Advanced glycation end products inhibit the osteogenic differentiation potential of adipose-derived stem cells by modulating Wnt/β-catenin signalling pathway via DNA methylation

Abstract

Objectives: Advanced glycation end products (AGEs) are considered a cause of diabetic osteoporosis. Although adipose-derived stem cells (ASCs) are widely used in the research of bone regeneration, the mechanisms of the osteogenic differentiation of ASCs from diabetic osteoporosis model remain unclear. This work aimed to investigate the influence and the molecular mechanisms of AGEs on the osteogenic potential of ASCs. Materials and methods: Enzyme-linked immunosorbent assay was used to measure the change of AGEs in diabetic osteoporotic and control C57BL/6 mice. ASCs were obtained from the inguinal fat of C57BL/6 mice. AGEs, 5-aza2′-deoxycytidine (5-aza-dC) and DKK-1 were used to treat ASCs. Real-time cell analysis and cell counting kit-8 were used to monitor the proliferation of ASCs within and without AGEs. Real-time PCR, Western blot and Immunofluorescence were used to analyse the genes and proteins expression of osteogenic factors, DNA methylation factors and Wnt/β-catenin signalling pathway among the different groups. Results: The AGEs and DNA methylation were increased in the adipose and bone tissue of the diabetic osteoporosis group. Untreated ASCs had higher cell proliferation activity than AGEs-treatment group. The expression levels of osteogenic genes, Opn and Runx2, were lower, and mineralized nodules were less in AGEs-treatment group. Meanwhile, DNA methylation was increased, and the Wnt signalling pathway markers, including β-Catenin, Lef1 and P-GSK-3β, were inhibited. After treatment with 5-aza-dC, the osteogenic differentiation capacity of ASCs in the AGEs environment was restored and the Wnt signalling pathway was activated during this process. Conclusions: Advanced glycation end products inhibit the osteogenic differentiation ability of ASCs by activating DNA methylation and inhibiting Wnt/β-catenin pathway in vitro. Therefore, DNA methylation may be promising targets for the bone regeneration of ASCs with diabetic osteoporosis.