Moringa oleifera leaf extracts protect BMSC osteogenic induction following peroxidative damage by activating the PI3K/Akt/Foxo1 pathway

Abstract

Objective: We aimed to investigate the therapeutic effects of Moringa oleifera leaf extracts on osteogenic induction of rat bone marrow mesenchymal stem cells (BMSCs) following peroxidative damage and to explore the underlying mechanisms.

Methods: Conditioned medium was used to induce osteogenic differentiation of BMSCs, which were treated with H₂O₂, Moringa oleifera leaf extracts-containing serum, or the phosphatidyl inositol-3 kinase (PI3K) inhibitor wortmannin, alone or in combination. Cell viability was measured using the MTT assay. Cell cycle was assayed using flow cytometry. Expression levels of Akt, phosphorylated (p)Akt, Foxo1, and cleaved caspase-3 were analyzed using western blot analysis. The mRNA levels of osteogenesis-associated genes, including alkaline phosphatase (ALP), collagen І, osteopontin (OPN), and Runx2, were detected using qRT-PCR. Reactive oxygen species (ROS) and malondialdehyde (MDA) levels, as well as superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and ALP activity were detected using commercially available kits. Osteogenic differentiation capability was determined using alizarin red staining.

Results: During osteogenic induction of rat BMSCs, H₂O₂ reduced cell viability and proliferation, inhibited osteogenesis, increased ROS and MDA levels, and decreased SOD and GSH-PX activity. H₂O₂ significantly reduced pAkt and Foxo1 expression, and increased cleaved caspase-3 levels in BMSCs. Additional treatments with Moringa oleifera leaf extracts partially reversed the H₂O₂-induced changes. Wortmannin partially attenuated the effects of Moringa oleifera leaf extracts on protein expression of Foxo1, pAkt, and cleaved caspase-3, as well as mRNA levels of osteogenesis-associated genes.

Conclusion: Moringa oleifera leaf extracts ameliorate peroxidative damage and enhance osteogenic induction of rat BMSCs by activating the PI3K/Akt/Foxo1 pathway.

Keywords: Bone marrow mesenchymal stem cells; Foxo1; Hydrogen peroxide; Moringa oleifera leaf extracts; Osteogenic induction; PI3K/Akt pathway.

Fig. 1

Moringa oleifera attenuates H₂O₂-induced dysregulation of cell viability and cell cycle progression in BMSCs during osteogenic differentiation. a The effects of H₂O₂ at indicated concentrations and treatment times on cell viability in P3 cells. H₂O₂50, cells treated with 50 μmol/L H₂O₂; H₂O₂100, cells treated with 100 μmol/L H₂O₂; H₂O₂200, cells treated with 200 μmol/L H₂O₂. *P < 0.05 compared with the control group; ^#P < 0.05 compared with the H₂O₂50 group; ^@P < 0.05 compared with the H₂O₂100 group. b, c The effects of H₂O₂ and Moringa oleifera on cell viability and cell cycle progression of BMSCs during osteogenic differentiation. Cell viability (b) and cell cycle progression (c) of the indicated groups were measured at 48 h after initiation of osteogenic differentiation. Representative flow profile of cell cycle progression and percentage of cells in the G2 + S phases are shown. N = 3 for each group; *P < 0.05 compared with the control group; ^#P < 0.05 compared with the MO group; ^@P < 0.05 compared with the OS + H₂O₂ group

Fig. 2

The effects of H₂O₂ and Moringa oleifera on intracellular peroxide markers during osteogenic differentiation. a–d At 48 h after initiation of osteogenic differentiation, ROS levels (a), MDA levels (b), SOD activity (c), and GSH-PX activity (d) of BMSCs under the indicated osteogenic induction conditions were measured. N = 3 for each group; *P < 0.05 compared with the control group; ^#P < 0.05 compared with the MO group; ^@P < 0.05 compared with the OS + H₂O₂ group

Fig. 3

The effects of H₂O₂ and Moringa oleifera on expression of Foxo1 and cleaved caspase-3 and pAkt/Akt in BMSCs during osteogenic differentiation. a, b At 48 h after initiation of osteogenic differentiation, protein expression of Foxo1, Akt, and pAkt in BMSCs under the indicated osteogenic induction conditions were measured using western blot assays. a n = 3 for each group; *P < 0.05 compared with the control group; ^#P < 0.05 compared with the MO group; ^@P < 0.05 compared with the OS + H₂O₂ group. b n = 3 for each group; *P < 0.05 compared with the MO + H₂O₂ + DMSO group, ^#P < 0.05 compared with the MO + H₂O₂ group. c Protein expression of cleaved caspase-3 in BMSCs treated with 100 μmol/L H₂O₂ for the indicated duration was measured using western blot assay. n = 3 for each group; *P < 0.05 compared with the control group. d At 48 h after initiation of osteogenic differentiation, protein expression of cleaved caspase-3 in BMSCs under the indicated osteogenic induction conditions was measured using western blot assays. N = 3 for each group; *P < 0.05 compared with the MO group; ^#P < 0.05 compared with the OS + H₂O₂ group; ^@P < 0.05 compared with the MO + H₂O₂ group

Fig. 4

The effect of H₂O₂ and Moringa oleifera on expression of osteogenic markers in BMSCs during osteogenic differentiation. a At 7 days after initiation of osteogenic differentiation, mRNA levels of ALP, collagen I, OPN, and Runx2 in BMSCs under the indicated osteogenic induction conditions were measured using qRT-PCR. N = 3 for each group; *P < 0.05 compared with the control group; ^#P < 0.05 compared with the MO group; ^@P < 0.05 compared with the OS + H₂O₂ group; ^&P < 0.05 compared with the MO + H₂O₂ group. b Representative images of ALP staining at 7 days and alizarin red staining at 21 days after the initiation of osteogenic differentiation in BMSCs. Magnification, × 40; scale bar, 200 μm

Fig. 5

Characterization of BMSCs with immunofluorescence staining. The expression levels of CD44, CD90, CD31, and CD34 in the differentiated cells were characterized by indirect immunofluorescence assays using primary monoclonal antibodies against the indicated antigens and fluorescent secondary antibodies. The data are representative images with the indicated magnifications obtained from three independent experiments. a CD44 expression in P3 BMSCs. b CD90 expression in P3 BMSCs. c Negative CD31 expression in BMSCs. d Negative CD34 expression in BMSCs. Scale bar, 100 μm

Fig. 6

Non-target UHPLC-QE-MS analysis confirmed the existence of Moringa oleifera in the serum of rats that received oral dosing of Moringa leaf solution. HPLC analysis shows the differences in drug-containing serum from rats administrated Moringa leaf solution for three consecutive days (right panels) and blank serum components from rats administrated control saline (left panels). The differences in components between the two sets of samples are significant in both the negative ion mode (upper panels) and the positive ion mode (lower panels), as indicated by the different patterns of peaks. Further targeted analyses are needed to identify what ingredients the specific peaks are