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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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Effect of 2-Cl-IB-MECA on TNF-α-induced IκB-α and p-IκB-α protein expression in HT-29 cells. HT29 cells were treated with various concentrations (10 nM, 30 nM, and 50 nM) of 2-Cl-IB-MECA for 30 min before TNF-α (10 ng/mL) stimulation for 30 min. Then total protein was extracted and analyzed by western blot. (a, c) TNF-α resulted in a reduction in IκB-α expression and an increase in p-IκB-α expression in HT-29 cells. (b, d) Compared with the TNF-α-only-treated group, 2-Cl-IB-MECA pretreatment enhanced the expression of IκB-α and attenuated the expression of p-IκB-α in a concentration-dependent manner. (e, f) Representative pictures of IκB-α protein expression in indicated groups detected by western blot. (g, h) Representative pictures of p-IκB-α protein expression in indicated groups detected by western blot. Data are shown as the mean ± SD from three independent experiments. **P < 0.05 compared with the NC group; #P < 0.05 compared with the TNF-α-only-treated group; §P < 0.05 between the indicated groups.
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fig5: Effect of 2-Cl-IB-MECA on TNF-α-induced IκB-α and p-IκB-α protein expression in HT-29 cells. HT29 cells were treated with various concentrations (10 nM, 30 nM, and 50 nM) of 2-Cl-IB-MECA for 30 min before TNF-α (10 ng/mL) stimulation for 30 min. Then total protein was extracted and analyzed by western blot. (a, c) TNF-α resulted in a reduction in IκB-α expression and an increase in p-IκB-α expression in HT-29 cells. (b, d) Compared with the TNF-α-only-treated group, 2-Cl-IB-MECA pretreatment enhanced the expression of IκB-α and attenuated the expression of p-IκB-α in a concentration-dependent manner. (e, f) Representative pictures of IκB-α protein expression in indicated groups detected by western blot. (g, h) Representative pictures of p-IκB-α protein expression in indicated groups detected by western blot. Data are shown as the mean ± SD from three independent experiments. **P < 0.05 compared with the NC group; #P < 0.05 compared with the TNF-α-only-treated group; §P < 0.05 between the indicated groups.

Mentions: To determine whether A3AR activation participates in TNF-α-induced NF-κB activation, we estimated the level of NF-κB p65, IκB-α, and phosphorylated-IκB-α using western blot after HT29 cells were treated with various concentrations (10 nM, 30 nM, and 50 nM) of 2-Cl-IB-MECA for 30 min and then incubated with TNF-α (10 ng/mL) for 30 min. As the results show, NF-κB p65 mainly resided in the cytoplasm and was almost absent from the nucleus (Figure 4.). Stimulation of HT-29 cells with TNF-α resulted in a significant shift of NF-κB p65 towards the nucleus, as p65 protein expression increased in the nucleus of the cells and decreased in the cytoplasm. We also observed a decrease in IκB-α and an increase in phosphorylated-IκB-α expression, compared to the control groups (P < 0.05, Figures 4 and 5). 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, compared to TNF-α-only-treated groups (P < 0.05, Figures 4 and 5). These results were consistent with our IF results. Moreover, the suppressive effects of 2-Cl-IB-MECA on TNF-α-induced NF-κB activation occurred in a concentration-dependent manner (Figures 4 and 5). These results show that 2-Cl-IB-MECA inhibited TNF-α-induced NF-κB activation.


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)

Effect of 2-Cl-IB-MECA on TNF-α-induced IκB-α and p-IκB-α protein expression in HT-29 cells. HT29 cells were treated with various concentrations (10 nM, 30 nM, and 50 nM) of 2-Cl-IB-MECA for 30 min before TNF-α (10 ng/mL) stimulation for 30 min. Then total protein was extracted and analyzed by western blot. (a, c) TNF-α resulted in a reduction in IκB-α expression and an increase in p-IκB-α expression in HT-29 cells. (b, d) Compared with the TNF-α-only-treated group, 2-Cl-IB-MECA pretreatment enhanced the expression of IκB-α and attenuated the expression of p-IκB-α in a concentration-dependent manner. (e, f) Representative pictures of IκB-α protein expression in indicated groups detected by western blot. (g, h) Representative pictures of p-IκB-α protein expression in indicated groups detected by western blot. Data are shown as the mean ± SD from three independent experiments. **P < 0.05 compared with the NC group; #P < 0.05 compared with the TNF-α-only-treated group; §P < 0.05 between the indicated groups.
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Related In: Results  -  Collection

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fig5: Effect of 2-Cl-IB-MECA on TNF-α-induced IκB-α and p-IκB-α protein expression in HT-29 cells. HT29 cells were treated with various concentrations (10 nM, 30 nM, and 50 nM) of 2-Cl-IB-MECA for 30 min before TNF-α (10 ng/mL) stimulation for 30 min. Then total protein was extracted and analyzed by western blot. (a, c) TNF-α resulted in a reduction in IκB-α expression and an increase in p-IκB-α expression in HT-29 cells. (b, d) Compared with the TNF-α-only-treated group, 2-Cl-IB-MECA pretreatment enhanced the expression of IκB-α and attenuated the expression of p-IκB-α in a concentration-dependent manner. (e, f) Representative pictures of IκB-α protein expression in indicated groups detected by western blot. (g, h) Representative pictures of p-IκB-α protein expression in indicated groups detected by western blot. Data are shown as the mean ± SD from three independent experiments. **P < 0.05 compared with the NC group; #P < 0.05 compared with the TNF-α-only-treated group; §P < 0.05 between the indicated groups.
Mentions: To determine whether A3AR activation participates in TNF-α-induced NF-κB activation, we estimated the level of NF-κB p65, IκB-α, and phosphorylated-IκB-α using western blot after HT29 cells were treated with various concentrations (10 nM, 30 nM, and 50 nM) of 2-Cl-IB-MECA for 30 min and then incubated with TNF-α (10 ng/mL) for 30 min. As the results show, NF-κB p65 mainly resided in the cytoplasm and was almost absent from the nucleus (Figure 4.). Stimulation of HT-29 cells with TNF-α resulted in a significant shift of NF-κB p65 towards the nucleus, as p65 protein expression increased in the nucleus of the cells and decreased in the cytoplasm. We also observed a decrease in IκB-α and an increase in phosphorylated-IκB-α expression, compared to the control groups (P < 0.05, Figures 4 and 5). 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, compared to TNF-α-only-treated groups (P < 0.05, Figures 4 and 5). These results were consistent with our IF results. Moreover, the suppressive effects of 2-Cl-IB-MECA on TNF-α-induced NF-κB activation occurred in a concentration-dependent manner (Figures 4 and 5). These results show that 2-Cl-IB-MECA inhibited TNF-α-induced NF-κB activation.

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