Allopurinol Protects against Ischemia/Reperfusion-Induced Injury in Rat Urinary Bladders

Abstract

Bladder ischemia-reperfusion (I/R) injury results in the generation of reactive oxygen species (ROS) and markedly elevates the risk of lower urinary tract symptoms (LUTS). Allopurinol is an inhibitor of xanthine oxidase (XO) and thus can serve as an antioxidant that reduces oxidative stress. Here, a rat model was used to assess the ability of allopurinol treatment to ameliorate the deleterious effects of urinary bladder I/R injury. I/R injury reduced the in vitro contractile responses of longitudinal bladder strips, elevated XO activity in the plasma and bladder tissue, increased the bladder levels of tumor necrosis factor-α (TNF-α), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase, reduced the bladder levels of extracellular regulated kinase (ERK), and decreased and increased the bladder levels of Bcl-2 and Bax, respectively. I/R injury also elevated lipid peroxidation in the bladder. Allopurinol treatment in the I/R injury was generated significantly ameliorating all I/R-induced changes. Moreover, an in situ fluorohistological approach also showed that allopurinol reduces the generation of intracellular superoxides enlarged by I/R injury. Together, the beneficial effects of allopurinol reducing ROS production may be mediated by normalizing the activity of the ERK, JNK, and Bax/Bcl-2 pathways and by controlling TNF-α expression.

Figure 1

Concentration-contraction curves obtained by cumulative addition of carbachol (CCh) to bladder strips. Each point represents the mean ± SEM of 15 bladder strips. ∗ indicates that I/R + Allo group is significantly different from I/R + S group (P < 0.05). † indicates that I/R + S group is significantly different from sham-operated groups (P < 0.01). Values are expressed as gram tension per 100 mg wet tissue. Sham-operated plus saline-pretreated group (Sham + S), ischemia-reperfusion and saline-pretreated group (I/R + S), and ischemia-reperfusion and allopurinol-pretreated group (I/R + Allo).

Figure 2

(a) Results of xanthine oxidase (XO) in plasma and bladder tissue. Values are expressed as mU/mg protein. In each of the experiments, XO activity obtained with Sham + S group was normalized as 1.0. (b) Protein levels of XO in bladder tissue. Expression level of xanthine oxidase was normalized to GAPDH. Bars indicated mean ± SEM of 15 rats. ∗ indicates that I/R + S group is significantly different from sham-operated groups (P < 0.01). † indicates that I/R + Allo group is significantly different from I/R + S group (P < 0.05).

Figure 3

TNF-α protein level in the rat urinary bladder. Each bar represents the mean ± SEM. Values are expressed as pg/mg protein. ∗ indicates that TNF-α level in I/R + S group is significantly different from Sham + S group (P < 0.01). † indicates that I/R + Allo group is significantly different from I/R + S group (P < 0.05).

Figure 4

Effect of allopurinol on MAPKs and apoptosis pathway. (a) Western blot analysis of ERK, JNK1, p38, Bax, and Bcl-2. Expression levels of each protein were normalized to GAPDH. (b) Western blot analysis of ERK, Phospho-ERK, JNK, Phospho-JNK, p38, and Phospho-p38. ^∗ P < 0.05, I/R + S group compared with the sham-operated group. ^† P < 0.05, ^†† P < 0.01, I/R + S group versus I/R + Allo. Data show mean ± SEM of n = 15 replicates.

Figure 5

Malondialdehyde (MDA) levels in bladder tissue. ∗ indicates that I/R + S group is significantly different from sham-operated groups (P < 0.01). † indicates that I/R + Allo group is significantly different from I/R + S groups (P < 0.05). Bars indicate mean ± SEM. Values are expressed as nmol/mg protein.

Figure 6

Detection of in situ superoxide in bladder tissue by DHE staining. I/R + S group showed significantly increased signal intensities compared with Sham + S group. Compared with I/R + S group, the DHE fluorescence intensity in the bladder of I/R + Allo group was significantly decreased. Images were acquired at identical settings and are representative of similar results obtained from three independent experiments. (a) Sham + S; (b) I/R + S; (c) I/R + Allo. The scale bar indicates 50 mm (bottom). (d) Intensity values are expressed as arbitrary fluorescence units. ∗ indicates that I/R + S group is significantly different from sham-operated groups (P < 0.05). † indicates that I/R + Allo group is significantly different from I/R + S groups (P < 0.05). Bars indicate mean ± SEM.