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.
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.
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.
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.
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.
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.