Influence of transforming growth factor-β1 on nucleus pulposus cell apoptosis

English Keywords:Transforming growth factor-β1(TGF-β1)  Drosophila mothers against decapentaplegic protein/ Runt-related transcription factor 2(Smad/Runx2)  Tumor necrosis factor-α(TNF-α)  Nucleus pulposus cells  Apoptosis

English Abstract:

  【Abstract】 Objectives: To explore the effect of TGF-β1 on nucleus pulposus cell apoptosis and the potential mechanisms. Methods: Nucleus pulposus cells(NPCs) were isolated from Sprague Dawley rats and cultured by the sequential enzyme method. Cells at passage 3 were divided into six groups: group A, cultured in DMEM with no other treatment; group B, cultured in DMEM with 20ng/ml TNF-α; group C, cultured in DMEM with 50ng/ml TNF-α; group D, cultured in DMEM with 100ng/ml TNF-α; group E, cultured in DMEM with 100ng/ml TNF-α and 10ng/ml TGF-β1; group F, cultured in DMEM with 100ng/ml TNF-α, 10ng/ml TGF-β1 and 10μmol/L SB431542. The cell counting Kit-8(CCK-8) was used to detect the cell proliferation activity after culturing for 12h, transcription-polymerase chain reaction(RT-PCR) was reversed and Western blot were used to detect the expression of matrix metalloproteinases 3(MMP3), bcl-2-associated x protein(Bax), ACAN, Collagen Ⅱ, phosphorylate drosophila mothers against decapentaplegic protein 3(p-smad3) and runt-related transcription factor 2(Runx2). Results: Compared with group A, the different concentrations of TNF-α groups(group B-D) promoted the expressions of MMP3(group B-D respectively: 0.652+0.015, 0.899+0.018, 1.026+0.023 respectively) and Bax(0.725+0.058, 0.928+0.018, 1.138+0.019 respectively), and promoted apoptosis in a dose-dependent manner. Compared with group D, the expressions of MMP3(0.568+0.015) and Bax(0.626+0.024) were significantly inhibited in group E, while the expressions of ACAN(1.056+0.014), Collagen Ⅱ（1.098+0.032）, p-smad3 and Runx2 significantly increased, the difference was statistically significant(P<0.05). Compared with group E, the expressions of MMP3(1.015+0.015) and Bax(1.126+0.024) significantly increased in group F, while the expression of ACAN（0.314+0.023）, Collagen Ⅱ(0.299+0.0.19), p-smad3 and Runx2 were significantly inhibited, the difference was statistically significant(P<0.05). Conclusions: TGF-β1 can activate smad/Runx2 pathway and antagonize TNF-α induced nucleus pulposus cell apoptosis.