| Objective: Intervertebral disc degeneration refers to the tissues of disc occur aging and degeneration with age.It is the premise and foundation of many spinal degenerative diseases,such as spinal stenosis,intervertebral disc herniation and spinal instability.Although the exact mechanism of intervertebral disc degeneration is unclear,most scholars believe that the intervertebral disc degeneration is the outcome of multiple factors.Intervertebral disc cells reduction and extracellular matrix components change are pathophysiological basis of intervertebral disc degeneration,and the apoptosis of nucleus pulposus and annulus fibrosus(AF) cells is the main reason of intervertebral disc cells reduction.The study found that the degenerative intervertebral disc tissue can produce a large number of inflammatory cytokines.Interleukin-1β(IL-1β) is a pleiotropic cytokine that mediates inflammatory and cell death activities and plays an important role in intervertebral disc cells apoptosis.Clinical observation have shown that compared with matched males, female subjects have a higher incidence of intervertebral disc degeneration,so it indicates that estrogen may have a close association with intervertebral disc degeneration.In recent years,researchers have found that estrogen can protect pancreaticβ-cells and chondrocytes from apoptosis.Whether estrogen can protect intervertebral disc cells from apoptosis, however, has not been reported.The present study aims to examine whether 17β-estradiol(17 β-E2) inhibits IL-1 β-induced apoptosis of rat AF cells.Additionally, the dose-response effect of 17β-E2 on cell apoptosis was investigated.Methods: Rat AF cells were primarily cultured by methods of enzyme digestion, and subcultured to next generation by digestion of 0.25% collagenase type II and 0.2% typsin(including 0.02% EDTA) when cells were confluent. AF cells of the passage 3 were cultured in DMEM/F12 without fetal bovine serum(FBS) and phenol red.The experiment was divided into six groups:(1) Group 1,the control group;(2) Group 2,IL-1βinduction group;(3) Group 3,0.1μmol/L estrogen group;(4) Group 4,1μmol/L estrogen group;(5) Group 5,10 μ mol/L estrogen group;(6) Group 6,ICI182, 780 inhibitor group,and each group contains 6 samples.Cells were synchronized growth after cultured for 24 h and harvested after cultured with corresponding drugs for another 24 h.The apoptotic cells were observed under inverted microscope;cell apoptosis was determined by flow cytometry and Caspase-3 activity assay;cell proliferation activity was detected by MTT assay.Results:(1)Apoptotic AF cells were characterized by plasma membrane blebbing, cell shrinkage and nuclei condensing under inverted microscope.(2) AF cells apoptotic rate by flow cytometry revealed as follows:3.47%±0.25% in Group 1;18.80%±0.99% in Group 2;12.97%±0.61% in Group 3;18.67%±0.74% in Group 4;6.00%±0.61% in Group 5;18.57%±0.51% in Group 6,respectively.Data difference among Group 1,Group 2,Group 5 and Group 6 was statistically significant(F=237.21,P<0.001).The apoptotic rate in Group 5 was lower than that of Group 2(P<0.001) and Group 6(P<0.001). Difference of apoptotic rate between Group 2 and Group 6 was no statistically significance;Data difference among Group 1,Group 2,Group 3,Group 4 and Group 5 was statistically significant(F=474.78,P<0.001).Group 3,Group 4 and Group 5 apoptotic rate gradually decreased.Caspase-3 activity assay revealed as follows: Caspase-3 activity in Group 2 decreased obviously,Group 3,Group 4 and Group 5 Caspase-3 activity gradually increased,Caspase-3 activity in Group 2 decreased.(3)Cell proliferation activity showed that OD value was 71.31%±1.31% in Group 1;26.74%±1.85% in Group 2;42.13%±2.10% in Group 3;53.68%±2.71% in Group 4;62.89%±3.12% in Group 5 and 32.14%±2.25% in Group 6,respectively.Data difference among Group 1,Group 2,Group 5 and Group 6 was statistically significant(F=340.95,P<0.001).The cell proliferation activity in Group 5 was greater than that of Group 2(P<0.001) and Group 6(P<0.001). Difference of cell proliferation activity between Group 2 and Group 6 was no statistically significance;Data difference among Group 1,Group 2,Group 3,Group 4 and Group 5 was statistically significant(F=474.78,P<0.001).Group 3,Group 4 and Group 5 cell proliferation activity gradually increased.Conclusion: 17β-E2 inhibited IL-1β-induced apoptosis in rat AF cells, and pretreatment of cells with 17β-E2 could decrease AF cell apoptosis in a concentration-dependent manner. |
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