Role Of HIF-1α In Nucleus Pulposus Cells Senescence Induced By Serum Starvation

Objective:Human degenerated and non-degenerated nucleus pulposus were performed by staining the samples with senescence-associated β-galactosidase(SA-β-Gal)solution and then observing cell positivity for SA-β-Gal.Primary rat NP cells were isolated and cultured in media supplement with different fetal bovine serum(FBS)concentrations to induce NP cells senescence.To explore the relationship between serum starvation and nucleus pulposus cell senescence.And the relationship between nucleus pulposus cell senescence and HIF-1α was clarified by examining the HIF-1α expression.Methods:Non-degenerated lumbar nucleus pulposus were separated from adolescent with scoliosis,degenerated nucleus pulposus were separated from lumbar disc degeneration and then were performed by SA-β-Gal staining.Nucleus pulposus cells were isolated from adult male rats(weighing 200-250g)and cultured in vitro.Cells were randomized to3 groups,control cells were maintained with 10% of FBS,low-concentration cells were maintained with 5% of FBS,no-concentration cells were maintained with 1% of FBS.After 72 hours,cells were performed by SA-β-Gal staining,observed changes in cell morphology and total NP cells and SA-β-Gal-positive cells were counted.The p53,p21,HIF-1α mRNA and their protein was detected by qPCR and western blot.Western blot was qualitative analysis and qPCR was quantitative analysis.Results:We found that cells were blue-stained in both non-degenerated nucleus pulposus and degenerated nucleus pulposus,but there were significant differences.Cells in non-degenerated nucleus pulposus were lightly stained,and the morphology of cells was slightly round,not very regular,and the contour was relatively clear.Cells in degenerated nucleus pulposus were significantly stained,and the morphology of cells was more regular,round,and clearly outlined.Control,low-concentration and no-concentration had SA-β-Gal-positive cells,which were assessed under a microscope for the development of blue color.But the number of blue-stained cells was significantly different,with statistical significance(F = 30.023,P < 0.01).And the multiple comparisons showed,there was no significant difference between the control group and the low concentration group,but there was significant difference between the low concentration group and the no concentration group(P < 0.01),there was significant difference between the control group and the no concentration group(P < 0.01).Western blot qualitative analysis showed that proteins of p53,p21,and HIF-1α were expressed in the cells of the three groups,but there were significant differences in the expression contents among the three groups.qPCR results were consistent with WB results.Multiple comparisons showed that the expression of p21 was statistically significant in the control and low-concentration(P < 0.05),and there was a significant difference between the control and no-concentration(P < 0.01),while there was a significant difference between the low-concentration group and the no-concentration(P < 0.05).The expression of p53 was statistically significant in the control and low-concentration(P < 0.01),and there was a significant difference between the control and no-concentration(P<0.01),while there was no significant difference between the low-concentration and the no-concentration.HIF-1 was statistically significant in the control and low-concentration(P < 0.05),statistically significant in the control and no-concentration(P < 0.01),and not statistically significant in the low-concentration and no-concentration.Conclusion:Serum starvation can induce NPCs senescence,and serum starvation is one of the factors of NPCs senescence,both normal NPCs and senescence NPCs have senescence gene expression(p53,p21),but the expression of senescence NPCs is higher than the normal NPCs,HIF-1α is expressed in both normal NPCs and senescence NPCs,but the expression of senescence NPCs is lower than the normal NPCs,both non-degenerated nucleus pulposus and degenerated nucleus pulposus have SA-β-Gal-positive cells,but degenerated nucleus pulposus shows darker staining.