Sildenafil improves radiation-induced oral mucositis by attenuating oxidative stress, NF-κB, ERK and JNK signalling pathways

Abstract

Radiation-induced oral mucositis is a common and dose-limiting complication of head and neck radiotherapy with no effective treatment. Previous studies revealed that sildenafil, a phosphodiesterase 5 inhibitor, has anti-inflammatory and anti-cancer effects. In this study, we investigated the effect of sildenafil on radiation-induced mucositis in rats. Two doses of radiation (8 and 26 Gy X-ray) were used to induce low-grade and high-grade oral mucositis, separately. A control group and three groups of sildenafil citrate-treated rats (5, 10, and 40 mg/kg/day) were used for each dose of radiation. Radiation increased MDA and activated NF-κB, ERK and JNK signalling pathways. Sildenafil significantly decreased MDA level, nitric oxide (NO) level, IL1β, IL6 and TNF-α. The most effective dose of sildenafil was 40 mg/kg/day in this study. Sildenafil also significantly inhibited NF-κB, ERK and JNK signalling pathways and increased bcl2/bax ratio. In addition, high-dose radiation severely destructed the mucosal layer in histopathology and led to mucosal cell apoptosis in the TUNEL assay. Sildenafil significantly improved mucosal structure and decreased inflammatory cell infiltration after exposure to high-dose radiation and reduced apoptosis in the TUNEL assay. These findings show that sildenafil can improve radiation-induced oral mucositis and decrease the apoptosis of mucosal cells via attenuation of inflammation and oxidative stress.

Keywords: apoptosis; inflammation; oxidative stress; radiation-induced oral mucositis; sildenafil.

FIGURE 1

Histopathological changes of tongue mucosal layer after exposure to 8 Gy X‐ray. Low‐dose radiation did not markedly change the mucosal structure in histopathology. Normal control (A), irradiated control (B) and sildenafil‐treated rats [5 (C), 10 (D) and 40 (E)] all had nearly similar mucosal integrity

FIGURE 2

Histopathological changes of tongue mucosal layer after exposure to 26 Gy X‐ray. Irradiation severely distorted tongue mucosal structure in the control group (A) and nearly destructed the mucosal barrier. Sildenafil 5 (B), 10 (C) and 40 (D) mg/kg/day partly preserved the mucosal layer and significantly decreased (*p < 0.05, *, p < 0.05 and **p < 0.01, respectively) tissue pathological score. In addition, sildenafil 10 and 40 mg/kg significantly (*p < 0.05) decreased inflammatory cell infiltration

FIGURE 3

TUNEL assay of the tongue tissue after irradiation with 26 Gy X‐ray. Sildenafil 5 (B), 10 (C) and 40 (D) mg/kg/day could significantly (*p < 0.05, **p < 0.01 and ***p < 0.001, respectively) improve mucosal cells survival and decrease apoptosis, compared with saline‐treated controls (A). Red arrows are indicating apoptotic cells

FIGURE 4

Effects of sildenafil on oxidative stress and inflammatory cytokines. Radiation‐induced oral mucositis led to remarkable (&&&&p < 0.0001) increase in IL1β, IL6, TNF‐α and MDA levels. Low‐dose radiation also increased nitrite level (&&p < 0.01). Sildenafil significantly decreased MDA (***, p < 0.001), TNF‐α (**p < 0.01 for 5 mg/kg/day and ***p < 0.001 for 10 and 40 mg/kg/day), IL1β (**p < 0.01 for 5 mg/kg and ***, p < 0.001 for 10 and 40 mg/kg), IL6 (**p < 0.01 for 5 mg/kg and,***, p < 0.001 for 10 mg/kg/day and p < 0.0001,**** 40 mg/kg/day) and nitrite (*p < 0.05 for 10 mg/kg/day), compared to the control groups

FIGURE 5

Effects of sildenafil on NF‐κB and ERK signalling pathways. Induction of mucositis was associated with a significant (&&&&p < 0.0001) increase in the expression of NF‐κB and its phosphorylation. P‐ERK/β‐Actin markedly (&&&&p < 0.0001) increased after irradiation, as well. Treatment with sildenafil was associated with significant decrease in the expression of NF‐κB (***p < 0.001) p65‐NF‐κB/β‐Actin (*p < 0.05 for 10 mg/kg/day and ***p < 0.001 for 40 mg/kg/day) and p‐ERK/β‐Actin (**p < 0.01 for 5 and 10 mg/kg/day and ***p < 0.001 for 40 mg/kg/day). The same band is shown for β‐Actin of the control group and sildenafil 40 mg/kg in Figures 5 and 6 because the protein bands presented for the control group and sildenafil 40 mg/kg in Figures 5 and 6 belong to the same samples

FIGURE 6

Effects of sildenafil on JNK phosphorylation and bcl2/bax ratio. Irradiation was associated with conceivably increase (&&&&p < 0.0001) in the phosphorylation of JNK and decrease in bcl‐2/bax ratio (&&&&p < 0.0001) Treatment with sildenafil was associated with significant decrease in p‐JNK/JNK (**p < 0.01 for 10 mg/kg/day and ***p < 0.001 for 40 mg/kg/day). Sildenafil could also increase bcl2/bax ratio (*p < 0.05 for 10 mg/kg/day and **p < 0.01 for 40 mg/kg/day). The same band is shown for β‐Actin of the control group and sildenafil 40 mg/kg in Figures 5 and 6 because the protein bands presented for the control group and sildenafil 40 mg/kg in Figures 5 and 6 belong to the same samples