Journal: Biomolecules

Article Title: Coumarin Ameliorates Impaired Bone Turnover by Inhibiting the Formation of Advanced Glycation End Products in Diabetic Osteoblasts and Osteoclasts

doi: 10.3390/biom10071052

Figure Lengend Snippet: Inhibition of glucose-induced advanced glycation end products (AGE) secretion ( A ) and receptor for AGE (RAGE) expression ( B ), and elevation of AGE-induced osteoclast differentiation ( C ), and bone resorption ( D ) by coumarin in Raw 264.7 cells. Cells were cultured in α-MEM media containing 5.5 mM glucose, 33 mM glucose, or 100 μg/mL AGE with 50 ng/mL RANKL in the absence and presence of 1–20 μM coumarin for five days. Cell media and lysates were subject to western blot analysis with a primary antibody against AGE and RAGE. Representative blot data were obtained from three independent experiments, and β-actin protein was used as an internal control. The bar graphs (mean ± SEM) in the bottom panel represent quantitative results obtained from a densitometer. Values not sharing a letter are different at p < 0.05. After cells were cultured with 100 μg/mL AGE–bovine serum albumin (BSA) for five days, cells were fixed and stained using a leukocyte acid phosphatase kit ( C ). TRAP-positive multinucleated osteoclasts were visualized under light microscopy. The osteoclast bone resorption was assayed by using a commercially available bone resorption assay kit ( D ). The resorbed areas on the plate were visualized under light microscopy. Magnification: 20-fold.

Article Snippet: Advanced glycation end product-bovine serum albumin (AGE-BSA) protein was obtained from Merck Millipore (Burlington, MA, USA).

Techniques: Inhibition, Expressing, Cell Culture, Western Blot, Control, Staining, Light Microscopy

Journal: Biomolecules

Article Title: Coumarin Ameliorates Impaired Bone Turnover by Inhibiting the Formation of Advanced Glycation End Products in Diabetic Osteoblasts and Osteoclasts

doi: 10.3390/biom10071052

Figure Lengend Snippet: Inhibition of advanced glycation end-product (AGE) secretion and receptor for AGE (RAGE) induction ( A ) in osteoblastic MC3T3-E1 cells by coumarin. MC3T3-E1 cells were cultured for nine days in the presence of 1–20 μM of coumarin. Cell media and lysates were subject to western blot analysis with a primary antibody against AGE and RAGE. Representative blot data were obtained from three independent experiments, and β-actin protein was used as an internal control. The bar graphs (mean ± SEM) in the bottom panel represent quantitative results obtained from a densitometer. MC3T3-E1 cells were cultured in differentiation media containing 5.5 mM glucose or 100 μg/mL AGE–bovine serum albumin (BSA) in the presence of 1–20 μM coumarin on glass chamber plates for six days ( B ). Alkaline phosphatase (ALP) staining was proportional to the content of ALP enzyme activity from MC3T3-E1 cells. The intensity of ALP staining (mean ± SEM, n = 4) was measured using an optical Axiomager microscope system ( B ). The ALP staining was visualized under light microscopy. Magnification: 10-fold. Values not sharing a letter are different at p < 0.05.

Article Snippet: Advanced glycation end product-bovine serum albumin (AGE-BSA) protein was obtained from Merck Millipore (Burlington, MA, USA).

Techniques: Inhibition, Cell Culture, Western Blot, Control, Staining, Activity Assay, Microscopy, Light Microscopy