HO-1 Induction by CO-RM2 Attenuates TNF-α-Induced Cytosolic Phospholipase A2 Expression via Inhibition of PKCα-Dependent NADPH Oxidase/ROS and NF-κB
Figure 7
Effect of CORM-2 on TNF-α-induced NF-κB activation and cPLA2 expression. (a) Cells were pretreated with CORM-2 or iCORM-2 for 16 h and then incubated with TNF-α for 2 h. Chromatin immunoprecipitation (ChIP) assays were performed using an anti-p65 antibody. (b) Cells were transfected with NF-κB-luc reporter gene, pretreated with or without CORM-2 for 16 h, and then incubated with TNF-α for 4 h. NF-κB promoter activity was determined. ((c), (d), and (f)) Cells were pretreated with or without CORM-2 or iCORM-2 for 16 h and then treated with TNF-α for the indicated time intervals. The levels of phospho-p65, phospho-IKKα/β, phospho-JNK1/2, phospho-p38 MAPK, and HO-1 were determined by Western blotting. (e) Cells were pretreated with or without CORM-2 for 16 h and then incubated with TNF-α for 90 min. The cells were added with (5 μM) DHE and images acquired to determine the ROS generation using a fluorescence microscopy. All analyses were performed on samples from 4 RA patients. (f) Immunohistochemical staining for HO-1, cPLA2, or vimentin and hematoxylin and eosin (H&E) staining of serial sections of ankle joints from mice injected with phosphate-buffered saline (sham) ((A)–(E)), mice injected with TNF-α ((F)–(J)), and mice injected with CORM-2 for 16 h followed by TNF-α for 24 h ((K)–(O)) were performed. Arrowheads indicate positive staining. Results are representative of 3 mice per experimental group. Results are representative of 3 independent experiments. In (a), (b), (c), and (g), values are the mean ± SEM. ; versus TNF-α alone.
