degeneration rats.ResultsThe expression of TNFAIP3 in normal human NP tissue is significantly higher than that of degenerated NP tissue;The expressions of TNFAIP3,TNF-α,TRAF6,p-NF-κB and NF-κB were significantly increased in degenerat Ive disc of 20 weeks compared with normal disc of 20 weeks(p<0.05).There was no significant difference in the expression of TNFAIP3 in the NP tissue of rats at 4 weeks and 20 weeks.The expression of TNFAIP3 in the NP tissue of rats at 50 weeks was significantly lower than that of 4 and 20 weeks(p<0.05);In addition,the expression of TRAF6 in the NP tissue of rats at 4 weeks was very low(p<0.05),However,the expression of TRAF6 was significantly increased in the NP tissue of rats at 20 weeks,20 weeks of acupuncture degeneration and 50 weeks(p <0.05).With the occurrence of IVDD,the activation of the NF-κB signaling pathway continued to increase.Compared with 4-week and20-week rats,the expression of p-NF-κB of NP tissue was significantly increasedin 20-week acupuncture and 50-week rats(p <0.05).The activation of NF-κB pathway in the NP tissue at50 weeks was significantly higher than that of normal groups at 4 and 20 weeks(p<0.05).ConclusionIn the process of IVDD caused by puncture,the expression levels of TNF-α,TNFAIP3,TRAF6,and p-NF-κB in the NP tissue of the degeneration group were significantly higher than those of normal NP tissue(p<0.05).Comparing the expression levels of TNFAIP3 in NP tissues of rats at 4 weeks,20 weeks and 50 weeks,it was found that as the age of rats increased,the expression of TNFAIP3 gradually decreased.The above molecules are involved in the process of IVDD,and their related molecular mechanisms and effects can be further studied.Part II Expression and mechanism of TNFAIP3 during senescence of nucleus pulposus cells under TNF-α inflammation microenvironmentObjective Inflammatory response plays a pivotal role in the pathophysiological process of IVDD.TNFAIP3 is a ubiquitin-editing enzyme that restrics NF-κB signaling.TNFAIP3 prevents the occurrence of multiple inflammatory diseases.However,the role of TNFAIP3 in the initiation of IVDD has not been elucidated.The aim of the study was to investigate the effect of TNFAIP3 in senescence of TNF-α-induced NP cells.Methods The NP cells were dissected from the tail vertebra of healthy male SD rats and were cultured in the incubator.In the experiment,TNF-α was used to mimic the inflammatory environment of IVDD.The experiment was divided into three groups: normal control group,TNF-α intervention group(10ng/ml,50ng/ml,100ng/ml),and natural aging group(P20).To assess the senescence of NP cells and the changes of TNFAIP3,NF-κB pathways from the level of gene,protein and cellular function,CCK-8/SA-β-Gal staining/q PCR/Western Blot/IF were applied.To further study the relationship between TNFAIP3 and NF-κB pathway in intervertebral discs,the experiment was further divided into four groups: normal control group,TNF-α intervention group,TNFAIP3 gene silencing group,and TNFAIP3 gene silencing control group.Using the above experimental methods and flow cytometry techniques to detect the changes of senescence-related phenotypes(cell proliferation activity,SA-β-Gal staining,cell cycle,p53,p16)and extracellular matrix synthesis function(Aggrecan,Collagen)II)of NP cells in different test compounds.Additionally,detected the activation of NF-κB pathway-related molecules in each experimental group.To clarify the mechanism of TNFAIP3 in TNF-α-induced senescence of NP cells.Results TNF-α could induce the senescence of NP cells and the expression of TNFAIP3 and p-NF-κBwere significantly increased in senescent NP cells induced by TNF-α(p<0.05);Compared with the control group,The expression of TNFAIP3 and p-NF-κB were increased in TNF-α-induced NP cells(p<0.05),while the expression of TNFAIP3 in Replicativesenescence NP cellswas decreased(p<0.05),the expression of p-NF-κB increased(p<0.05);The positive rate of β-galactosidase staining and the expression of p53 and p16 were significantly increased in TNFAIP3 gene-silenced NP cells induced by TNF-α(p<0.05),while the expression of aggrecan and collagen II were significantly reduced(p<0.05);Compared with the control group,the cell cycle arrest in the TNF-α group was significantly enhanced(p<0.05);Compared with the TNF-α group,the cell cycle arrest in the TNFAIP3 gene silencing group was significantly enhanced(p<0.05),while there was no significant difference in cell cycle arrest in the TNFAIP3 gene silencing control group;TNF-α could reduce the proliferation activity of NP cells.The cell proliferation activity in the TNFAIP3 gene silencing group was significantly lower than that in the TNF-αgroup(p<0.05);The expression of senescence indicators of TNFAIP3 gene silencing group treated with TNF-α were significantly up-regulated compared to TNF-α-treated normal NP cells.The expression of TRAF6、p-NF-κB in the TNFAIP3 gene silencing group was more higher than that in the normal group and TNF-α group(p <0.05).Conclusion There also is a certain dose relationship between the senescence of NP cells and TNF-α.TNFAIP3 has a self-protective effect on the senescence of NP cells induced by TNF-α via TRAF6/NF-κB pathway.The down-regulation of TNFAIP3 in NP cells exacerbated the senescence of NP cells induced by TNF-α via NF-κB signaling.Moreover,There was a dose relationship between the expression of TNFAIP3 and TNF-α.High concentration of TNF-α could reduce the expression of TNFAIP3,and low concentration of TNF-α(10ng/ml)significantly increase the expression of TNFAIP3. |
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