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Activation of adenosine A3 receptor alleviates TNF-α-induced inflammation through inhibition of the NF-κB signaling pathway in human colonic epithelial cells.

Ren T, Qiu Y, Wu W, Feng X, Ye S, Wang Z, Tian T, He Y, Yu C, Zhou Y - Mediators Inflamm. (2014)

Bottom Line: Pretreatment with 2-Cl-IB-MECA prior to stimulation with TNF-α attenuated NF-κB p65 nuclear translocation as p65 protein decreased in the nucleus of cells and increased in the cytoplasm, inhibited the degradation of IκB-α, and reduced phosphorylated-IκB-α level significantly, compared to TNF-α-only-treated groups.Furthermore, 2-Cl-IB-MECA significantly decreased TNF-α-stimulated IL-8 and IL-1β mRNA expression and secretion, compared to the TNF-α-only treated group.These results confirm that A3AR is expressed in human colonic epithelial cells and demonstrate that its activation has an anti-inflammatory effect, through the inhibition of NF-κB signaling pathway, which leads to inhibition of downstream IL-8 and IL-1β expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, The Affiliated Hospital of Guangdong Medical College, Zhanjiang 524001, China.

ABSTRACT
To investigate the expression of adenosine A3 receptor (A3AR) in human colonic epithelial cells and the effects of A3AR activation on tumor necrosis factor alpha (TNF-α-) induced inflammation in order to determine its mechanism of action in human colonic epithelial cells, human colonic epithelial cells (HT-29 cells) were treated with different concentrations of 2-Cl-IB-MECA prior to TNF-α stimulation, followed by analysis of NF-κB signaling pathway activation and downstream IL-8 and IL-1β production. A3AR mRNA and protein were expressed in HT-29 cells and not altered by changes in TNF-α or 2-Cl-IB-MECA. Pretreatment with 2-Cl-IB-MECA prior to stimulation with TNF-α attenuated NF-κB p65 nuclear translocation as p65 protein decreased in the nucleus of cells and increased in the cytoplasm, inhibited the degradation of IκB-α, and reduced phosphorylated-IκB-α level significantly, compared to TNF-α-only-treated groups. Furthermore, 2-Cl-IB-MECA significantly decreased TNF-α-stimulated IL-8 and IL-1β mRNA expression and secretion, compared to the TNF-α-only treated group. These results confirm that A3AR is expressed in human colonic epithelial cells and demonstrate that its activation has an anti-inflammatory effect, through the inhibition of NF-κB signaling pathway, which leads to inhibition of downstream IL-8 and IL-1β expression. Therefore, A3AR activation may be a potential treatment for gut inflammatory diseases such as inflammatory bowel disease.

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Cell survival rate at various concentrations of 2-Cl-IB-MECA. HT-29 cells were treated with various concentrations of 2-Cl-IB-MECA (5 nM to 1000 nM) for 12 hours, before cell viability was measured by using a CCK-8 assay. Compared with the NC group, the cell viability of each group showed no significant difference (P > 0.05), except for the 1000 nM group (*P < 0.05). Data are shown as the mean ± SD from three independent experiments.
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fig1: Cell survival rate at various concentrations of 2-Cl-IB-MECA. HT-29 cells were treated with various concentrations of 2-Cl-IB-MECA (5 nM to 1000 nM) for 12 hours, before cell viability was measured by using a CCK-8 assay. Compared with the NC group, the cell viability of each group showed no significant difference (P > 0.05), except for the 1000 nM group (*P < 0.05). Data are shown as the mean ± SD from three independent experiments.

Mentions: To determine whether the A3AR agonist 2-Cl-IB-MECA affected the viability of HT-29 cells, we performed a CCK-8 assay. As shown in Figure 1, no significant effect on cell viability was observed at test concentrations of up to 400 nM and the cell survival rate of each group exceeded 90%. However, when concentration was increased to 1000 nM, the cell viability was significantly downregulated compared to the control group (P < 0.05). These data demonstrated that concentrations of 2-Cl-IB-MECA that were less than 400 nM did not affect the rate of HT-29 cell survival. Based on previous reports and our preliminary experiments, the concentration of 2-Cl-IB-MECA used in subsequent studies was 10 nM, 30 nM, and 50 nM.


Activation of adenosine A3 receptor alleviates TNF-α-induced inflammation through inhibition of the NF-κB signaling pathway in human colonic epithelial cells.

Ren T, Qiu Y, Wu W, Feng X, Ye S, Wang Z, Tian T, He Y, Yu C, Zhou Y - Mediators Inflamm. (2014)

Cell survival rate at various concentrations of 2-Cl-IB-MECA. HT-29 cells were treated with various concentrations of 2-Cl-IB-MECA (5 nM to 1000 nM) for 12 hours, before cell viability was measured by using a CCK-8 assay. Compared with the NC group, the cell viability of each group showed no significant difference (P > 0.05), except for the 1000 nM group (*P < 0.05). Data are shown as the mean ± SD from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4016939&req=5

fig1: Cell survival rate at various concentrations of 2-Cl-IB-MECA. HT-29 cells were treated with various concentrations of 2-Cl-IB-MECA (5 nM to 1000 nM) for 12 hours, before cell viability was measured by using a CCK-8 assay. Compared with the NC group, the cell viability of each group showed no significant difference (P > 0.05), except for the 1000 nM group (*P < 0.05). Data are shown as the mean ± SD from three independent experiments.
Mentions: To determine whether the A3AR agonist 2-Cl-IB-MECA affected the viability of HT-29 cells, we performed a CCK-8 assay. As shown in Figure 1, no significant effect on cell viability was observed at test concentrations of up to 400 nM and the cell survival rate of each group exceeded 90%. However, when concentration was increased to 1000 nM, the cell viability was significantly downregulated compared to the control group (P < 0.05). These data demonstrated that concentrations of 2-Cl-IB-MECA that were less than 400 nM did not affect the rate of HT-29 cell survival. Based on previous reports and our preliminary experiments, the concentration of 2-Cl-IB-MECA used in subsequent studies was 10 nM, 30 nM, and 50 nM.

Bottom Line: Pretreatment with 2-Cl-IB-MECA prior to stimulation with TNF-α attenuated NF-κB p65 nuclear translocation as p65 protein decreased in the nucleus of cells and increased in the cytoplasm, inhibited the degradation of IκB-α, and reduced phosphorylated-IκB-α level significantly, compared to TNF-α-only-treated groups.Furthermore, 2-Cl-IB-MECA significantly decreased TNF-α-stimulated IL-8 and IL-1β mRNA expression and secretion, compared to the TNF-α-only treated group.These results confirm that A3AR is expressed in human colonic epithelial cells and demonstrate that its activation has an anti-inflammatory effect, through the inhibition of NF-κB signaling pathway, which leads to inhibition of downstream IL-8 and IL-1β expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, The Affiliated Hospital of Guangdong Medical College, Zhanjiang 524001, China.

ABSTRACT
To investigate the expression of adenosine A3 receptor (A3AR) in human colonic epithelial cells and the effects of A3AR activation on tumor necrosis factor alpha (TNF-α-) induced inflammation in order to determine its mechanism of action in human colonic epithelial cells, human colonic epithelial cells (HT-29 cells) were treated with different concentrations of 2-Cl-IB-MECA prior to TNF-α stimulation, followed by analysis of NF-κB signaling pathway activation and downstream IL-8 and IL-1β production. A3AR mRNA and protein were expressed in HT-29 cells and not altered by changes in TNF-α or 2-Cl-IB-MECA. Pretreatment with 2-Cl-IB-MECA prior to stimulation with TNF-α attenuated NF-κB p65 nuclear translocation as p65 protein decreased in the nucleus of cells and increased in the cytoplasm, inhibited the degradation of IκB-α, and reduced phosphorylated-IκB-α level significantly, compared to TNF-α-only-treated groups. Furthermore, 2-Cl-IB-MECA significantly decreased TNF-α-stimulated IL-8 and IL-1β mRNA expression and secretion, compared to the TNF-α-only treated group. These results confirm that A3AR is expressed in human colonic epithelial cells and demonstrate that its activation has an anti-inflammatory effect, through the inhibition of NF-κB signaling pathway, which leads to inhibition of downstream IL-8 and IL-1β expression. Therefore, A3AR activation may be a potential treatment for gut inflammatory diseases such as inflammatory bowel disease.

Show MeSH
Related in: MedlinePlus