Adenosine A2B receptor modulates intestinal barrier function under hypoxic and ischemia/reperfusion conditions

Abstract

Background: Intestinal barrier function failure from ischemia/reperfusion (I/R) and acute hypoxia has been implicated as a critical determinant in the predisposition to intestinal inflammation and a number of inflammatory disorders. Here, we identified the role of Adenosine A2B receptor (A2BAR) in the regulation of intestinal barrier function under I/R and acute hypoxic conditions.

Methods: C57BL/6J mice were used, and were randomized into three groups: Sham, I/R, IR+PSB1115 (a specific A2BAR antagonist) groups. After surgery, the small bowel was harvested for immunohistochemical staining, RNA and protein content, and intestinal permeability analyses. Using an epithelial cell culture model, we investigated the influence of hypoxia on the epithelial function, and the role of A2BAR in the expressions of tight junction and epithelial permeability. The expressions of Claudin-1, occludin and ZO-1 were detected by RT-PCR and Western-Blot. Epithelial barrier function was assessed with transepithelial resistance (TER).

Results and conclusions: The A2BAR antagonist, PSB1115, significantly increased tight junction protein expression after intestinal I/R or acute hypoxia conditions. PSB1115 also attenuated the disrupted distribution of TJ proteins. Furthermore, inhibition of A2BAR attenuated the decrease in TER induced by I/R or acute hypoxic conditions, and maintained intestinal barrier function. Antagonism of A2BAR activity improves intestinal epithelial structure and barrier function in a mouse model of intestinal I/R and a cell model of acute hypoxia. These findings support a potentially destructive role for A2BAR under intestinal I/R and acute hypoxic conditions.

Keywords: Ischemia/reperfusion; TER; ZO-1; adenosine A2B receptor; claudin-1; hypoxia; occludin; tight junction.

Figure 1

Changes in A2BAR expression under hypoxia and intestinal I/R models. A. Caco-2 cells were exposed to normoxia or hypoxia for indicated time. Total RNA was isolated, and A2BAR mRNA levels were determined by RT-PCR. Data were calculated relative to β-actin and expressed as fold change relative to normoxia. B. Induction of intestinal epithelia A2BAR protein by hypoxia. Shown here is a representative Western blot of A2BAR following incubation for indicated periods of hypoxia, with β-actin as a control. C. Induction of intestinal epithelial A2BAR mRNA levels by IR. Results are derived from three experiments (*: Different from normoxia and Sham group, P < 0.01). F-H. Immunohistochemistry for A2BAR. A2BAR expression was detected in I/R 6 h, compared with the Sham group. F. Negative control; G. Sham group exhibit weak staining; H) The structures of intestinal villus were significantly damaged of I/R injury when compared with the sham group (n = 6 per group). I. Induction of A2BAR in Caco-2 cell lines in normoxic or hypoxic conditions. Immunocytofluorescence labeling of A2BAR (red) in Caco-2 cells cultured for 6 h under normoxia, hypoxia and hypoxia plus PSB1115 (10 μM) is shown. Cellular nuclei are labeled with DAPI (blue). The positive fluorescence of A2BAR (red) is higher under the hypoxic condition than under the normoxic condition.

Figure 2

Role of an A2BAR antagonist on TJ mRNA levels under hypoxia and I/R. Caco-2 cells were pretreated with or without PSB1115, and the monolayers were then subjected to hypoxia for 6 h. The mRNA levels of the TJs were determined using RT-PCR. The data are the mean ± SD (n = 6). A-C. Hypoxia down-regulated claudin-1, occludin and ZO-1 mRNA expression, and PSB1115 attenuated the down-regulation of these TJs as induced by hypoxia. D-F. I/R down-regulated claudin-1, occludin and ZO-1 mRNA expression, and PSB1115 attenuated the down-regulation of TJ mRNA expression induced by I/R (*P < 0.05, **P < 0.01 compared to the Nx group and Sham group; ***P < 0.05, compared to the Hx. #P < 0.01, compared to the I/R group).

Figure 3

Effect of an A2BAR antagonist on the abundance of TJ proteins in Caco-2 cells exposed to hypoxia and I/R intestinal tissue. A-D. Caco-2 cells were cultured in MEM for 7 days under normoxia, incubated for 24 h in serum-free medium, and then exposed to hypoxia in the absence or presence of PSB1115 (10 μM) for 6 h. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies against claudin-1, occludin and ZO-1. The amount of each TJ protein was normalized to the GAPDH and then expressed relative to the corresponding normalized value for the normoxic condition. E-H. Claudin-1, occludin and ZO-1 was detected in Sham, I/R and I/R+PSB1115 groups. The amount of each TJ protein was normalized to the GAPDH and then expressed relative to the corresponding normalized value for the Sham group. The data are the means ± SD from three independent experiments. (*P < 0.05, **P < 0.01, compared to the Nx group and Sham group; ***P < 0.05, compared to the Hx group and IR group).

Figure 4

A2BAR inhibition prevents the disruption of TJs induced by hypoxia. Caco-2 cells were cultured in MEM for 7 days under the normoxic condition, incubated for 24 h in serum-free medium, then exposed to hypoxia in the absence or presence of PSB1115 (10 μM) and then exposed to hypoxia for 6 h. The cells were subjected to an immunofluorescence analysis using antibodies against claudin-1 (A), occludin (B) and ZO-1 (C). The data are representative of three independent experiments.

Figure 5

Effect of an A2BAR antagonist on the hypoxia-induced the changes in the TER in Caco-2 monolayers and I/R-induced changes in TER in intestinal epithelia. Hypoxia-induced reduction in the TER in Caco-2 monolayers and IR-induced reduction the TER in intestinal epithelia can be prevented by A2BAR antagonist pretreatment. The results are expressed as the mean ± SD (n = 6). A. **P < 0.01, compared to the Nx group; ***P < 0.05, compared to the Hx group. B. **P < 0.01, compared to the Sham group; #P < 0.01, compared to I/R group.