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

Jiang W, Zhang X, Hao J, Shen J, Fang J, Dong W, Wang D, Zhang X, Shui W, Luo Y, Lin L, Qiu Q, Liu B, Hu Z - Sci Rep (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, the First Affiliated Hospital of Chongqing Medical University, Chongqing, China.

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.

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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).
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f5: 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).

Mentions: Resveratrol (8 μM) or nicotinamide (12 mM) was used as activator or inhibitor of SIRT1 to treat degenerative human NP cells. Meanwhile, endogenous SIRT1 of degenerative human NP cells was silenced by siRNA transfection. Western blotting was used to measure the effects of up- or down-regulation of SIRT1.Western blotting results showed that the level of SIRT1 protein expression was increased after treatment with resveratrol, and it was reduced after treatment with nicotinamide or siRNA transfection for SIRT1. Moreover, the level of SIRT1 protein expression was reduced more obviously after siRNA transfection for SIRT1 compared with that after treatment with nicotinamide (Figure 5A).


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

Jiang W, Zhang X, Hao J, Shen J, Fang J, Dong W, Wang D, Zhang X, Shui W, Luo Y, Lin L, Qiu Q, Liu B, Hu Z - Sci Rep (2014)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4264007&req=5

f5: 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).
Mentions: Resveratrol (8 μM) or nicotinamide (12 mM) was used as activator or inhibitor of SIRT1 to treat degenerative human NP cells. Meanwhile, endogenous SIRT1 of degenerative human NP cells was silenced by siRNA transfection. Western blotting was used to measure the effects of up- or down-regulation of SIRT1.Western blotting results showed that the level of SIRT1 protein expression was increased after treatment with resveratrol, and it was reduced after treatment with nicotinamide or siRNA transfection for SIRT1. Moreover, the level of SIRT1 protein expression was reduced more obviously after siRNA transfection for SIRT1 compared with that after treatment with nicotinamide (Figure 5A).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, the First Affiliated Hospital of Chongqing Medical University, Chongqing, China.

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.

Show MeSH
Related in: MedlinePlus