Objective To explore the mechanism by which ω-3 polyunsaturated fatty acids (hereinafter referred to as “ω-3”) exert antioxidant stress protection and promote osteogenic differentiation in MC3T3-E1 cells, and to reveal the relationship between ω-3 and the key antioxidant stress pathway involving nuclear factor E2-related factor 2 (Nrf2) and NAD (P) H quinone oxidoreductase 1 (NQO1) in MC3T3-E1 cells. Methods The optimal concentration of H2O2 (used to establish the oxidative stress model of MC3T3-E1 cells in vitro) and the optimal intervention concentrations of ω-3 were screened by cell counting kit 8. MC3T3-E1 cells were divided into blank control group, oxidative stress group (H2O2), low-dose ω-3 group (H2O2+low-dose ω-3), and high-dose ω-3 group (H2O2+high-dose ω-3). After osteoblastic differentiation for 7 or 14 days, the intracellular reactive oxygen species (ROS) level was measured by fluorescence staining and flow cytometry, and the mitochondrial morphological changes were observed by biological transmission electron microscope; the expression levels of Nrf2, NQO1, heme oxygenase 1 (HO-1), Mitofusin 1 (Mfn1), and Mfn2 were detected by Western blot to evaluate the cells’ antioxidant stress capacity; the expression levels of Runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) were detected by immunofluorescence staining and Western blot; osteogenic potential of MC3T3-E1 cells was evaluated by alkaline phosphatase (ALP) staining and alizarin red staining. Results Compared with the oxidative stress group, the content of ROS in the low and high dose ω-3 groups significantly decreased, and the protein expressions of Nrf2, NQO1, and HO-1 significantly increased (P<0.05). At the same time, the mitochondrial morphology of MC3T3-E1 cells improved, and the expressions of mitochondrial morphology-related proteins Mfn1 and Mfn2 significantly increased (P<0.05). ALP staining and alizarin red staining showed that the low-dose and high-dose ω-3 groups showed stronger osteogenic ability, and the expressions of osteogenesis-related proteins RUNX2 and OCN significantly increased (P<0.05). And the above results showed a dose-dependence in the two ω-3 treatment groups (P<0.05). Conclusion ω-3 can enhance the antioxidant capacity of MC3T3-E1 cells under oxidative stress conditions and upregulate their osteogenic activity, possibly through the Nrf2/NQO1 signaling pathway.
Citation：
HUANG Jiahui, CHEN Long, XU Chen, YU Haojie, ZHOU Shishuai, GUAN Jianzhong. Mechanism analysis of ω-3 polyunsaturated fatty acids in alleviating oxidative stress and promoting osteogenic differentiation of MC3T3-E1 cells through activating Nrf2/NQO1 pathway. Chinese Journal of Reparative and Reconstructive Surgery, 2025, 39(11): 1459-1467. doi: 10.7507/1002-1892.202506037
Copy
Copyright © the editorial department of Chinese Journal of Reparative and Reconstructive Surgery of West China Medical Publisher. All rights reserved
| 1. |
|
| 2. |
|
| 3. |
|
| 4. |
|
| 5. |
|
| 6. |
|
| 7. |
|
| 8. |
|
| 9. |
|
| 10. |
|
| 11. |
|
| 12. |
|
| 13. |
|
| 14. |
|
| 15. |
|
| 16. |
|
| 17. |
|
| 18. |
|
| 19. |
|
| 20. |
|
| 21. |
|
| 22. |
|
| 23. |
|
| 24. |
|
| 25. |
|
- 1.
- 2.
- 3.
- 4.
- 5.
- 6.
- 7.
- 8.
- 9.
- 10.
- 11.
- 12.
- 13.
- 14.
- 15.
- 16.
- 17.
- 18.
- 19.
- 20.
- 21.
- 22.
- 23.
- 24.
- 25.
