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. 2013 Jun 28;8(6):e67504.
doi: 10.1371/journal.pone.0067504. Print 2013.

VSports app下载 - Hesperetin alleviates the inhibitory effects of high glucose on the osteoblastic differentiation of periodontal ligament stem cells

So Yeon Kim  1 , Jin-Yong Lee, Yong-Duk Park, Kyung Lhi Kang, Jeong-Chae Lee, Jung Sun Heo
Affiliations

"V体育2025版" Hesperetin alleviates the inhibitory effects of high glucose on the osteoblastic differentiation of periodontal ligament stem cells

So Yeon Kim et al. PLoS One. .

Abstract

Hesperetin (3',5,7-trihydroxy-4-methoxyflavanone) is a metabolite of hesperidin (hesperetin-7-O-rutinoside), which belongs to the flavanone subgroup and is found mainly in citrus fruits. Hesperetin has been reported to be an effective osteoinductive compound in various in vivo and in vitro models. However, how hesperetin effects osteogenic differentiation is not fully understood. In this study, we investigated the capacity of hesperetin to stimulate the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and to relieve the anti-osteogenic effect of high glucose. Osteogenesis of PDLSCs was assessed by measurement of alkaline phosphatase (ALP) activity, and evaluation of the mRNA expression of ALP, runt-related gene 2 (Runx2), osterix (OSX), and FRA1 as osteogenic transcription factors, as well as assessment of protein expression of osteopontin (OPN) and collagen type IA (COLIA). When PDLSCs were exposed to a high concentration (30 mM) of glucose, osteogenic activity decreased compared to control cells. Hesperetin significantly increased ALP activity at doses of 1, 10, and 100 µM. Pretreatment of cells with hesperetin alleviated the high-glucose-induced suppression of the osteogenic activity of PDLSCs. Hesperetin scavenged intracellular reactive oxygen species (ROS) produced under high glucose condition. Furthermore, hesperetin increased the activity of the PI3K/Akt and β-catenin pathways. Consistent with this, blockage of Akt or β-catenin diminished the protective effect of hesperetin against high glucose-inhibited osteogenic differentiation. Collectively, our results suggest that hesperetin alleviates the high glucose-mediated suppression of osteogenic differentiation in PDLSCs by regulating ROS levels and the PI3K/Akt and β-catenin signaling pathways. VSports手机版.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Effect of high glucose (HG) and hesperetin on osteogenic differentiation of PDLSCs.
Cells were incubated in osteogenic medium with (A) 30 mM glucose (HG) or (B) hesperetin (0.1, 1, 10, 100 µM) for 4 and 7 days, respectively, and then ALP activity was assessed as described in the Materials and Methods. (C) Cells were pretreated with hesperetin at concentrations of 10 and 100 µM for 2 hr before HG exposure and ALP activity was determined after 4, 7, 14 days of osteogenic induction. The values reported are the means ± S.D. of five independent experiments. *P<0.05, **P<0.001 vs. control value, or # P<0.05 vs. HG treatment alone.
Figure 2
Figure 2. Effects of hesperetin on the high glucose-suppressed mRNA expression of ALP, Runx2, OSX, and FRA1.
Cells were pretreated with hesperetin at concentrations of 10 and 100 µM for 2 hr before HG exposure and the mRNA levels of (A) ALP, (B) Runx2, (C) OSX, and (D) FRA1 were analyzed using real time RT-PCR after 4 days of osteogenic induction. The values reported are the means ± S.D. of three independent experiments. *P<0.05, **P<0.001 vs. control values, or # P<0.05 vs. HG treatment alone.
Figure 3
Figure 3. Effects of hesperetin on the high glucose-mediated suppression of Runx2, OSX, OPN, and COLIA protein levels.
(A) Cells were pretreated with hesperetin at concentrations of 10 and 100 µM for 2 hr before HG incubation and then protein levels of (A) Runx2, OSX, (B) OPN, and COLIA were determined by western blot analysis using total protein lysates. Cells were incubated with hesperetin in the presence of HG for 4 days then (C) OPN and (D) COLIA were detected by immunostaining. Negative control staining using secondary antibody alone to assess nonspecific fluorescence. Nuclei were stained with DAPI (blue staining). A representative result from four independent experiments is shown.
Figure 4
Figure 4. Effect of hesperetin on intracellular ROS levels and PI3K/Akt signaling.
(A) Cells were preincubated with hesperetin at different concentrations for 2 hr then 10 µM CM-H2DCF-DA and high glucose were added. After 40 min, DCF fluorescence was determined using a spectrofluorophotometer. (B) Protein levels of PI3K p110α and γ isoforms as well as p-Akt were determined after cells were incubated with hesperetin or vitamin C in the presence of HG for 4 days. (C) Phosphorylation of Akt by hesperetin (10 µM) was assessed with cells of 7 day-osteogenic induction. (D, E) OPN and COLIA levels were determined after incubation of cells with Akt inhibitor in the presence of HG, hesperetin, or HG+hesperetin. (F–I) The mRNA levels of osteogenic target genes were determined after incubation with Akt inhibitor in the presence of HG or HG+hesperetin. The values reported are the mean ± S.D. of three independent experiments. *P<0.05 vs. control values, **P<0.05 vs. HG treatment alone, or # P<0.05 vs. HG+hesperetin.
Figure 5
Figure 5. Effect of hesperetin on Wnt/β-catenin signaling.
(A) Protein levels of β-catenin and (B, C) nuclear translocation of β-catenin were assessed by Western blot analysis or immunofluorescence staining after cells were incubated with hesperetin. Protein levels of β-catenin were determined after cells were conditioned with (D) hesperetin in the presence of HG and (E) hesperetin+Akt inhibitor. (F) Changes in β-catenin levels were determined according to transfection. (G) Protein levels of OPN and COLIA, (H) ALP activity, and (I) the mRNA levels of Runx2 and OSX were measured after cells were transfected with β-catenin siRNA for 24 hr and further incubated with hesperetin and HG for 24 hr. The values reported are the mean ± S.D. of three independent experiments. *P<0.05 vs. control values, **P<0.05 vs. HG treatment alone, or # P<0.05 vs. HG+hesperetin.
Figure 6
Figure 6. Hypothesized model of the signaling pathways underlying the rescuing effects of hesperetin on high glucose-exposed PDLSCs.
High glucose increases ROS generation, which inhibits PI3K/Akt signaling and Wnt/β-catenin. Hesperetin suppresses ROS production and activates PI3K/Akt signaling and Wnt/β-catenin to induce the translocation of β-catenin into the nucleus, which leads to the osteogenic differentiation of PDLSCs. In this scheme, grey lines are proposed pathways affected by high glucose concentrations and black lines are hesperetin-stimulated pathways.
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