HUANG Jiahui 1,2 , CHEN Long 1,2 , XU Chen 1,2 , YU Haojie 1,2 , ZHOU Shishuai 1,2 , GUAN Jianzhong 1,2
  • 1. Department of Orthopaedics, the First Affiliated Hospital of Bengbu Medical College, Bengbu Anhui, 233000, P. R. China;
  • 2. Key Laboratory of Anhui Province for Tissue Transplantation, Bengbu Anhui, 233000, P. R. China;
GUAN Jianzhong, Email: Guanjianzhong@bbmc.edu.cn
ω-3 polyunsaturated fatty acids; nuclear factor E2-related factor 2; NAD (P) H: quinone oxidoreductase 1; oxidative stress; MC3T3-E1
10.7507/1002-1892.202506037
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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 high and low 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 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 was 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 expression 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.

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