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.


| INTRODUC TI ON
Oral mucositis is an acute inflammatory disease with specific causes.
Radiation-induced oral mucositis is a dose-limiting complication of head and neck cancer radiotherapy. Radiation-induced oral mucositis happens in more than 80% of patients with head and neck cancer who undergo radiotherapy. Female sex, older age, malnutrition, smoking, chronic kidney disease, poor oral hygiene and decreased secretion of salvia are associated with a higher prevalence of radiotherapy-induced oral mucositis. 1  5-Healing and proliferation. 2,3 However, numerous studies have attempted to introduce an effective remedy for oral mucositis; still, there is not a consensus regarding the management of oral mucositis and current treatment protocols focus on symptomatic management. 2 High-dose radiation leads to the release of a large amount of ROS and inflammatory cytokines and the production of pro-apoptotic proteins which endangers mucosal integrity. 4 Therefore, several groups of drugs with different mechanisms of action are presumed to mitigate the inflammatory response or contribute to the recovery of the mucosal barrier.
In this regard, growth factors, immunosuppressive agents, antioxidants and even herbal drugs with anti-inflammatory properties were investigated in animal models and clinical studies. 5 Recently, a genome-wide study has shown that perturbation of JNK-related stress can increase the susceptibility to radiation-induced oral mucositis. 6 Similarly, several studies have shown that attenuating impaired and uncontrolled NF-κB signalling can partly alleviate the inflammatory phase of oral mucositis. 7,8 JNK and NF-κB activation disrupts mucosal cells tight junction and endangers mucosal integrity. 9 Stimulation of ERK may be followed by an acceleration of the healing phase in the oral mucositis, but can also be associated with the proliferation of neutrophils and macrophages and a more severe inflammatory response. 10,11 Sildenafil is a phosphodiesterase 5 inhibitor and a vasodilator potentiating the nitric oxide system. It is commonly used in erectile dysfunction and pulmonary arterial hypertension. 12 Recently, it has been shown that the drug can improve several inflammatory and non-inflammatory diseases other than its conventional indications. 12 Phosphodiesterase 5 inhibition can alleviate inflammation and downregulate NF-κB signalling pathway. 13 In addition to its suppressive effect on NF-κB, sildenafil can attenuate JNK-and ERK-mediated inflammation, apoptosis and cell injury. 14 Sildenafil increases the bioavailability of cyclic guanosine monophosphate (cGMP) to suppress inflammation. 15 Sildenafil also enhances the healing process. 16 The drug can partly attenuate oxidative stress, prevent inflammatory cytokines release and protect against apoptosis. 12 In addition, sildenafil can protect endothelial cells against radiation-induced oxidative stress. 17 Interestingly, numerous studies have shown that sildenafil possesses anti-tumour properties that can be useful when added to radiotherapy. 12,18,19 Because of its protective effects against oxidative stress and inflammation, we hypothesized that sildenafil citrate might ameliorate radiation-induced oral mucositis.

| Animals and grouping
Forty-five male Wistar rats weighing 180-220 g were used in this study. Animals were provided by the Department of Pharmacology, Faculty of Medicine, Tehran University of Medical Sciences (TUMS).
They were housed in a temperature-controlled room (25 ± 2°C) with free access to food and water. We anaesthetised rats with ketamine (87 mg/kg) and xylazine (13 mg/kg) before irradiation and tissue sample collection. 20 They were killed with a CO 2 chamber at the end of the study.
The effect of sildenafil was measured on both low-dose (8 Gy) and high-dose (26 Gy) radiation-induced mucositis. Sildenafil citrate was dissolved in normal saline, and three doses of sildenafil citrate 5, 10 and 40 mg/kg/day were administered intraperitoneally in this study. These doses of sildenafil are usually used in rats and do not show significant adverse effects. 21,22 Rats were divided into 9 groups including one healthy control group (intact) and 8 irradiated groups as follows: low-dose X-ray control group receiving 8 Gy X-ray + normal saline, high-dose X-ray control group receiving 26 Gy X-ray + normal saline, sildenafil citrate 5 mg/kg/day +8 Gy X-ray, sildenafil citrate 5 mg/kg/day +26 Gy Xray, sildenafil citrate 10 mg/kg/day +8 Gy X-ray, sildenafil citrate 10 mg/kg/day +26 Gy X-ray, sildenafil citrate 40 mg/kg/day +8 Gy Xray and sildenafil citrate 40 mg/kg/day +26 Gy X-ray ( Table 1). Each group consisted of 5 rats.
Treatment with sildenafil or normal saline started 1 day before radiotherapy and animals were treated with a daily dose of them for 4 days. Animals were followed and their tongues specimens were collected 7 days after irradiation. Haematoxylin and eosin (H&E) staining and molecular assessments such as Western blotting, and enzyme-linked immunosorbent assay (ELISA) were performed in low-dose groups. Furthermore, H&E staining and TUNEL assay were performed in high-dose groups.

| Radiation protocol for inducing oral mucositis
We performed irradiation in a single session. The head and neck of rats (above their upper extremities) were exposed to radiation.
They received an accumulating dose of 8 Gy (4 groups) or 26 Gy (4 groups) using a conventional high-energy linear accelerator (6MV Elekta Compact Energy), at a dose rate of 2 Gy/minute, SSD (source to surface distance) 101 cm. 4 Rats were placed side-by-side in the prone position along the borders of a 40 cm × 40 cm square cone.
Four groups of rats (low-dose groups or high-dose groups) were irradiated all at the same time, and their heads were placed on the isocenter plane of the device.

| Histopathology
H&E staining was used to determine the severity of mucosal damage and measure the extent of epithelial defects, distortion of the normal mucosal structure and inflammatory cells infiltration. Furthermore, we used histopathological grading criteria for oral mucositis, as proposed by Sunavala-Dossabhoy et al. 23 (Table 2). Seven days after irradiation, rats were anaesthetised and their tongues were cut.
Tongue tissues were fixed in formaldehyde 4%. After the fixation process, slices with 5 μm thickness were prepared. Each slice was stained with H&E. An expert pathologist blinded to the samples interpreted them.

| MDA
MDA kit was used to measure the oxidative stress induced by radiation. To assess MDA level in the tongue, specimens were snapfrozen after collection. The specimens were kept at −80°C until assay. A Biocore Diagnostik (ZellBio) MDA assay Kit was used to measure the lipid peroxidation rate in tissue homogenates. The kit was used according to manufacturer's instructions. Tissue specimens or standard (100 μl) were added to wells. Then, reagent and chromogenic solution were added, respectively. After 1 h of boiling, the mixture was cooled and the absorbance of the supernatant was read at 535 nm. MDA level was measured according to the standard curve.

| Griess test
Griess test was used to assess the concentration of nitrite, a product of NO, in the mucosal tissue. Tissue homogenate was prepared, and the supernatant was separated for measuring nitrite level. To measure the tissue level of NO, a Griess reagent assay kit (Sigma-Aldrich, G4410) was used. First, tissue supernatant and an equal volume of Griess reagents (100 μl) were mixed. After 15 min, the pink colour was metered at 570 nm by a microplate spectrophotometer. Nitrite concentration was calculated against a nitrite standard.

| Western blotting
To measure the effect of radiation on the signalling pathways and apoptosis, we used Western blotting. Tongue tissue homogenate was prepared using the following chemicals as the lysis buffer (Tris-HCl  Focal or diffuse alteration of basal cell layer with nuclear atypia and ≤2 dyskeratotic squamous cells.

3
Loss of epithelium without a break in keratinization or presence of atrophied eosinophilic epithelium. An open-source image processing software, Image J, was used to assess the optical density of each band.

| Statistical analysis
Data were analysed by GraphPad Prism version 7. One-way anova followed by post hoc Tukey's test was utilized for the analysis of data. Differences were interpreted as significant when p < 0.05.
Charts are shown as mean ± SEM.

| Sildenafil decreased apoptosis of mucosal cells and could partly preserve the integrity of the mucosal barrier against radiotherapy
Rats were sacrificed 7 days after irradiation, and tissue specimens (n = 5) were collected. H&E staining was performed, and the histopathological view of specimens was interpreted. It was shown that low-grade radiation (8 Gy) could not lead to a significant change in histopathology and all groups had nearly the same normal tissue structure ( Figure 1). No defect was found in the mucosal layer integrity, and inflammatory cell infiltration into the tissue was negligible.

| Sildenafil significantly decreased the increased level of inflammatory cytokines, nitrite and MDA
However, low-dose radiation (8 Gy) showed no significant impact on the histologic view of the mucosal layer (n = 5); further measurements were performed to uncover the molecular alterations caused by irradiation and sildenafil in the tissue. We found that low-dose radiation significantly (&&&&p < 0.0001) enhanced the production of inflammatory cytokines including TNFα, IL1β, IL6 and increased MDA levels, as a marker of oxidative stress. Low-dose radiation also significantly (&&p < 0.01) increased nitrite level, as measured by the Griess test. In contrast, sildenafil partly reversed these changes and significantly decreased MDA (***p < 0.001), TNFα (**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/day and ***p < 0.001 for 10 and 40 mg/kg/ day), IL6 (**p < 0.01 for 5 mg/kg/day, ***p < 0.001 for 10 mg/kg/day and ****p < 0.0001 for 40 mg/kg/day) and nitrite level (*p < 0.05 for 10 mg/kg/day). Sildenafil 40 mg/kg was the most effective dose in this study regarding its effects on the inflammatory mediators ( Figure 4).

| Sildenafil decreased JNK phosphorylation and increased bcl-2/bax ratio
In addition to the TUNEL assay, we measured the levels of apoptosis markers and activation of the JNK pathway as a major apoptosis pathway in oral mucositis (n = 5). Western blotting showed that exposure to low-dose radiation was associated F I G U R E 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 with a significant increase (&&&&p < 0.0001) in p-JNK/JNK ratio and a significant decrease (&&&&p < 0.0001) in bcl-2/bax ratio.

| DISCUSS ION
The present study has shown that sildenafil can alleviate inflammation and decrease oxidative stress after head and neck irradiation.
Consistently, sildenafil decreased the apoptosis of mucosal cells.
In addition, the drug partly preserved mucosal structure after irradiation. The current study has shown that sildenafil partly attenuated oxidative stress, and inhibited NF-κB, ERK and JNK signalling pathways. The drug significantly decreased inflammatory cytokines including TNFα, IL1β, IL6 and nitrite levels. In addition, sildenafil also prevented mucosal cell apoptosis.
Radiotherapy leads to the production of a large amount of ROS, which can cause mucosal injury. The burst of oxidative stress can promote the inflammatory response. 24 Soon, several inflammatory mediators are produced. DNA breaks and cell damage finally upregulates pro-apoptotic pathways. 24 ROS are a major driver for radiotherapy-induced oral mucositis, and inhibition of mitochondrial ROS production was shown to be protective against postradiotherapy oral mucositis. 25 Consistently, anti-oxidants could effectively ameliorate radiation-induced oral mucositis. 26 The current study showed that sildenafil can significantly decrease the tissue concentration of MDA, a marker of oxidative stress. In addition, sildenafil decreased NO production mainly produced by iNOS (inflammatory nitric oxide) in oral mucositis to augment inflammation. 27,28 Previously, it was shown that decreased function of iNOS and decreased levels of NO metabolites are associated with a better outcome of oral mucositis. 27,28 Hence, sildenafil may help to inhibit the initiation of oral mucositis.

F I G U R E 4
Effects of sildenafil on oxidative stress and inflammatory cytokines. Radiation-induced oral mucositis led to remarkable (&&&&p < 0.0001) increase in IL1β, IL6, TNFα and MDA levels. Low-dose radiation also increased nitrite level (&&p < 0.01). Sildenafil significantly decreased MDA (***, p < 0.001), TNFα (**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 It was uncovered that head and neck cancer patients who have undergone chemoradiation therapy have higher salivary levels of inflammatory cytokines such as IL1β, IL6 and TNFα. 29 Meanwhile, higher levels of these cytokines predicted more severe oral mucositis. 29 Similarly, radiation-induced oral mucositis is associated with a significant increase in inflammatory cytokines such as IL1β and TNFα and alleviation of mucositis led to a significant decrease in inflammatory cytokines levels. 30 Successful treatment of oral mucositis is associated with a conceivable reduction in the production of inflammatory cytokines such as IL1β and TNFα. [31][32][33] Increased levels of inflammatory cytokines result in the apoptosis of mucosal cells after radiotherapy and endanger the integrity of the mucosal barrier. 34 Treatment with sildenafil effectively decreased IL1β, IL6 and TNFα in this study.
NF-κB is strongly involved in the progression and amplification of inflammatory response. Its phosphorylation and nuclear F I G U R E 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 F I G U R E 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 translocation are followed by the production of numerous inflammatory cytokines. 35 ROS can activate NF-κB to increase the production of inflammatory cytokines. 3 Hence, the NF-κB signalling pathway is responsible for amplification of inflammatory response in radiationinduced oral mucositis. 3 Downregulation of NF-κB signalling protects against oral mucositis and is associated with improvement in mucosal histology. 4,36 Radiation-induced mucositis is associated with increased ERK phosphorylation. 4 Indeed, ERK phosphorylation is a sign of inflammation and mucosal damage. 37,38 Also, decreased ERK phosphorylation is associated with the alleviation of mucositis. 39 In addition, overactivation of the ERK pathway is involved in cell cycle arrest and apoptotic cell death and attenuation of the ERK signalling pathway may prevent apoptosis. 40,41 In this study, the protective effects of sildenafil on radiation-induced oral mucositis have been associated with a significant decrease in the expression of NF-κB and phosphorylation of NF-κB and ERK.
Radiation and inflammation enhance the JNK signalling pathway. 42,43 JNK participates in intrinsic and extrinsic pathways of apoptosis. In response to environmental stress such as radiation, JNK upregulates the expression of pro-apoptotic proteins. 44 Even, JNK deletion prevents apoptotic cell death in response to ultraviolet (UV) radiation. 44 JNK decreases bcl-2/bax ratio to promote apoptotic cell death. 45 Sildenafil decreased JNK phosphorylation and increased bcl-2/bax ratio. As a result, sildenafil-treated rats showed lower apoptosis in the TUNEL assay.

| CON CLUS ION
Taken together, our findings show that sildenafil can improve radiation-induced oral mucositis and prevent apoptosis of mucosal cells. Its effect on oral mucositis has been associated with attenuation of NF-κB, ERK and JNK signalling and reduction of oxidative stress and inflammatory cytokines release. Conceptualization (equal); supervision (equal); validation (equal).

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data analyzed for this article will be available from the corresponding author upon a reasonable request.