Recombinant human SIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human intervertebral disc nucleus pulposus cells

Shingo Miyazaki, Kenichiro Kakutani, Takashi Yurube, Koichiro Maeno, Toru Takada, Zhongying Zhang, Takuto Kurakawa, Yoshiki Terashima, Masaaki Ito, Takeshi Ueha, Takehiko Matsushita, Ryosuke Kuroda, Masahiro Kurosaka, Kotaro Nishida

Nutrient deprivation is a likely contributor to intervertebral disc (IVD) degeneration. Silent mating type information regulator 2 homolog 1 (SIRT1) protects cells against limited nutrition by modulation of apoptosis and autophagy. However, little evidence exists regarding the extent to which SIRT1 affects IVD cells. Therefore, we conducted an in vitro study using human IVD nucleus pulposus (NP) cells. Thirty-two IVD specimens were obtained from patients who underwent surgical intervention and were categorized based on Pfirrmann IVD degeneration grades. Cells were isolated from the NP and cultured in the presence of recombinant human SIRT1 (rhSIRT1) under different serum conditions, including 10 % (v/v) fetal bovine serum (FBS) as normal nutrition (N) and 1 % (v/v) FBS as low nutrition (LN). 3-Methyladenine (3-MA) was used to inhibit autophagy. Autophagic activity was assessed by measuring the absorbance of monodansylcadaverine and immunostaining and Western blotting for light chain 3 and p62/SQSTM1. Apoptosis and pathway analyses were performed by flow cytometry and Western blotting. Cells cultured under LN conditions decreased in number and exhibited enhanced autophagy compared with the N condition. Medium supplementation with rhSIRT1 inhibited this decrease in cell number and induced an additional increase in autophagic activity (P < 0.05), whereas the combined use of rhSIRT1 and 3-MA resulted in drastic decreases in cell number and autophagy (P < 0.05). The incidence of apoptotic cell death increased under the LN condition, which was decreased by rhSIRT1 (P < 0.05) but increased further by a combination of rhSIRT1 and 3-MA (P < 0.05). Under LN conditions, NP cells showed a decrease in antiapoptotic Bcl-2 and an increase in proapoptotic Bax, cleaved caspase 3, and cleaved caspase 9, indicating apoptosis induction via the mitochondrial pathway. These changes were suppressed by rhSIRT1 but elevated further by rhSIRT1 with 3-MA, suggesting an effect of rhSIRT1-induced autophagy on apoptosis inhibition. Furthermore, the observed autophagy and apoptosis were more remarkable in cells from IVDs of Pfirrmann grade IV than in those from IVDs of Pfirrmann grade II. SIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human IVD NP cells, suggesting that rhSIRT1 may be a potent treatment agent for human degenerative IVD disease.