Effects of adiponectin on the phosphorylation of adenosine monophosphate-activated protein kinase and the cell proliferation of osteosarcoma MG-63 cells / 中国医师杂志

ABSTRACT

Objective To investigate the effect of adiponectin on the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and the cell proliferation of osteosarcoma MG-63 cells.Methods The MG-63 cells were seeded in 6-cm board with a inoculum density of 1 × 105 cells/ml.When the cells grew up to about 80％,a volume (5 ml) of medium containing adiponectin (concentration 1 μg/ml) was added to each plate and incubated for 0 min,15 min,30 min,60 min,and 120 min,respectively ; and Western Blotting was used to detect the levels of AMPK phosphorylation,and the optimal time point processed by adiponectin was selected.The control and adiponectin groups were set (0,0.001,0.01,0.1,1 μg/ml) according to the optimal processing time of adiponectin,respectively; and Western blotting was used to detect the levels of AMPK phosphorylation and determine the optimal concentration of adiponectin.Based on the optimal processing time and optimal concentration,PBS was used as a control,the corresponding concentrations of adiponectin were used as the experimental group.Western blotting was used to detect the levels of AMPK phosphorylation to determine the effect of adiponectin on AMPK phosphorylation of osteosarcoma MG-63 cells.CCK-8 assay was used to investigate the effect of adiponectin on the cell proliferation of MG-63 cells.The MG-63 cells were seeded in the 96-well plates (5,000 cells/well) and cultured overnight.The experiment was set as blank control group and adiponectin-recombinant groups with 5 different concentration gradients (0.001,0.01,0.1,1,10 μg/ml).Five parallel wells were set for each group,and the cells were cultured for 24 h.During the last 4 h of culturing,a volume (10 μl) of CCK-8 reagent was added to each well,and the cells were cultured for another 2.5 h.The optical density (OD490) was obtained.Results When the concentration of adiponectin was greater than 0.01 μg/ml,the level of AMPK phosphorylation in MG-63 cells were significantly elevated (t =5.894,P =0.007).The short-time stimulation of adiponectin did not inhibit the proliferation of MG-63 cells (F =6.335,P =0.072).Conclusions Adiponectin can enhance the AMPK phosphorylation.The short-time stimulation of adiponectin did not inhibit the proliferation of MG-63 cells.