The Senescence Of Human Intervertebral Disc Cartilage End-plate Cells And Its Regulation Mechanism Study

Objective To investigate whether senescence is more prominent in human degenerative CEP and whether SIRT1-regulated CEP cells senescence in degenerative IVD as well as identify the signaling pathways that control that the cell fate decision in oxidative stress.Method Degenerative CEP samples were donated by the patients with DDD during discectomy and intervertebral fusion surgery.Mild-degenerated CEP samples were donated by the patients with LVF undergoing posterior discectomy.CEP samples collected were compared according to their macroscopic morphologies,and specimens were stained with hematoxylin and eosin,and detected by immunohistochemical analysis to determine protein expression of SIRT1,p53,p21,and p16.CEP cells were isolated and cultured,and they were subsequently incubated with H₂O₂ for oxidative stress-indcued senescence.The cell cycle,cell apoptosis and the expression of senescence-related signal pathway genes were detected by flow cytometry,SA-β-Gal staining,Real-time PCR,and Western blotting.Ad-SIRT1 were generated using Ad Easy technology to increase SIRT1 expression,and then determine the CEP cells infected with Ad-SIRT1 whether exhibited high expression of SIRT1,and Ad-GFP as the control.Nicotinamide was used to inhibit SIRT1 activity.PTF-α was used to inhibit p53 activity.Flow cytometry,SA-β-Gal staining,cell immunofluorescence staining,Real-time PCR,and WB were used to detect the cell cycle,cell apoptosis and the expression and location of senescence-related signal pathway genes,to explore the role and mechanism of SIRT1 on CEP cell senescence in oxidative stress.Results The samples obtained from the DDD patients were yellow in color and exhibited varying levels of calcification according to their macroscopic morphologies.The borders of the CEP cells obtained from all the IVF patients were rounded,while the borders of the CEP cells obtained from the all DDD patients were platy in the HE staining.Both the percentage of SA-β-Gal-positive cells（blue）and staining intensity of the CEP cells obtained from all the DDD patients were significantly greater than those of the CEP cells obtained from all the LVF patients.The immunohistochemical detection results indicated that the CEP samples obtained from the DDD patients exhibited a significantly lower number of SIRT1-postive cells than the CEP samples obtained from the LVF patients（p<0.05）.In contrast,a higher number of p53-and p21-positive cells was observed in the DDD samples than in the LVF samples（p<0.05）.No significant differences in the number of p16-positive cells in the LVF and DDD groups existed（p>0.05）.The sublethal oxidative stress induced by H₂O₂ did not cause increased levels of the early-and late-stage apoptosis in the CEP cells（p>0.05）.However,the H₂O₂-induced oxidative stress did significantly increase the population of G1 phase cells（p<0.05）.The percentage SA-β-Gal-positive cells（blue）,and staining intensity.The expression of collagen II was decreased and matrix metalloproteinase 13（MMP13）was increased in oxidative stress（p<0.05）.Significant differences in the p53 mRNA levels were observed under oxidative stress（p<0.05）.In addition,the levels of p21 mRNA and protein increased significantly under H₂O₂-induced oxidative stress（p<0.05）.The levels of acetylated p53 were also higher under oxidative stress.In contrast,there was no significant increase for p38 and p16 levels of mRNA（p>0.05）and levels of phosphorylated p38 and p16 level of protein under oxidative stress（p>0.05）.The overexpression of SIRT1 significantly decreased the levels of acetylated p53 as well as the levels of its pro-senescence effector p21（p<0.05）.The overexpression of SIRT1 also decreased the population of G1 phase cells（p<0.05）,the percentege of SA-β-Gal-positive cells（blue）,and staining intensity of the CEP cells.In contrast,the addition of nicotinamide significantly increased the levels of acetylated p53 as well as the levels of its pro-senescence effector p21（p<0.05）.The nicotinamide also increased the population of G1 phase cells（p<0.05）,the percentage of SA-β-Gal-positive cells（blue）,and staining intensity of the CEP cells.Both SIRT1 and p53 were located in cell nucleus by immunofluorescence staining under sublethal oxidative stress.Nicotinamide was also used in this study to inhibit endogenous SIRT1 activity in order to further activate the p53/p21 pathway under sublethal oxidative stress,and PTF-α was used to inhibit p53 activity.PTF-α significantly inhibited p21 protein expression,and decreased the population of G1 phase cells（p<0.05）and the percentage of SA-β-Gal-positive cells（blue）,and staining intensity with or without the addition of nicotinamide,alleviating CEP cell senescence.Conclusion The cell senescence phenotype was found to be more prominent in the CEP cells obtained from DDD patients than in the CEP cells obtained from age-matched LVF patients.The p53/p21 pathway plays an important role in the senescence of CEP cells in vivo and vitro.Furthermore,SIRT1 was found to be capable of alleviating the oxidative stress-induced senescence of CEP cells in humans via p53/p21 pathway.Thus,the information presented in this study could be used to further investigate the underlying mechanisms of CEP degeneration.