Arbutin promotes MC3T3‑E1 mouse osteoblast precursor cell proliferation and differentiation via the Wnt/β‑catenin signaling pathway

Abstract

Arbutin is a natural compound extracted from various plants, including bearberry leaves, that exerts multiple effects including skin whitening, anti‑inflammatory and oxidative stress‑protective properties. However, the effects of arbutin on osteoblasts remain unknown. The aim of the present study was to investigate the function and the mechanisms of arbutin on the proliferation and differentiation of MC3T3‑E1 mouse osteoblast precursor cells in vitro. The proliferation of MC3T3‑E1 cells treated with arbutin was assessed using a Cell Counting Kit‑8 assay and a 5‑ethynyl‑2'‑deoxyuridine labeling assay. Additionally, cell cycle and apoptosis were examined using flow cytometry analysis. The effects of arbutin on osteoblast differentiation were investigated using alkaline phosphatase (ALP) staining and by examining the mRNA expression levels of collagen type I α1 chain (COL1A1), bone γ‑carboxyglutamate protein (BGLAP) and Sp7 transcription factor (SP7). To further investigate the molecular mechanism underlying arbutin function in promoting osteogenesis, the mRNA and protein expression levels of runt‑related transcription factor 2 (RUNX2) and β‑catenin were analyzed by reverse transcription‑quantitative polymerase chain reaction and western blotting. Arbutin significantly promoted MC3T3‑E1 cell proliferation and increased the ratio of cells in S‑phase. Treatment with arbutin increased ALP activity and the mRNA expression levels of COL1A1, BGLAP and SP7 in MC3T3‑E1 cells. Furthermore, the protein and the mRNA expression levels of RUNX2 and β‑catenin increased significantly following treatment with arbutin. Collectively, the present findings suggested that arbutin was able to promote proliferation and differentiation of MC3T3‑E1 cells via the Wnt/β‑catenin signaling pathway.

Figure 1.

Chemical structure of arbutin.

Figure 2.

Effects of arbutin on MC3T3-E1 cell proliferation. (A-C) MC3T3-E1 mouse osteoblast precursor cells were treated with arbutin at 0 (control), 10, 50 or 100 µM for 24, 48 or 72 h. (A) MC3T3-E1 cell proliferation was analyzed using Cell Counting Kit-8 assay. (B) Percentage of EdU-positive MC3T3-E1 cells, indicating the number of proliferating cells. (C) EdU (red) and Hoechst (blue; nucleus) staining results. (n=3); *P<0.05, **P<0.01 and ***P<0.001 vs. untreated control. EdU, 5-ethynyl-2′-deoxyuridine.

Figure 3.

Arbutin accelerates cell cycle progression without affecting apoptosis of MC3T3-E1 cells. (A-E) MC3T3-E1 mouse osteoblast precursor cells were treated with 0 (control), 10, 50 or 100 µM arbutin for 3 days. The effects of arbutin on the (A) cell cycle and (B) cell apoptosis were assessed by flow cytometry analysis. (C and D) Percentage of cells in (C) S-phase and (D) G1-phase in each group (n=3). (E) Quantification of apoptotic rate (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. untreated control. PI, propidium iodide; FITC, fluorescein isothiocyanate.

Figure 4.

Effects of arbutin on osteogenic differentiation. (A-F) MC3T3-E1 mouse osteoblast precursor cells were cultured in differentiation medium and treated with 0 (control), 10, 50 or 100 µM arbutin for (A) 9 or (B-F) 3 days. (A) ALP-positive cells are stained in blue or purple. The effects of arbutin on the mRNA expression levels of (B) COL1A1, (C) β-catenin, (D) RUNX2, (E) BGLAP and (F) SP7 in MC3T3-E1 cells. mRNA expression levels were assessed by reverse transcription-quantitative polymerase chain reaction analysis; β-actin served as the internal control. *P<0.05 and **P<0.01 vs. untreated control. ALP, alkaline phosphatase; COL1A1, collagen type I α 1 chain; BGLAP, bone γ-carboxyglutamate protein; SP7, Sp7 transcription factor; RUNX2, runt-related transcription factor 2.

Figure 5.

Arbutin promotes MC3T3-E1 cell differentiation via the Wnt/β-catenin signaling pathway. (A) Cells were treated with 0 (control), 10, 50 or 100 µM arbutin for 3 days, and the protein expression levels of β-catenin and RUNX2 were detected by western blot analysis. (B) MC3T3-E1 cells were treated with 0.5 µg/ml DKK1 for 6 h prior to treatment with 100 µM arbutin. Protein expression levels of (C) β-catenin and (D) RUNX2, from Part A, were quantified by densitometric analysis. *P<0.05 and **P<0.01 vs. untreated control. (E) Densitometric analysis of RUNX2 following combined treatment with DKK1 and arbutin, from Part B. *P<0.05 vs. untreated control; ^##P<0.01 vs. arbutin alone. RUNX2, runt-related transcription factor 2; DKK1, dickkopf WNT signaling pathway inhibitor 1; Wnt, wingless/integrated.