Leonurine Protects Bone Mesenchymal Stem Cells from Oxidative Stress by Activating Mitophagy through PI3K/Akt/mTOR Pathway

Abstract

Osteoporosis bears an imbalance between bone formation and resorption, which is strongly related to oxidative stress. The function of leonurine on bone marrow-derived mesenchymal stem cells (BMSCs) under oxidative stress is still unclear. Therefore, this study was aimed at identifying the protective effect of leonurine on H₂O₂ stimulated rat BMSCs. We found that leonurine can alleviate cell apoptosis and promote the differentiation ability of rat BMSCs induced by oxidative stress at an appropriate concentration at 10 μM. Meanwhile, the intracellular ROS level and the level of the COX2 and NOX4 mRNA decreased after leonurine treatment in vitro. The ATP level and mitochondrial membrane potential were upregulated after leonurine treatment. The protein level of PINK1 and Parkin showed the same trend. The mitophage in rat BMSCs blocked by 3-MA was partially rescued by leonurine. Bioinformatics analysis and leonurine-protein coupling provides a strong direct combination between leonurine and the PI3K protein at the position of Asp841, Glu880, Val882. In conclusion, leonurine protects the proliferation and differentiation of BMSCs from oxidative stress by activating mitophagy, which depends on the PI3K/Akt/mTOR pathway. The results showed that leonurine may have potential usage in osteoporosis and bone defect repair in osteoporosis patients.

Keywords: leonurine; mitophagy; osteoporosis.

Figure 1

(A) CCK-8 essay for BMSCs co-cultured with different concentrations of H₂O₂. (B) Evaluation of leonurine protection at different concentrations in skeptical ROS burden. (C) Double live/dead staining (scale bar = 200 μM). (D) Distribution of apoptotic BMSCs observed under flow cytometry (FITC-Annexin V apoptotic detection assay). (E) Expression of apoptosis-related protein marker. Leonurine can help BMSCs survive from an overload ROS environment. (&& p < 0.01 vs. Control group. * p < 0.05, ** p < 0.01, vs. H₂O₂ group).

Figure 2

Effect of leonurine protection on BMSCs from ROS damage. (A) ALP staining of leonurine treated groups (0–10 μM) at day 6 (scale bar = 200 μM). (B) Aliza red staining of leonurine treated groups (0–10 μM) at day 14 (scale bar = 200 μM). (C) Osteogenic-related mRNA expression level. (D) Osteogenic-related protein expression level. (& p < 0.05, && p < 0.01 vs. control group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. H₂O₂ group).

Figure 3

Effect of leonurine function on ameliorating ROS level. (A) Intracellular ROS measurement under microscopy (scale bar = 100 μM). (B) Intracellular ROS measurement by flow cytometry. (C) Quantitative analysis of flow cytometry results. (D) Intracellular ROS-related marker expression level. &&& p < 0.001 vs. control group ** p < 0.01, *** p < 0.001 vs. H₂O₂ group).

Figure 4

Leonurine activate mitophagy during ROS damage. (A) Intracellular ATP level (B) Measurement of mitochondrial membrane potential by microscopy (scale bar = 100 μM). (C) Measurement of mitochondrial membrane potential by flow cytometry. (D) Quantitative analysis of flow cytometry results. (E) Mitochondrial and lysosome colocalization analysis (scale bar = 50 μM). (F) Expression level of mitophagy-related proteins. (&&& p < 0.001 vs. control group. ** p < 0.01, *** p < 0.001 vs. H₂O₂ group).

Figure 5

Effects of leonurine on mitophagy-inhibited BMSCs from ROS damage. (A) ALP staining on day six (scale bar = 200 μM). (B) Aliza red staining on day 14 (scale bar = 200 μM). (C) Osteogenic-related mRNA expression level. (D) Intracellular ROS-related marker expression level. (E) Related protein expression change (& p < 0.05, && p < 0.01, &&& p < 0.001 vs. control group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. H₂O₂ group. # p < 0.05 vs. 3-MA+H₂O₂ group).

Figure 6

Leonurine can directly moderate PI3K-AKT-mTOR activation. (A) Research on the analysis of the leonurine and mitophagy pathway. (B) Relevance score on the analysis of the leonurine and mitophagy pathway. (C) 2D-molecular docking and 3D-molecular docking between leonurine and the PI3K protein. (D) The effect of leonurine on the PI3K-AKT-mTOR pathway and change in mitophagy.

Figure 7

Leonurine ameliorates osteoporosis and contributes to osteogenesis in vivo. (A) Body weight. (B) Organic weight. (C) MicroCT of the femur on longitudinal section. (D) MicroCT of the femur on transection; (E) H&E staining of the femur on cancellous bone. (F) H&E staining of the femur in cortical bone. (G) MicroCT assessment evaluated for: Tb.N trabecular number; Tb.S trabecular spacing; Tb.Th trabecular thickness; BV/TV trabecular bone volume fraction; (H) Skull bone defect healing condition. (I) H&E staining of new bone formation. (J) Quantitative analysis of the new bone formation area. (&& p < 0.01, &&& p < 0.001 vs. Control group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. H₂O₂ group).

Figure 8

Mechanisms underlying our results. Leonurine ameliorates ROS damage to BMSCs by blocking ROS generation cycle by mitophagy to keep mitochondrial quality control. The mechanism probably involves the moderate activation of the PI3K-AKT-mTOR pathway. Therefore, leonurine can activate mitophagy to alleviate damage to BMSCs form ROS overload by the moderate PI3K-AKT-mTOR pathway.