Recombinant human SIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human intervertebral disc nucleus pulposus cells.
Recombinant human SIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human intervertebral disc nucleus pulposus cells.
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DOI：
10.1186/s13075-015-0763-6
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发表时间：
2015-09-15
影响因子：
4.9
通讯作者：
Nishida K
中科院分区：
文献类型：
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作者：
Miyazaki S;Kakutani K;Yurube T;Maeno K;Takada T;Zhang Z;Kurakawa T;Terashima Y;Ito M;Ueha T;Matsushita T;Kuroda R;Kurosaka M;Nishida K
营养缺乏可能是导致椎间盘（IVD）退化的一个因素。沉默交配型信息调节器 2 同源物 1 (SIRT1) 通过调节细胞凋亡和自噬来保护细胞免受有限营养的影响。然而，关于 SIRT1 对 IVD 细胞影响程度的证据很少。因此，我们使用人类 IVD 髓核 (NP) 细胞进行了一项体外研究。 32 份 IVD 标本取自接受手术干预的患者，并根据 Pfirrmann IVD 变性等级进行分类。从 NP 中分离细胞，并在不同血清条件下在重组人 SIRT1 (rhSIRT1) 存在下培养，包括 10% (v/v) 胎牛血清 (FBS) 作为正常营养 (N) 和 1% (v/v) FBS 作为低营养 (LN)。 3-甲基腺嘌呤（3-MA）用于抑制自噬。通过测量单丹磺酰尸胺的吸光度以及轻链 3 和 p62/SQSTM1 的免疫染色和蛋白质印迹来评估自噬活性。通过流式细胞术和蛋白质印迹法进行细胞凋亡和途径分析。与 N 条件相比，LN 条件下培养的细胞数量减少，自噬增强。培养基中添加rhSIRT1可抑制细胞数量的减少，并诱导自噬活性进一步增加（P < 0.05），而rhSIRT1和3-MA的联合使用导致细胞数量和自噬急剧减少（P < 0.05）。 LN条件下细胞凋亡的发生率增加，rhSIRT1使凋亡细胞死亡发生率降低（P<0.05），但rhSIRT1和3-MA联合使用则进一步增加（P<0.05）。在 LN 条件下，NP 细胞显示抗凋亡 Bcl-2 减少，促凋亡 Bax、cleaved caspase 3 和 cleaved caspase 9 增加，表明通过线粒体途径诱导细胞凋亡。这些变化被rhSIRT1抑制，但被rhSIRT1和3-MA进一步升高，表明rhSIRT1诱导的自噬对细胞凋亡抑制有影响。此外，在 Pfirrmann IV 级 IVD 的细胞中观察到的自噬和凋亡比 Pfirrmann II 级 IVD 的细胞更显着。 SIRT1 通过在人类 IVD NP 细胞中诱导自噬来防止营养剥夺诱导的线粒体凋亡，这表明 rhSIRT1 可能是人类退行性 IVD 疾病的有效治疗剂。
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.