Diallyl Disulfide Prevents Cyclophosphamide-Induced Hemorrhagic Cystitis in Rats through the Inhibition of Oxidative Damage, MAPKs, and NF-κB Pathways

Abstract

This study investigated the possible effects and molecular mechanisms of diallyl disulfide (DADS) against cyclophosphamide (CP)-induced hemorrhagic cystitis (HC) in rats. Inflammation response was assessed by histopathology and serum cytokines levels. We determined the protein expressions of nuclear transcription factor kappa-B (NF-κB), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and tumor necrosis factor-α (TNF-α), oxidative stress, urinary nitrite-nitrate, malondialdehyde (MDA), and 8-hydroxy-2'-deoxyguanosine (8-OHdG). Finally, we studied the involvement of mitogen-activated protein kinases (MAPKs) signaling in the protective effects of DADS against CP-induced HC. CP treatment caused a HC which was evidenced by an increase in histopathological changes, proinflammatory cytokines levels, urinary nitrite-nitrate level, and the protein expression of NF-κB, COX-2, iNOS, TNF-α, p-c-Jun N-terminal kinase (JNK), and p-extracellular signal regulated kinase (ERK). The significant decreases in glutathione content and glutathione-S-transferase and glutathione reductase activities, and the significant increase in MDA content and urinary MDA and 8-OHdG levels indicated that CP-induced bladder injury was mediated through oxidative DNA damage. In contrast, DADS pretreatment attenuated CP-induced HC, including histopathological lesion, serum cytokines levels, oxidative damage, and urinary oxidative DNA damage. DADS also caused significantly decreased the protein expressions of NF-κB, COX-2, iNOS, TNF-α, p-JNK, and p-ERK. These results indicate that DADS prevents CP-induced HC and that the protective effects of DADS may be due to its ability to regulate proinflammatory cytokines production by inhibition of NF-κB and MAPKs expressions, and its potent anti-oxidative capability through reduction of oxidative DNA damage in the bladder.

Keywords: Cyclophosphamide; Diallyl disulfide; Hemorrhagic cystitis; MAPKs; NF-κB; Oxidative damage.

Fig. 1.

Representative photographs of bladder sections treated with CP and/or DADS. Bladder from vehicle control (A) and DADS treated (D) rats showing normal appearance. However, bladder from a CP treated rat (B) showing inflammatory cells infiltration (black arrow) and epithelial ulceration (white arrow). Bladder from a CP&DADS treated rat (C) showing mild inflammatory cells infiltration. H&E stain. Bar=40 μm.

Fig. 2.

Effects of DADS on CP-induced changes in serum IL-1β, IL-6, and TNF-α levels. Statistical analysis was performed using one-way ANOVA followed by the Tukey’s multiple comparison test. Data are expressed as means ± SD (n=6). *p<0.05 compared with the control group. **p<0.01 compared with the control group. ^†p<0.05 compared with the CP group.

Fig. 3.

Effects of DADS on CP-induced changes in urine NOx (A), MDA (B), and 8-OHdG (C) levels. Statistical analysis was performed using one-way ANOVA followed by the Tukey’s multiple comparison test. Data are expressed as means ± SD (n=6). *p<0.05 compared with the control group. **p<0.01 compared with the control group. ^††p<0.01 compared with the CP group.

Fig. 4.

Antioxidant enzymes, GSH, and LPO in the urinary bladder of male rats treated with CP and/or DADS. Statistical analysis was performed using one-way ANOVA followed by the Tukey’s multiple comparison test. Data are expressed as means ± SD (n=6). *p<0.05 compared with the control group. **p<0.01 compared with the control group. ^†p<0.05 compared with the CP group. ^††p<0.01 compared with the CP group.

Fig. 5.

(A) Western blot analysis of NF-κB, iNOS, COX-2, and TNF-α expression in the urinary bladder of male rats treated with CP and/or DADS. Detection of β-actin expression was used a loading control. The bar graphs show quantitative relative levels of NF-κB (B), iNOS (C), COX-2 (D), and TNF-α (E) protein expression for vehicle, CP, CP&DADS, and DADS-treated rats. Values are presented as means ± SD (n=3). **p<0.01 compared with the control group. ^††p<0.01 compared with the CP group.

Fig. 6.

(A) Western blot analysis of p-JNK, p-ERK, and p-p38 expression in the urinary bladder of male rats treated with CP and/or DADS. The bar graphs show quantitative relative levels of p-JNK (B), p-ERK (C), and p-p38 (D) protein expression for vehicle, CP, CP&DADS, and DADS-treated rats. Values are presented as means ± SD (n=3). *p<0.05 compared with the control group. **p<0.01 compared with the control group. ^††p<0.01 compared with the CP group.