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.