N-acetyl cysteine inhibits H2O2-mediated reduction in the mineralization of MC3T3-E1 cells by down-regulating Nrf2/HO-1 pathway

Abstract

There are controversial findings regarding the roles of nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway on bone metabolism under oxidative stress. We investigated how Nrf2/HO-1 pathway affects osteoblast differentiation of MC3T3-E1 cells in response to hydrogen peroxide (H2O2), N-acetyl cysteine (NAC), or both. Exposing the cells to H2O2 decreased the alkaline phosphatase activity, calcium accumulation, and expression of osteoblast markers, such as osteocalcin and runt-related transcription factor-2. In contrast, H2O2 treatment increased the expression of Nrf2 and HO-1 in the cells. Treatment with hemin, a chemical HO-1 inducer, mimicked the inhibitory effect of H2O2 on osteoblast differentiation by increasing the HO-1 expression and decreasing the osteogenic marker genes. Pretreatment with NAC restored all changes induced by H2O2 to near normal levels in the cells. Collectively, our findings suggest that H2O2-mediated activation of Nrf2/HO-1 pathway negatively regulates the osteoblast differentiation, which is inhibited by NAC.

Fig. 1.. H₂O₂ inhibits the ALP activity, calcium accumulation, and viability in a dose-dependent manner. Cells were exposed to increasing concentrations (0–400 μM) of H₂O₂. (A) ALP activity and calcium content, and (B) viability of the cells, were measured after 7 days of exposure. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the control values without H₂O₂.

Fig. 2.. H₂O₂ upregulates the induction of Nrf2 and HO-1 with attendant corresponding reduction of Runx2 in MC3T3-E1 cells. Cells were exposed to the indicated doses of H₂O₂. The HO-1 protein (A) and mRNA levels (B), and the nuclear protein levels of Nrf2 and Runx2 (C) were determined after 24 h of incubation by immunoblotting or RT-PCR. (D) Relative intensities of Nrf2 and Runx2 proteins from triplicate experiments are shown. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the control values without H₂O₂.

Fig. 3.. NAC prevents the H₂O₂-mediated decreases in OCN expression, ALP activity, and calcium accumulation, by restoring the levels of Nrf2, HO-1, and Runx2 to those of non-treated control values. Cells were incubated with 200 μM H₂O₂ in the presence and absence of 2.5 mM or 5 mM NAC. (A, B) Protein levels of HO-1, Nrf2, and Runx2 were measured by immunoblotting after 24 h of incubation. (C) OCN mRNA expression, (D) ALP activity, and (E) calcium content, were also determined after 7 days of incubation. **P < 0.01 and ***P < 0.001 vs. the control values without H₂O₂. ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. 200 μM H₂O₂ treatment only.

Fig. 4.. The increase of HO-1 induction by hemin reduces the expression of osteogenic markers and mineralization of MC3T3-E1 cells. Cells were pretreated with the indicated concentrations of hemin in osteogenic medium for 24 h; the medium was replaced with fresh medium without hemin, followed by additional incubation for various times. Mineralization of the cells was evaluated by alizarin red staining (A) and ALP activity assay (B) at 7 days of incubation, or by calcium content determination (C) at 14 day post-incubation. The mRNA expressions of HO-1 (D), Runx2 (E), and OCN (F), and the nuclear level of Runx2 (G), were determined after 24 h of incubation by real time RT-PCR and Western blot analyses, respectively. Panel H shows viability of the cells exposed to 20 μM and 50 μM hemin for 1 or 3 days in osteogenic medium. (I) A proposed mechanism involved in the H₂O₂-mediated reduction of osteoblast differentiation, and its inhibition by NAC. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the control cells without hemin.