Adiponectin Inhibits LPS-Induced HMGB1 Release through an AMP Kinase and Heme Oxygenase-1-Dependent Pathway in RAW 264 Macrophage Cells
Effect of recombinant adiponectin on LPS-induced HMGB1 release and HMGB1 cellular translocation. Raw 264 cells were cultured in DMEM supplemented with 10% FBS and were cultured in serum-free OPTI-MEM I medium for additional 12 h. The cells were treated with increasing concentrations of adiponectin for 18 h, then stimulated with LPS (200 ng/mL) for another 24 h. (a) Culture medium was collected and analyzed by HMGB1 western blotting, followed by quantification of the intensity of the chemiluminescent HMGB1 band. The results are expressed as means ± SE of three independent experiments ( significance compared with control; significance compared with LPS treated cells). (b and c) Cellular HMGB1 was immunostained with an anti-HMGB1 rabbit primary and Alexa Fluor 488 anti-rabbit secondary antibodies. The nucleus was stained with DAPI. Merge indicates the combination of both HMGB1 (Green) and nuclear (Blue) fluorescence. The fluorescence intensities of cytosolic and nuclear HMGB1 in (b) were separately analyzed and the ratio of cytosolic HMGB1 to nuclear HMGB1 is shown in (c) ( significance compared with control; significance compared with LPS treated cells).