SIRT1 protects against apoptosis by promoting autophagy in degenerative human disc nucleus pulposus cells

Abstract

SIRT1 could protect degenerative human NP cells against apoptosis, and there were extensive and intimate connection between apoptosis and autophagy. Up to now, the role of autophagy in the process of human IVD degeneration is unclear. We sought to explore the relationship between autophagy and human IVD degeneration and to understand whether autophagy is involved in the protective effect of SIRT1 against apoptosis in NP cells. Our results showed that the autophagosomes number, the mRNA level of LC3 and Beclin-1, the protein expression of LC3-II/I and Beclin-1, decreased in NP from DDD. Resveratrol could increase the protein expression of LC3-II/I and Beclin-1, and reduce apoptosis in degenerative NP cells. In contrast, the protein levels of LC3-II/I and Beclin-1 were down-regulated and apoptosis level was significantly up-regulated in treatment with nicotinamide or SIRT1-siRNA transfection. Further analysis identified that the expression of cleaved Caspase3 and apoptosis incidence significantly increased with the pretreatment of bafilomycin A, whether resveratrol was added or not. These suggested that autophagy may play an important role in IVD degeneration, and SIRT1 protected degenerative human NP cells against apoptosis via promoting autophagy. These findings would aid in the development of novel therapeutic approaches for degenerative disc disease treatment.

Figure 1. Autophagosomes were detected by TEM in NP cells from patients with LVF and DDD.

(A) Autophagosomes were detected by TEM (30,000×) in NP cells from LVF (black arrow). Double-limiting membrane could be observed in some autophagosomes. (B) One autophagosome was detected by TEM (50,000×) in NP cells from DDD (black arrow).

Figure 2. Real-time PCR analysis for SIRT1, LC3 and Beclin-1 gene expression in NP from patients with LVF and DDD.

The mRNA levels of SIRT1, LC3 and Beclin-1 are decreased in NPs from patients with DDD (n = 3, *P<0.05).

Figure 3. Western blot analysis for SIRT1, LC3II/I and Beclin-1 expression in NPs from patients with LVF and DDD.

The protein levels of SIRT1, LC3II/I and Beclin-1 are decreased in NPs from patients with DDD (n = 3, *P<0.05).

Figure 4. Immunohistochemistry analysis of SIRT1, Caspase3 and Collegan II expression in human NP from patients with LVF and DDD.

(A) SIRT1 levels are decreased in NPs from patients with DDD (n = 3, 200×, *P<0.05). (B) Caspase3 levels are elevated in NPs from patients with DDD (n = 3, 200×, *P<0.05). (C) Collegan II levels are decreased in NPs from patients with DDD (n = 3, 200×, *P<0.05). (D) Apoptotic cells in the NPs from LVF and DDD were detected by TUNEL. The percentages of apoptotic cells are elevated in NPs from patients with DDD (n = 3, 400×, *P<0.05).

Figure 5. SIRT1 enhances autophagy and inhibits apoptosis in degenerative human NP cells.

(A) SIRT1 protein levels were detected by western blot. SIRT1 expression was significantly elevated in degenerative human NP cells which were treated with resveratrol (8 μM) for 48 h. Conversely, SIRT1 expression was reduced in NP cells treated with nicotinamide (12 μM) or SIRT1-siRNA for 48 h. Protein expression levels are normalized against β-actin (*P<0.05 versus control). (B) Autophagy related-protein expressions were assessed by Western blot. Autophagy related-protein expression was significantly elevated in degenerative human NP cells which were treated with resveratrol (8 μM) for 48 h. Conversely, autophagy related-protein expression was reduced in NP cells treated with nicotinamide (12 μM) or SIRT1-siRNA for 48 h. Protein expression levels are normalized against β-actin (*P<0.05 versus control). (C) The apoptosis incidence of degenerative human NP cells evaluated by flow cytometry. The apoptosis incidence decreased significantly in NP cells treated with resveratrol (8 μM) and increased significantly in NP cells treated with nicotinamide (12 μM) or SIRT1-siRNA for 48 h (*P<0.05 versus control).

Figure 6. SIRT1 inhibits apoptosis of degenerative human NP cells by promoting autophagy.

(A) LC3-II/I and cleaved caspase3 protein detected by western blot. LC3-II/I enhanced in human degenerative NP cells treated with bafilomycin A (100 nM) in combination with resveratrol (8 μM) or not. The levels of cleaved caspase3 decreased in NP cells treated with resveratrol (8 μM) and increased in NP cells treated with bafilomycin A (100 nM) in combination with resveratrol (8 μM) or not (*P<0.05 versus control, #P<0.05 versus resveratrol). (B) The apoptosis incidence of degenerative human NP cells evaluated by flow cytometry. The apoptosis incidence increased significantly in NP cells with bafilomycin A (100 nM) treatment independently whether resveratrol (8 μM) was added or not (*P<0.05 versus control, #P<0.05 versus resveratrol).

Figure 7. Representative MRI of patients.

(A) Patient with lumbar LVF undergoing posterior discectomy, spinal fusion, decompression, and stability within 24 h of trauma, without formerly documented clinical history of LBP. The red arrow indicates the experimental material position. The L1 vertebrae is fractured and dislocated, and the disc of T12/L1 is classified as Grade I according to the Pfirrmann classification. (B) Patient with DDD undergoing discectomy and intervertebral fusion surgery. The red arrow indicates the experimental material position. The disc of L4/5 is herniated and classified as Grade IV according to the Pfirrmann classification.