Zhou Lu, Wang Sung Il, Moon Young Jae, Kim Kyoung Min, Lee Kwang Bok, Park Byung-Hyun, Jang Kyu Yun, Kim Jung Ryul
Department of Sports Medicine, Taishan Medical University, Taian, Shandong 271000, P.R. China.
Department of Orthopaedic Surgery, Chonbuk National University Medical School, Research Institute of Clinical Medicine of Chonbuk National University, Biomedical Research Institute of Chonbuk National University Hospital and Research Institute for Endocrine Sciences, Jeonju 54896, Republic of Korea.
Mol Med Rep. 2017 Sep;16(3):2969-2975. doi: 10.3892/mmr.2017.6917. Epub 2017 Jul 4.
Hypoxic‑ischemic injury of the bone results in osteonecrosis. Nicotinamide adenosine dinucleotide (NAD)‑dependent deacetylase sirtuin‑1 (SIRT1), a type of NAD‑dependent deacetylase, is involved in multiple biological functions, particularly in anti‑apoptosis. However, the effects of SIRT1 in osteoblasts remain unclear and whether SIRT1 protects osteoblasts in hypoxic conditions remains to be elucidated. In the present study, the role of SIRT1 in the osteoblast cells under hypoxia and the underlying mechanism of the anti‑apoptotic activity of SIRT1 were investigated. MC3T3‑E1 osteoblast cells were used for the present study and oxygen‑absorbing packs were used to induce cell hypoxia and apoptosis. MC3T3‑E1 cells were overexpressed SIRT1 by transfection with a SIRT1 adenovirus. The small interfering RNA of SIRT1 to was used to transfect cells to decrease the protein level. An MTT assay was used to estimate cell viability. Apoptosis was examined with the APOPercentage apoptosis assay kit and the activity of caspases was measured by a caspase 3 and 7 activity kit. Co‑immunoprecipitation was used to investigate protein binding ability. The mRNA and protein expression levels were quantified with reverse transcription‑quantitative polymerase chain reaction and immunoblotting. It was demonstrated that the expression of SIRT1 mRNA and protein were elevated, and peaked at 12 h under hypoxic conditions. The data demonstrated that SIRT1 overexpression in cells significantly increased cell viability and markedly decreased the percentage of apoptosis compared with the control and knockdown groups. Furthermore, overexpression of SIRT1 significantly activated anti‑apoptotic effects by deacetylating lysine residue binding to protein kinase B and decreasing the activity of caspases 3, 9 and subsequent pathways. The results from the present study suggested that SIRT1 may serve a protective function in hypoxia‑induced apoptosis in MC3T3‑E1 cells, and that SIRT1 intervention may potentially aid in the treatment of ischemic bone disease.
骨的缺氧缺血性损伤会导致骨坏死。烟酰胺腺嘌呤二核苷酸（NAD）依赖性脱乙酰酶沉默调节蛋白1（SIRT1），作为一种NAD依赖性脱乙酰酶，参与多种生物学功能，尤其在抗凋亡方面。然而，SIRT1在成骨细胞中的作用仍不清楚，且SIRT1在缺氧条件下是否能保护成骨细胞仍有待阐明。在本研究中，探究了SIRT1在缺氧条件下对成骨细胞的作用以及SIRT1抗凋亡活性的潜在机制。本研究采用MC3T3-E1成骨细胞，使用吸氧包诱导细胞缺氧和凋亡。通过用SIRT1腺病毒转染使MC3T3-E1细胞过表达SIRT1。使用SIRT1的小干扰RNA转染细胞以降低蛋白水平。采用MTT法评估细胞活力。使用APOPercentage凋亡检测试剂盒检测凋亡情况，并用caspase 3和7活性试剂盒测量caspase的活性。采用免疫共沉淀法研究蛋白结合能力。用逆转录定量聚合酶链反应和免疫印迹法定量mRNA和蛋白表达水平。结果表明，缺氧条件下SIRT1 mRNA和蛋白表达升高，并在12小时达到峰值。数据表明，与对照组和敲低组相比，细胞中SIRT1过表达显著提高了细胞活力，并显著降低了凋亡百分比。此外，SIRT1的过表达通过使与蛋白激酶B结合的赖氨酸残基去乙酰化并降低caspase 3、9及后续途径的活性，显著激活了抗凋亡作用。本研究结果表明，SIRT1可能在MC3T3-E1细胞缺氧诱导的凋亡中发挥保护作用，且SIRT1干预可能有助于缺血性骨病的治疗。
