Activation of Adenosine A3 Receptor Alleviates TNF-α-Induced Inflammation through Inhibition of the NF-κB Signaling Pathway in Human Colonic Epithelial Cells
Figure 2
Qualitative expression of A3AR, NF-κB p65, IκB-α, and phosphorylated-IκB-α (p-IκB-α) in HT-29 cells following different treatments. HT-29 cells were treated with 30 nM 2-Cl-IB-MECA (CIM) for 30 min prior to TNF-α (10 ng/mL) stimulation for 30 min for the immunofluorescence (IF) assay. IF was performed by using specific antibodies for target proteins and DAPI for counterstaining of nuclei. ((a)1–3) The strong green fluorescence signal representing A3AR was observed mainly on the HT-29 cell membrane. However, cells stimulated by TNF-α alone or pretreated with 2-Cl-IB-MECA had no obvious change in A3AR expression. ((b)1–3) In untreated cells, NF-κB p65 was limited to the cytoplasm, and nuclear localization was observed in TNF-α-treated HT-29 cells. However, a significant reduction in p65 nuclear translocalization was seen in 2-Cl-IB-MECA + TNF-α cells. ((c)1–3) There was a tendency for IκB-α to decrease in the cytoplasm of TNF-α-treated cells. This effect was inhibited in 2-Cl-IB-MECA + TNF-α cells. ((d)1–3) There was a tendency for p-IκB-α to increase in the cytoplasm of TNF-α-treated cells. This effect was inhibited in 2-Cl-IB-MECA + TNF-α cells. Magnification 600; scale bars indicated 50 μm.
