2,4'-Dihydroxybenzophenone Exerts Bone Formation and Antiosteoporotic Activity by Stimulating the β-Catenin Signaling Pathway

Abstract

2,4'-Dihydroxybenzophenone (DHP) is an organic compound derived from Garcinia xanthochymus, but there have been no reports on its biochemical functions and bioavailability. In this study, we evaluated whether DHP affects osteoblast differentiation and activation in MC3T3-E1 preosteoblast cells, as well as antiosteoporotic activity in zebrafish larvae. Nontoxic concentrations of DHP-treated MC3T3-E1 preosteoblast cells increased alkaline phosphatase (ALP) activation and mineralization in a concentration-dependent manner, accompanied by higher expression of osteoblast-specific markers, including Runt-related transcription factor 2 (RUNX2), osterix, and ALP. Consistent with the data in MC3T3-E1 preosteoblast cells, DHP upregulated osteoblast-specific marker genes in zebrafish larvae and simultaneously enhanced vertebral formation. We also revealed that DHP increased the phosphorylation of glycogen synthase kinase-3β (GSK-3β) at Ser9 and the total expression of β-catenin in the cytosol and markedly increased the localization of β-catenin into the nucleus. Furthermore, DHP restored the prednisolone (PDS)-induced marked decrease in ALP activity and mineralization, as well as osteoblast-specific marker expression. In PDS-treated zebrafish, DHP also alleviated PDS-induced osteoporosis by restoring vertebral formation and osteoblast-related gene expression. Taken together, these results suggest that DHP is a potential osteoanabolic candidate for treating osteoporosis by stimulating osteoblast differentiation.

Figure 1

Chemical structure of DHP.

Figure 2

DHP at a concentration of 100 μM reduces the viability of MC3T3-E1 preosteoblasts. MC3T3-E1 preosteoblasts (1 × 10⁴ cells/mL) were treated with DHP (0–100 μM) for 14 days, with the medium being replaced every 2 days. (A) Relative cell viability was determined using an MTT assay. (B) Morphological changes in MC3T3-E1 preosteoblasts were monitored using stereomicroscopy. Scale bar = 50 μm. (C) Viability in the nucleated cells was measured using flow cytometry. (D) Viable cell count and (E) dead cell population were determined using flow cytometry. The data represent the mean ± standard error of the mean of three independent experiments. Significant differences among groups were determined using one-way ANOVA with Bonferroni correction (***, p < 0.001 vs untreated cells).

Figure 3

DHP stimulates osteoblast differentiation. MC3T3-E1 preosteoblasts (1 × 10⁴ cells/mL) were treated with DHP (0–10 μM) for 14 days; β-glycerophosphate (GP, 2 mM) was used as the positive control. The media were replaced every 2 days with the specified chemicals. The cells were stained using (A) TRACP and ALP double-staining kit and (B) 2% Alizarin Red solution. Representative images were acquired using phase-contrast microscopy (×10). Scale bar = 50 μm. (C) On day 14, total mRNA was extracted, and RT-PCR was performed to detect the expression of osteoblast-related genes, including RUNX2, OSX, and ALP, with GAPDH as the loading control. (D) Total protein was isolated on day 14, and Western blotting was performed to detect the expression of osteoblast marker proteins, including RUNX2, OSX, and ALP, with β-actin as the loading control. The relative density was calculated using ImageJ and normalized to the expression of GAPDH and β-actin. The data represent the mean ± standard error of the mean of three independent experiments. Significant differences among groups were determined using one-way ANOVA with Bonferroni correction (^**p < 0.01 and ^***p < 0.001 vs untreated cells).

Figure 4

DHP promotes vertebral formation in zebrafish larvae. Zebrafish larvae at 3 dpf were treated with the indicated concentrations of DHP (0–10 μM) and until 9 dpf, with β-glycerophosphate (GP, 4 mM) as the positive control. The media were replaced with the specified chemicals every 2 days. (A) On day 9, zebrafish larvae were stained with 1% calcein solution to visualize vertebral formation, and the (B) number of mineralized vertebral bodies was manually counted. (C) Bone density was measured using ImageJ software and normalized to that of untreated zebrafish larvae. (D) In a parallel experiment, total mRNA was extracted, and RT-PCR was performed to detect the expression of osteoblast-related genes, including RUNX2a, OSX, and ALP, with β-actin as the loading control. The relative density was calculated using ImageJ software and normalized to the expression of β-actin. The data represent the mean ± standard error of the mean of three independent experiments. Significant differences among groups were determined using one-way ANOVA with Bonferroni correction (***, p < 0.001 vs untreated zebrafish larvae).

Figure 5

DHP enhances GSK-3β phosphorylation at Ser9 and subsequent β-catenin activation. MC3T3-E1 cells (1 × 10⁴ cells/mL) were cultured with the indicated concentrations of DHP for 14 days. β-Glycerophosphate (GP, 2 mM) was used as the positive control. The media were replaced every 2 days. (A) Total proteins were extracted, and Western blotting was performed to detect the expression of phosphor (p)-GSK-3β at Ser9, GSK-3β, and β-catenin. β-Actin was used as the loading control. (B) Nuclear proteins were extracted, and Western blotting was performed to detect the expression of β-catenin. Nucleolin was used as the loading control. The relative density was calculated using ImageJ and normalized to the density of β-actin and nucleolin. The data represent the mean ± standard error of the mean of three independent experiments. Significant differences among groups were determined using one-way ANOVA with Bonferroni correction (^**p < 0.01 and ^***p < 0.001 vs untreated cells).

Figure 6

DHP restored osteoblast differentiation impaired by prednisolone (PDS). MC3T3-E1 preosteoblasts (1 × 10⁴ cells/mL) were treated with varying concentrations of DHP (0–10 μM) for 14 days in the presence of PDS (10 μM). The media were replaced with the indicated chemicals every 2 days. Cells were stained using (A) TRACP and ALP double-staining kit and (B) 2% Alizarin Red solution. Representative images were acquired using phase-contrast microscopy (×10). Scale bar = 50 μm. (C) Expression of RUNX2, OSX, and ALP was detected by extracting total RNA and performing RT-PCR on day 14, with GAPDH as the loading control. (D) Total protein was isolated, and Western blotting was performed to detect the expression of osteoblast marker proteins, including RUNX2, OSX, and ALP, with β-actin as the loading control. The relative densities of genes and proteins were calculated by using ImageJ software and normalized to the densities of GAPDH and β-actin. The data represent the mean ± standard error of the mean of three independent experiments. Significant differences among groups were determined using the Student′s t test (^###p < 0.001 vs untreated cells) and one-way ANOVA with Bonferroni correction (^***p < 0.001 vs PDS-treated cells).

Figure 7

DHP recovers PDS-induced impaired vertebral formation. At 5 dpf, zebrafish larvae (n = 20) were pretreated with PDS (10 μM) for 2 h, followed by treatment with DHP (0–10 μM) until 9 dpf. (A) At 9 dpf, the larvae were stained with 1% calcein solution to visualize vertebral formation. (B) Number of vertebrae was counted and manually recorded (highlighted in yellow). (C) Bone density was measured using ImageJ software and normalized to that of untreated zebrafish larvae. (D) In a parallel experiment, total RNA was extracted, and RT-PCR was performed to detect the expression of RUNX2a, OSX, and ALP. β-Actin was used as the loading control. The relative density was calculated by using ImageJ and normalized to the density of β-actin. The data represent the mean ± standard error of the mean of three independent experiments. Significant differences among groups were determined using the Student′s t test (^###p < 0.001 vs untreated zebrafish larvae) and one-way ANOVA with Bonferroni correction (^***p < 0.001 vs PDS-treated zebrafish larvae).