Mechanism of SIRT3/MnSOD/ROS in mediating osteogenic differentiation of adipose derived stem cell in rats

Lü Yuanhang

doi:10.13406/j.cnki.cyxb.002809

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Mechanism of SIRT3/MnSOD/ROS in mediating osteogenic differentiation of adipose derived stem cell in rats

更新时间：2024-01-09

- Mechanism of SIRT3/MnSOD/ROS in mediating osteogenic differentiation of adipose derived stem cell in rats
- Journal of Chongqing Medical University Vol. 46, Issue 8, Pages: 875-881(2021)
- 作者机构：
  
  重庆医科大学附属口腔医院修复科、口腔疾病与生物医学重庆市重点实验室、重庆市高校市级口腔生物医学工程重点实验室,重庆,401147
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.002809
  CLC： Q254
- Published：2021，
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Lü Yuanhang. Mechanism of SIRT3/MnSOD/ROS in mediating osteogenic differentiation of adipose derived stem cell in rats. [J]. Journal of Chongqing Medical University 46(8):875-881(2021)
DOI：

Lü Yuanhang. Mechanism of SIRT3/MnSOD/ROS in mediating osteogenic differentiation of adipose derived stem cell in rats. [J]. Journal of Chongqing Medical University 46(8):875-881(2021) DOI： 10.13406/j.cnki.cyxb.002809.

摘要

目的：研究脂肪源性干细胞成骨分化过程中，SIRT3/MnSOD/ROS信号通路的调控作用。方法：提取SD大鼠脂肪源性干细胞（adipose derived stem cell，ADSC）并通过细胞表面标志物（CD29、CD44、CD34、CD45）及成骨、成脂多向分化鉴定。体外诱导其成骨分化，检测SIRT3表达情况；利用慢病毒转染抑制SIRT3表达，用茜素红染色检测ADSC成骨分化情况，qPCR检测成骨相关基因Runx2、ALP、OCN的表达，同时利用试剂盒检测锰超氧化物歧化酶（manganese superoxide dismutase，MnSOD）的活性及胞内活性氧簇（reactive oxygen species，ROS）水平。结果： ADSC成骨分化过程中，细胞内SIRT3 mRNA表达（7 d：1.71±0.03，15.21±0.17，P=0.014，n=5；14 d：1.44±0.04，9.70±0.18，P=0.002，n=5）及蛋白质表达（7 d：0.70±0.01，2.99±0.07，P=0.012，n=5；14 d：1.00±0.08，2.74±0.50，P=0.009，n=5）增加。转染SIRT3慢病毒可抑制SIRT3 mRNA表达（7 d：1.02±0.05，0.36±0.01，P=0.011，n=5；14 d：1.04±0.08，0.30±0.02，P=0.002，n=5）及蛋白质表达（7 d：0.88±0.04，0.29±0.01，P=0.034，n=5；14 d：1.18±0.03，0.43±0.01，P=0.014，n=5），导致ADSC成骨分化能力降低，MnSOD活性减弱（0.91±0.13，0.29±0.01，P=0.002，n=5），细胞内ROS水平升高（7 d：1.00±0.10，2.66±0.32，P=0.013，n=5；14 d：1.24±0.10，2.59±0.31，P=0.014，n=5），与成骨相关的相应靶基因Runx2、ALP、OCN表达下降（Runx2：1.07±0.02，0.19±0.01，P=0.001，n=5；ALP：1.09±0.02，0.53±0.01，P=0.001，n=5；OCN：0.95±0.02，0.33±0.01，P=0.004，n=5）。加入NAC预处理细胞，可逆转敲减SIRT3对ADSC成骨分化的抑制作用（ROS：2.63±0.20，1.07±0.19，P=0.010，n=5）。结论： ADSC成骨分化过程中，线粒体内SIRT3表达增加。SIRT3通过增强MnSOD的活性来降低胞内ROS水平，从而介导ADSC成骨分化。

Abstract

Objective: To study the regulatory effect of SIRT3/MnSOD/ROS signaling pathway on osteogenic differentiation of adipose derived stem cells (ADSC). Methods: In this study

we first cultured ADSC isolated from rat inguinal fat pads

and ADSC were identified by surface markers (CD29

CD44

CD34

CD45) and they could be induced to osteogenic and adipogenic differentiation. During osteogenic differentiation induction in vitro

expressions of SIRT3 were studied by western blotting. We used Lenti-SIRT3shRNA transfection to knockdown SIRT3 within ADSC

osteogenic capacities were identified by ARS staining

osteogenic differentiation-related genes Runx2

ALP

OCN mRNA expressions were assessed by qPCR

and the activity of MnSOD and reactive oxygen species (ROS) levels within ADSC were tested using kits. Results: The expression of SIRT3 mRNA (7 d: 1.71±0.03

15.21±0.17

P=0.014

n=5; 14 d: 1.44±0.04

9.70±0.18

P=0.002

n=5) and protein (7 d: 0.70±0.01

2.99±0.07

P=0.012

n=5; 14 d: 1.00±0.08

2.74±0.50

P=0.009

n=5) increased during osteogenic differentiation of ADSC. Lenti-SIRT3 shRNA transfection could inhibit SIRT3 mRNA expression (7 d: 1.02±0.05

0.36±0.01

P=0.011

n=5; 14 d: 1.04±0.08

0.30±0.02

P=0.002

n=5) and protein expression (7 d: 0.88±0.04

0.29±0.01

P=0.034

n=5; 14 d: 1.18±0.03

0.43±0.01

P=0.014

n=5)

with decreased MnSOD activities (0.91±0.13

0.29±0.01

P=0.002

n=5) and increased ROS levels (7 d: 1.00±0.10

2.66±0.32

P=0.013

n=5; 14 d: 1.24±0.10

2.59±0.31

P=0.014

n=5). The expression of corresponding target genes Runx2

ALP and OCN related to osteogenesis decreased (Runx2: 1.07±0.02

0.19±0.01

P=0.001

n=5; ALP: 1.09±0.02

0.53±0.01

P=0.001

n=5; OCN: 0.95±0.02

0.33±0.01

P=0.004

n=5). Inhibition of osteogenic differentiation

induced by knockdown of SIRT3

could be reversed by pretreatment of NAC (ROS: 2.63±0.20

1.07±0.19

P=0.010

n=5). Conclusion: SIRT3 within mitochondria plays vital roles in promoting osteogenic differentiation of ADSC

by mediating enhancement of MnSOD activities and reduction of intracellular ROS levels.

关键词

脂肪源性干细胞成骨分化SIRT3活性氧簇

Keywords

adipose derived stem cellosteogenic differentiationSIRT3reactive oxygen species

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