The effects of hypoxia and nutrition deprivation on cartilage endplate stem cells and the related mechanism

English Keywords:Cartilage endplate-derived stem cells  Hypoxia and nutrition deprivation  Apoptosis  BNIP3  Bax  Bak

English Abstract:

  【Abstract】 Objectives: To study the effects of hypoxia and nutrition deprivation on the apoptosis of cartilage endplate stem cells(CESCs) as well as the role of Bcl-2/adenovirus E1B 19-kDa-interacting protein 3(BNIP3) in the process. Methods: The CESCs were separated from the clinically acquired human intervertebral disc endplate cartilage tissue, and the third generation cells were cultured under normoxia with full medium(control group) or hypoxia with nutrition deprivation condition(experimental group) for 48h. Then, the cell apoptosis rate was detected by flow cytometry, the cell viability was detected by CCK-8, and the protein expressions of BNIP3, Bax, Bak and hypoxia-inducible factor-1(HIF-1α) were detected by Western Blot. Furthermore, BNIP3 small interfering RNA(siRNA) was used to knock down the BNIP3 expression in CESCs, and the interference-negative control group was treated with control siRNA. Thereafter, cells were treated as described previously and the apoptosis rate, cell viability and the expressions of BNIP3, Bax and Bak were detected again. Results: The CESCs obtained from 5 clinical specimens were identified by stem cell markers. The CD44, CD73, CD90 and CD105 were positive while the CD34, CD45, CD11b, CD19 and HLA-DR were negative, suggesting that the CESCs had the characteristics of stem cells. The apoptosis rate of the experimental group was (29.12±0.65)%, which was significantly higher than that of the control group(14.87±2.03)%(P<0.05). The cell viabilty in the experimental group was notably lower than that in the control group, which was about 56.18% of the control group(P<0.05). Compared with the control group, the expressions of BNIP3, Bax and Bak in the experimental group were significantly up-regulated(P<0.05). Knockdown of BNIP3 by RNA interference attenuated the increase of cell apoptosis and decrease of cell viability in CESCs caused by hypoxia and nutrition deprivation. Additionally, hypoxia and nutrition deprivation-induced expressions of BNIP3, Bax and Bak were also inhibited. Conclusions: Hypoxia and nutrition deprivation may induce CESCs apopotosis via up-regulating the expressions of BNIP3, Bax and Bak.