| Objective: Tumor necrosis factor-α(TNF-α)has been widely known to induce degeneration of nucleus pulposus cells(NPCs).17β-estradiol h as been broadly proven for its function of suppressing cell apoptosis.PI3K/AKT pathway could inhibit apoptosis of cell.The aim of this studyi s to explore whether 17β-E2 could protects apoptosis of human NPCs i nduced by TNF-α via the PI3K/AKT pathway.Material/Methods: Human NPCs were cultured in the laboratory.NPCs were divided into four groups: Group Ⅰ: control,Group Ⅱ: TNF-α(100 ng/ml),Group Ⅲ: TNF-α(100 ng/ml)with pretreated 17β-E2(10 μm/L),Group Ⅳ:TNF-α(100 ng/ml)with pretreated 17β-E2(10 μm/L)and MK2206(10 μm/L,inhibitor of the PI3K/AKT pathway).Every group has six samples.Apoptosis of NPCs were assessed by morphology,protein and nucleic acid levels.1Inverted phase-contrast microscopywas used to accomplish the morphological observation for apoptosis of treated cells.2 Flow cytometry was used to measure the apoptotic through quantitative analysis and sorting technology.3Cell Counting Kit 8(CCK-8)assay was used to detected cell proliferation.4Caspase-3 activity was used to evaluate activity for caspase-3 protein.5Western blot were applied to explore the expression of caspase-3/P17,pro-caspase-3,cleaved PARP,PARP,Akt and p-Akt.6 quantitative real-time PCR(qRT-PCR)were applied to explore the expression of caspase-3/p17,PARP and Akt.Results:1 The NPCs presented polygonal with clear outline.NPCs began to adhere to the bottom of culture flask in 6 or 8 hours and enter the log arithmic growth phase in 7 or 8 days.2 Results of Inverted phase-contrast microscopy showed that few ap optotic incidences for NPCs were observed in Group Ⅰ.Compared to Gr oup Ⅰ,TNF-α(100 ng/ml)induced marked apoptosis in Group Ⅱ,which was protected by 17β-E2(10 μm/L)in Group Ⅲ.However,the anti-ap optotic effect of 17β-E2 was inhibited by MK2206(inhibitor of PI3K/A KT pathway)in Group Ⅳ.3 FACS analysis presented that the incidence of early apoptosis of NPCs was 12.05%±2.27% in Group Ⅰ,the incidence was 30.8%±8.36%in Group Ⅱ,the incidence was 13.81%±1.97% in Group Ⅲ,the inciden ce was 30.01%±7.33% in Group Ⅳ,there were significant difference be tween Group Ⅰ and Group Ⅱ,Group Ⅱ and Group Ⅲ,Group Ⅲand Group Ⅳ(P<0.001).4 Cell Counting Kit 8(CCK-8)assay revealed that OD value for p roliferative activity was 90.32%±3.25% in Group Ⅰ,OD value was 37.78%±3.28% in Group Ⅱ,OD value was 88.67%±2.87% in Group Ⅲ,OD value was 38.97%±3.49% in Group Ⅳ,there were significant differenc e between Group Ⅰ and Group Ⅱ,Group Ⅱ and Group Ⅲ,GroupⅢ and Group Ⅳ(P<0.001).5 Caspase-3 activity,a crucial mediator of apoptosis,the caspase-3activity in Group Ⅱ,Ⅲ and Ⅳ were promoted to 3.9,1.1,3.7 times re spectively,compared to Group Ⅰ.There were significant difference betwe en Group Ⅰ and Group Ⅱ,Group Ⅱ and Group Ⅲ,Group Ⅲ and Grou p Ⅳ(P<0.001).6 As shown in result of Western blotting demonstrated that grey va lue for caspase-3/p17(/GAPDH)was 0.13±0.01,0.39±0.01,0.12±0.01,0.38±0.01 respectively in four groups,grey value for cleaved PARP(/GA PDH)was 0.13±0.01,0.33±0.01,0.14±0.01,0.32±0.01 respectively in fo ur groups,grey value for pro-caspase-3(/GAPDH)was 0.40±0.01,0.11±0.01,0.38±0.01,0.12±0.01 respectively in four groups,grey value for P ARP(/GAPDH)was 0.35±0.02,0.16±0.01,0.35±0.01,0.15±0.01 respecti vely in four groups,grey value for p-Akt(/GAPDH)was 0.14±0.02,0.30±0.01,0.43±0.02,0.13±0.02 respectively in four groups.There were sig nificant difference for above proteins between Group Ⅰ and Group Ⅱ,Gr oup Ⅱ and Group Ⅲ,Group Ⅲ and Group Ⅳ(P<0.001).However,gre y value for Akt(/GAPDH)was 0.49±0.02,0.48±0.01,0.49±0.02,0.48±0.01 respectively in four groups,there was no significant difference f-or g rey value of Akt in four groups.Figure 5d showed that TNF-α upregula ted p-Akt from 0 to 24 hours,while downregulated p-Akt from 24 to 48hours(0.10±0.01,0.18±0.01,0.33±0.02,0.18±0.01,0.10±0.01),there we re significant difference for grey value of p-Akt between 0 hour and 12 hours,12 hour and 24 hours,24 hour and 36 hours,36 hour and 48hours(all P<0.001).However,there was no significant difference for Akt in five time points(0.48±0.01,0.48±0.01,0.49±0.02,0.48±0.02,0.49±0.01,all P>0.05).Addtionally,17β-E2 upregulated p-Akt in a time-dependent manner(0.06±0.01,0.15±0.01,0.25±0.02,0.33±0.02,0.44±0.01,P<0.001),there were significant difference for grey value of p-Akt between 0 hour and 12 hours,12 hour and 24 hours,24 hour and 36 hours,36 hour and 48 hours.However,there was no significant differen ce for Akt in five time points(0.44±0.03,0.44±0.03,0.45±0.02,0.46±0.04,0.47±0.05,P>0.05).7 The relative ct value(/control group)for caspase-3/p17 was 2.45±0.23,0.98±0.15,2.50±0.13,the relative ct value(/control group)for P ARP was 0.30±0.03,0.95±0.03,0.27±0.04,there were siginificant differe nce for above mRNA between Group Ⅱ and Group Ⅲ,Group Ⅲ and Group Ⅳ(P<0.001).Whereas,mRNA levels of Akt,0.96±0.02,0.99±0.02,0.97±0.02,in remained obviously unchanged(P>0.05).Conclusions: TNF-a could induce apotosis of NPCs,but 17β-E2 iss hown to protect against apoptosis via the PI3K/AKT pathway. |
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