Docosahexaenoic acid improves altered mineralization proteins, the decreased quality of hydroxyapatite crystals and suppresses oxidative stress induced by high glucose

Abstract

Patients with type 2 diabetes mellitus (DM2) experience an increased risk of fractures and a variety of bone pathologies, such as osteoporosis. The suggested mechanisms of increased fracture risk in DM2 include chronic hyperglycaemia, which provokes oxidative stress, alters bone matrix, and decreases the quality of hydroxyapatite crystals. Docosahexaenoic acid (DHA), an omega-3 fatty acid, can increase bone formation, reduce bone loss, and it possesses antioxidant/anti-inflammatory properties. The present study aimed to determine the effect of DHA on altered osteoblast mineralisation and increased reactive oxygen species (ROS) induced by high glucose concentrations. A human osteoblast cell line was treated with 5.5 mM glucose (NG) or 24 mM glucose (HG), alone or in combination with 10 or 20 µM DHA. The collagen type 1 (Col1) scaffold, the expression of osteocalcin (OCN) and bone sialoprotein type-II (BSP-II), the alkaline phosphatase (ALP) specific activity, the mineral quality, the production of ROS and the mRNA expression of nuclear factor erythroid 2-related factor-2 (NRF2) were analysed. Osteoblasts cultured in HG and treated with either DHA concentration displayed an improved distribution of the Col1 scaffold, increased OCN and BSP-II expression, increased NRF2 mRNA, decreased ALP activity, carbonate substitution and reduced ROS production compared with osteoblasts cultured in HG alone. DHA counteracts the adverse effects of HG on bone mineral matrix quality and reduces oxidative stress, possibly by increasing the expression of NRF2.

Keywords: bone mineral matrix; bone quality; diabetic osteopathy; docosahexaenoic acid; omega 3.

Figure 1

Effect of treatment with DHA at 5.5 or 24 mM glucose concentrations on the production and distribution of Collagen type 1 and production of OCN. (A) Effect of DHA on the concentration and distribution of Collagen type 1 with different concentrations of glucose (magnification, x10; scale bar, 100 µm). (B) Effect of DHA on the production of OCN with different concentrations of glucose. Representative images. The bar graphs represent the mean ± SD of the mean fluorescence intensity of three independent experiments. ^*P<0.05 and ^**P<0.01 vs. control (5.5 mM glucose); ^#P<0.05 and ^##P<0.01 vs. 24 mM glucose. DHA, docosahexaenoic acid; OCN, osteocalcin; DMEM, Dulbecco's modified Eagle's medium; LG, low glucose; MFI, median fluorescence intensity.

Figure 2

Effect of DHA with 5.5 or 24 mM glucose concentrations on the production of BSP-II. Immunoblot of BSP-II detected by infrared. (A) Representative images of the results of the expression of BSP-II obtained by western blotting. (B) Densitometry results obtained by normalization of BSP-II expression to the control (5.5 mM of glucose) of three independent experiments expressed in a.u. The graphs represent the mean ± SD. ^*P<0.05 and ^**P<0.01 vs. control (5.5 mM glucose); ^#P<0.05 vs. HG group (24 mM glucose). DHA, docosahexaenoic acid; BSP-II, bone sialoprotein type-II; a.u., arbitrary units; NG, normal glucose (5.5 mM glucose); HG, high glucose (24 mM glucose).

Figure 3

Effect of DHA with 5.5 or 24 mM glucose concentrations on the specific activity of ALP. Results of the enzymatic activity of ALP expressed in IU/µg of protein. The graphs represent the mean ± SD. ^**P<0.01 vs. control (5.5 mM glucose); ^##P<0.01 vs. 24 mM glucose. DHA, docosahexaenoic acid; ALP, alkaline phosphatase.

Figure 4

Effect of DHA with 5.5 or 24 mM glucose concentrations on biomineralization. (A) Effect of DHA on the mineral matrix formation under 5.5 mM or 24 mM glucose. Representative microphotographs are shown. (B) Extraction of the dye was performed to quantify the amount of calcium (Ca) in the culture (scale bars, 100 µm). (C) Effect of DHA on crystal morphology formed by osteoblasts under conditions of 5.5 mM or 24 mM glucose by scanning electron microscopy (magnification, x250). The graphs represent the mean ± SD. ^*P<0.05 and ^**P<0.01 vs. control (5.5 mM glucose); ^##P<0.01 vs. 24 mM glucose. DHA, docosahexaenoic acid.

Figure 5

Effect of DHA with 5.5 or 24 mM glucose concentrations on relative mineralization and carbonate substitution. Representative spectra obtained by Fourier-transform infrared spectroscopy of the mineral matrix. Bands of amide I in 1,599-1,701 cm^-1, amide III in 1,250-1,371 cm^-1, phosphate group (v₁, v₃ PO₄^3-) in 900-1,212 cm^-1 and carbonate (v₂ CO₃^2-) in 854-892 cm^-1 are shown. DHA, docosahexaenoic acid.

Figure 6

Effect of DHA with 5.5 or 24 mM glucose concentrations on oxidative stress caused by 24 mM glucose. (A) Results of intracellular measurement of reactive oxygen species by flow cytometry using the DCF fluorescent probe. MFI graphs and histograms representative of the results obtained are shown. (B) Effect of DHA on the expression of NRF2 mRNA in the presence of different glucose concentrations. All graphs represent the mean ± SD. ^*P<0.05 and ^**P<0.01 vs. control (5.5 mM glucose); ^##P<0.01 vs. 24 mM glucose. DHA, docosahexaenoic acid; DCF, 2',7'-Dichlorofluorescein; MFI, median fluorescence intensity; NRF2, nuclear factor erythroid 2-related factor-2.

Figure 7

Effect of DHA with 5.5 or 24 mM glucose concentrations on the extracellular production of RANK-L. The graph represents the mean ± SD of three independent experiments. ^**P<0.01 vs. control (5.5 mM glucose); ^##P<0.01 vs. 24 mM glucose. DHA, docosahexaenoic acid; RANK-L, receptor activator of nuclear factor κ-β ligand.