Metformin and Sildenafil Attenuate Inflammation and Suppress Apoptosis After Ischemia/Reperfusion Injuries in Rat Urinary Bladder

Abstract

Purpose: Although metformin and sildenafil can protect various organs against ischemia/reperfusion (I/R) injuries, their effects and mechanisms of action in bladder I/R injuries remain unknown. This study investigated the effects and mechanisms of action of metformin and sildenafil against bladder I/R insults in rats.

Methods: One hundred male Sprague-Dawley rats were randomly divided into 5 groups, each of which contained 20 rats: a sham-operated group, a bladder I/R group, and bladder I/R groups treated with metformin, sildenafil, or both agents. Ischemia was induced by clamping the bilateral common iliac arteries with atraumatic vascular clamps for 2 hours, followed by reperfusion for 7 days. During this period, rats were injected once daily with 4-mg/kg metformin and/or 1-mg/kg sildenafil.

Results: I/R injuries induced increased malondialdehyde levels and myeloperoxidase activity and decreased superoxide dismutase activity. These changes were attenuated by treatment with metformin and/or sildenafil. The I/R group had significantly higher Jun N-terminal kinase, p38 mitogen-activated protein kinase (MAPK), Bax, caspase-3, and nuclear factor-kappa B (NF-κB) levels, and lower extracellular signal-regulated kinase, and Bcl-2 levels in the bladder than the sham-operated group; these changes were significantly ameliorated by metformin and/or sildenafil treatment. No differences in the levels of these markers were observed between rats coadministered metformin and sildenafil and those treated with either agent alone.

Conclusion: Metformin and sildenafil protected the rat bladder against I/R injuries. This effect may have been due to the inhibition of reactive oxygen species production through MAPK, Bax, and Bcl-2 activation, and the restoration of inflammation through NF-κB inhibition. However, the combination of metformin and sildenafil was not more effective than either agent alone.

Keywords: Ischemia-reperfusion; Metformin; Urinary bladder.

Fig. 1.

The effects of metformin and sildenafil on mitogen-activated protein kinase expression in bladder tissue. (A) Images and graphs of reverse-transcription polymerase chain reaction analyses of relative levels of extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK), and p38 mRNAs in bladder tissue samples. (B) Images and graphs of Western blot analyses of relative levels of ERK, JNK, and p38 proteins in bladder tissue. Results presented as mean±standard error of the mean. p-ERK, phospho-ERK; p-JNK, phospho-JNK. ^*P<0.05, ^**P<0.01 compared with the sham-operated group. ^†P<0.05, ^‡P<0.01 compared with the ischemia/ reperfusion (I/R) group.

Fig. 2.

The effects of metformin and sildenafil on Bax and Bcl-2 expression in bladder tissue. (A) Image and graphs of reverse-transcription polymerase chain reaction (RT-PCR) analysis of the relative expression of Bax and Bcl-2 mRNA and the Bax/Bcl-2 mRNA ratio in bladder tissue samples. (B) Images and graphs of Western blot analysis of the relative expression of Bax and Bcl-2 proteins and the Bax/Bcl-2 protein ratio in bladder tissue samples. Results are presented as mean±standard error of the mean. ^*P<0.05, ^**P<0.01 compared with the sham-operated group. ^†P<0.05, ^‡P<0.01 compared with the ischemia/reperfusion (I/R) group.

Fig. 3.

The effects of metformin and sildenafil on nuclear factor-kappa B (NF-κB) expression in bladder tissue. (A) Representative images of reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting analyses of NF-κB expression. (B) Graphs of RT-PCR and Western blot analyses of the relative expression of NF-κB mRNA and protein. Results are presented as mean±standard error of the mean. ^**P<0.01 compared with the sham-operated group. ^‡P<0.01 compared with the ischemia/reperfusion (I/R) group.

Fig. 4.

The relative expression of ERK, JNK, p38, Bax, Bcl-2, NF-κB, and caspase-3. (A) mRNA levels (analyzed by reverse-transcription polymerase chain reaction) and (B) protein levels (analyzed by Western blotting) in bladder tissues of ischemia/reperfusion-subjected rats treated with metformin or metformin and sildenafil. The expression of 7 factors was analyzed. ERK, extracellular signalregulated kinase; JNK, Jun N-terminal kinase; NF-κB, nuclear factor-kappa B; IR, ischemia/reperfusion.

Fig. 5.

The activity of malondialdehyde (MDA), myeloperoxidase (MPO), and superoxide dismutase (SOD) enzymes in the 5 groups of rats. ^*P<0.05 compared with the control group. ^†P< 0.05 compared with the ischemia/reperfusion (I/R) group.

Fig. 6.

The effects of metformin and sildenafil on the expression of caspase-3 and cleaved caspase-3 in bladder tissue. (A) Immunohistochemical analysis of cleaved caspase-3 expression in the sham-operated (a), ischemia/reperfusion (I/R) (b), I/R plus metformin (c), I/R plus sildenafil (d), and I/R plus metformin and sildenafil (e) groups. The brown staining indicates caspase-3 expression. The scale bar indicates 100 μm (bottom). (B) Images and graphs of reverse-transcription polymerase chain reaction and Western blot analyses of the relative expression of caspase-3 mRNA and protein. Results are presented as mean±standard error of the mean. ^**P<0.01 compared with the sham-operated group. ^††P<0.01 compared with the I/R group.