Edited by: Stephen J. Pandol, Cedars-Sinai Medical Center, United States
Reviewed by: Sarbjeet Makkar, Washington University in St. Louis, United States; Michael Hickey, Monash University, Australia
This article was submitted to Gastrointestinal Sciences, a section of the journal Frontiers in Physiology
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Oral mucositis (OM) is one of the main side effects of the head and neck cancer treatment, particularly radiotherapy and/or chemotherapy. OM is characterized by ulcers, erythema, dysphagia, xerostomia, and increased susceptibility to opportunistic infections. In the perspective of finding pharmacological therapies to prevent inflammation and ulceration of OM, the investigation of the pleiotropic effect of commercial drugs is needed, among them gliclazide, an antidiabetic drug. This study aimed to evaluate the effect of gliclazide in an experimental OM model induced by 5-fluorouracil. Male hamsters were pre-treated with oral gliclazide (1, 5, or 10 mg/kg) for 10 days. Cheek pouch samples were subjected to histopathological and immunohistochemical analysis (COX2, iNOS, MMP-2, NFκB P65, GPx) and imunofluorescence (P-selectin). IL-1β and TNF-α levels, Myeloperoxidase activity (MPO) and malondialdehyde (MDA) levels were investigated by ultraviolet-visible spectroscopy analysis. NFκB NLS P50 protein levels were analyzed by western blotting. The group treated with gliclazide at a dose of 10 mg/kg showed presence of erythema, no evidence of erosion, and absence of mucosal ulceration with a score of 1 (1–2) (
Oral mucositis (OM) is one of the main toxicities of cancer therapy, affecting up to 60–100% of patients undergoing radiotherapy and/or chemotherapy (
Among the chemotherapeutic agents, methotrexate, irinotecan and 5 Fluorouracil (5-FU) are associated with high prevalence of oral and intestinal mucositis, as well as anorexia, nausea and diarrhea (
The initiation phase of mucositis is characterized by radio or chemotherapy-induced direct DNA damage, that results in injury to the basal epithelial, submucosal, and endothelial cells. These cells release endogenous damage-associated molecular patterns (DAMPs), that further damage cell membranes, stimulate macrophages, and trigger molecules that activate transcription factors, including nuclear factor (NF)κB (
Treatment of OM symptoms includes anti-thermal, anti-inflammatory, antimicrobial, cryotherapy, and antiseptic analgesics, but there are still a few prophylactic therapies, namely laser therapy and keratinocyte growth factor intravenous administration (
In the perspective of finding pharmacological therapies to prevent inflammation and ulceration of OM, we highlight the need to investigate the pleiotropic effect of commercial drugs, specifically among them gliclazide, an antidiabetic drug classified as second generation sulfonylurea, with secondary anti-oxidant and anti-inflammatory effects (
Thirty (30) male Golden Sirian hamsters with a mean weight of 140–180 g were used. The hamsters were provided by the breeding room in the Department of Biophysics and Pharmacology under the appropriate hygiene conditions, at a temperature of 22°C and light/dark cycle 12 h. The animals were housed in individual cages and had free
The experimental model was performed according to the methodology described by
After euthanasia of the animals, their oral mucosa were photographed for macroscopic analysis. The evaluated parameters were the presence and intensity of erythema, hyperemia, hemorrhage, ulcers and abscesses, classified according to the following scores (
Score 0: completely healthy mucosa. No erosion or vasodilation.
Score 1: presence of erythema, but no evidence of mucosal erosion.
Score 2: severe erythema, vasodilatation and superficial erosion.
Score 3: ulcer formation on one or more faces, but not affecting more than 25% of the surface area of the pouch. Severe erythema and vasodilation.
Score 4: cumulative ulcer formation of about 50% of the pouch surface area.
Score 5: virtually complete ulceration of the mucosa of the pouch. Impossibility of mucosal exposure.
The oral mucosa was fixed in 10% buffered formaldehyde solution for 24 h, then dehydrated and embedded in paraffin. Each sample was sectioned in 4 μm thick serial sections followed by hematoxylin-eosin (H&E) staining. The specimens were analyzed by light microscopy of a simple-blind form for the inflammatory aspects, such as presence and intensity of the cellular infiltration, dilation and vascular engorgement, hemorrhage, edema, ulcers, and morphological characteristics suggestive of abscesses, classified according to the standardized scores (
Score 0: epithelium and connective tissue without vasodilation; absent or discrete cell infiltrate; absence of bleeding, edema, ulcers and abscesses.
Score 1: discrete vascular engorgement; areas of reepithelialization; discrete cell infiltrate, with greater number of mononuclear leukocytes, absence of hemorrhage, edema, ulcers and abscesses.
Score 2: moderate vascular engorgement; epithelial hydropic degeneration (vacuolization); moderate cellular infiltrate, with a predominance of mixed leukocytes; presence of hemorrhagic areas, edema, and occasional small ulcers; absence of abscesses.
Score 3: marked vascular engorgement; marked vasodilation; accentuated, mixed cell infiltrate; presence of hemorrhagic areas, edema, abscesses, and extensive ulcers.
Sections of 3-μm thick tissue embedded in paraffin were arranged on 3-aminopropyltriethoxysilane slides (Sigma Chemical Co., St. Louis, MO, United States) for immunohistochemical analysis. The samples were subsequently dewaxed with xylol, rehydrated in alcohols and washed in water and buffer solution. Antigen retrieval was performed using citrate and then blocking with bovine serum albumin (BSA) was performed at room temperature for 2 h. Endogenous peroxidase blockade was performed using 3% hydrogen peroxide for 10 min. Next, the slides were incubated for 18 h in a humidified chamber at 4°C with the following primary polyclonal antibodies (Santa Cruz Biotechnology, Interprise, Brazil): anti-COX-2 1:400; anti- metalloproteinase-2 (MMP-2) 1:400; anti-iNOS 1:400; anti-GPx 1:400, anti-NFκB P65 1:400. The sections were treated after washing with PBS.
Analyzed samples were considered positive for the expression of COX-2, MMP-2, iNOS, GPx, and NFκB P65 in mucosal tissues that exhibited brown cytoplasmic or membrane staining. A semi-quantitative analysis was performed for all antibodies following the methodology proposed by
The same immunohistochemistry protocol was used for confocal immunofluorescence, but the tissue sections were incubated overnight at 4°C with the primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, United States) to P-selectin 1:200 for 1 h. After washing in phosphate buffered saline/0.2% Triton X-100 for 5 min, the tissues were then incubated with Alexa Fluor 488-conjugated goat anti-rabbit (Abcam Inc., Cambridge, MA, United States) secondary antibody (1:500) for 1 h. Fluorescent images were obtained on a Carl Zeiss laser scanning microscope (LSM 710, 20 × objective, Carl Zeiss, Jena, Germany). Tissue reactivity in all groups was assessed by computerized densitometry analysis of the digital images captured with the confocal immunofluorescence microscope. Average densitometric values were calculated in Image J software (Wayne Rasband; National Institutes of Health, Bethesda, MD, United States).
The oral mucosa of animals treated with gliclazide (1, 5, 10 mg), 5-FU and saline were collected in 2 ml eppendorfs and conditioned under freezing at -80°C for subsequent cytokine dosing (TNF-α and IL-1β) following the protocol of
Malonyldialdehyde (MDA) is an end product of lipid peroxidation. To quantify the increase in free radicals in gingival sample, MDA content was measured via the assay previously described (
The extent of neutrophil accumulation in gingival tissue samples was measured by assaying MPO activity. Gingival tissue (4 samples per group) was harvested as described above and stored at -80°C until required for assay. After homogenization of tissue in Hexadecyltrimethylammonium bromide (1:20), 02 freeze/thaw cycles and centrifugation (5000 RPM for 20 min), the MPO activity in these samples (in units of MPO/mg tissue) was determined by a previously described colorimetric method 450 (
OM tissue samples were homogenized in RIPA lysis buffer (25 mmol/L Tris-HCL, pH 7.6; 150 mmol/L NaCl; 5 mmol/L EDTA; 1% NP40; 1% triton X-100; 1% sodium deoxycholate; 0.1% sodium dodecyl sulfatepolyacrylamide) and protease inhibitor (1 μl in 100 μl RIPA). After centrifugation (17 min, 4°C, 13,000 rpm), the supernatant was collected, and protein concentrations were determined by bicinchoninic acid assay (Thermo Fisher Scientific) according to the manufacturer’s protocol. Sodium dodecyl sulfatepolyacrylamide-polyacrylamide gel electrophoresis (10% or 8%) was performed with 40 μg of protein prepared in Laemmli sample buffer (BioRad) and denatured at 95°C for 5 min. The protein was transferred to a PVDF membrane (BioRad) for 2 h, blocked with 5% BSA for 1 h, incubated overnight with a primary antibody (mouse anti-β actin, sc-81178, 1:500, Santa Cruz Biotechnology; rabbit anti- NFKB NLS P50 -sc 114, 1:100, Santa Cruz Biotechnology and then incubated for 1 h and 30 min with a secondary antibody (goat anti-rabbit, 656120, 1:1000; or goat anti-mouse IgG, 626520, 1:500; Invitrogen). The bands were visualized with an ECL system applied according to the manufacturer’s instructions (BioRad). Chemiluminescence signal was detected with a ChemiDocTM XRS system (BioRad) and quantified densitometrically in the ImageJ software (NIH, Bethesda, MD, United States).
Data are presented as group means ± standard errors or as medians with ranges, as appropriate. Analysis of variance (ANOVA) followed by Bonferroni’s test was used to compare mean values across groups. The Kruskal–Wallis test followed by Dunn’s test was used to compare medians. The statistical analyses were conducted in Prism 5.0 software (GraphPad, La Jolla, CA, United States). A
The animals of the 5-FUT/saline group developed OM with clinical features such as severe erythema, extensive hyperemia, hemorrhagic areas, ulcers and abscesses, with a median of 4 (3–4), when compared to the normal group (
Macroscopic aspects of the samples. Normal hamster cheek pouch tissue
Photomicrograph showing the histopathological aspects of the mucosa of hamsters with 5-FU-induced oral mucositis treated with gliclazide. (SALINA): normal mucosa. (TRAUMA): discrete inflammatory infiltrate ∗. (5-FU): intense inflammatory infiltrate ∗∗∗; extensive ulceration with fibrinopurulent exudate ↔. (GLI-1FUT): focal areas of moderate inflammatory infiltrate ∗∗. (GLI-5FUT): focal areas with moderate inflammatory infiltrate ∗∗; engorged blood vessels.” (GLI-10FUT): Discrete inflammatory infiltrate ∗. (Scanner blades Panoramic Viewer – H&E, 200×). Histopathological scores of normal hamster cheek pouch tissue, tissue subjected to mechanic trauma/MT, 5FUT/saline, GLI 1-FUT, GLI 5-FUT, and GLI 10-FUT (∗
MDA showed a significant increase in the 5-FUT/saline group (
Levels of
Evaluation of inflammatory cytokines showed a significant reduction in TNF-α levels in the normal (
Representative images of NFκB P50 immunoblotting. The bands were visualized with an ECL system applied according to the manufacturer’s instructions (BioRad). The chemiluminescence signal was detected with a ChemiDocTM XRS system (BioRad) and densitometrically quantified in ImageJ software (NIH, Bethesda, MD). Normal hamster cheek pouch tissue, tissue subjected to mechanical trauma/MT, 5-FUT/saline, GLI 1-FUT, GLI 5-FUT, and GLI 10-FUT ∗∗
Immunohistochemical analysis was performed in the oral mucosa area, where immunostaining was detected in the cytoplasm and nucleus of epithelial, inflammatory, endothelial and fibroblast cells. The normal group exhibited a negative reaction for COX-2 (
Immunostaining of COX-2, iNOS, MMP-2, NFkB P65, GPx, in oral mucositis. Absent reaction for COX-2
Representative confocal photomicrographs P-selectin immunoreactivity (green) in hamster cheek pouch specimens from each group with DAPI nuclear counterstained (blue) (
Reactive oxygen species (ROS) produced by 5-FU are thought to account for many of the toxic effects, such as MDA production (
The cytotoxic effect of H2O2 is well known, including induction of apoptosis in human normal and cancer cells (
5-FU chemotherapy, which acts by the irreversible inhibition of thymidylate synthetase (TS), leads to deoxythymidine and monophosphate shortage (dTNP), damaging the DNA synthesis specifically in the S phase of the cell cycle, mainly acting on proliferative cells (
The GLI 10-FUT group showed a reduction of the anti-inflammatory activity demonstrated by the clinical findings and by the presence of erythema without evidence of mucosal erosion; as well as by histopathological findings with reepithelialization, discrete mononuclear inflammatory infiltration, absence of hemorrhage, edema, ulcers and abscesses. In relation to the inflammatory cytokines, a significant decrease in TNF-α levels was observed in the normal and GLI 10-FUT groups when compared to the 5-FUT/saline group. For IL-1β, a significant reduction was observed in the normal, MT and GLI 10-FUT groups when compared to the 5-FUT/saline group.
Another important marker of the inflammatory process is the cyclooxygenase-2 (Cox-2), which has direct participation in the formation of the prostaglandins involved in the inflammatory process (
Excessive production of nitric oxide (NO) in inflammatory processes alters microvascular permeability and plays a pro-inflammatory role (
Endothelial adhesion molecules, including E-selectin, P-selectin, ICAM-1 and vascular adhesion molecule 1 (VCAM-1), and leukocyte extravasation during inflammation are particularly regulated by pro-inflammatory NF-κB pathway, which controls transcription of adhesion molecules and mediators of inflammatory processes, such as IL-1β interleukins (
Adhesion of inflammatory cells to endothelial cells is linked to the process of radiation-induced damage. As radiation therapy continues, a steady state between mucosal cell death and regeneration may occur due to an increased cell production rate from the surviving cells, in a 05-stages process: (1) Initiation of tissue injury: radiation and/or chemotherapy induce cellular damage causing the death of the basal epithelial cells. The generation of ROS (free radicals) by radiation or chemotherapy is also believed to exert a role in the initiation of mucosal injury. These small highly reactive molecules are by-products of oxygen metabolism and can cause significant cellular damage. (2) Up-regulation of inflammation via generation of messenger signals: in addition to causing direct cell death, free radicals activate second messengers that transmit signals from receptors on the cellular surface to the inside of cell. This leads to up-regulation of pro-inflammatory cytokines, tissue injury, and cell death. (3) Signaling and amplification: up-regulation of pro-inflammatory cytokines, such as TNF-α, produced mainly by macrophages, causes injury to mucosal cells, and also activates molecular pathways that amplify mucosal injury. (4) Ulceration and inflammation: there is a significant inflammatory cell infiltration associated with the mucosal ulcerations, based in part on metabolic by-products of the colonizing oral microflora. Production of pro-inflammatory cytokines is also further up-regulated as a result of this secondary infection. (5) Healing: this phase is characterized by epithelial proliferation, as well as, cellular and tissue differentiation, restoring the integrity of the epithelium (
Recently, changes in the subepithelial ECM has been recognized as a hallmark of mucositis development. ECM is a complex structural network containing fibrous proteins, proteoglycans and glycoproteins. It provides structural support for the tissue and regulate epithelial cell kinetics. Dysregulation of ECM components, including collagen and fibronectin, has been demonstrated to closely correlate with the observation of maximal tissue damage in mucositis. The cellular receptors that bind to ECMs are referred to as CAMs and include integrins, cadherins and selectins. CAMs are well known for their role in regulation of cell kinetics through ECM regulation. Furthermore, they play a central role in inflammation, which is a key process in the pathogenesis of mucositis (
P-selectin belongs to the selectin family of leukocyte adhesion receptors and is a transmembrane protein stored in the α-granules of platelets and in the Weibel-Palade bodies of endothelial cells (
Gliclazide inhibits the adhesion of monocytes to endothelial cells, and in this way reduces the glycated albumin levels contributing to the decrease of NFκB activation, resulting in reduced transcription factors of the inflammatory process (
Several endothelial surface CAMs allow an initial selective adhesion of neutrophils to the tissue damage associated with infiltration of immunoinflammatory cells from the blood and/or lymphatic circulation (
Increase in secretion of ECM degrading enzymes, especially MMP-2 in tissue destruction of inflamed tissues in the jugal mucosa, is evidenced in the high immunolabeling of MMP-2 of the 5-FUT/saline group when compared to the absence of this protein in the GLI 10-FUT, and similar to the saline group. These results are directly due to the pathophysiology of the MMP-2 gelatinase enzyme that degrades denatured extracellular collagen of the type IV extracellular membrane in the presence of inflamed tissues and tissue destruction, differing from the physiological conditions of normal tissue that presents strict regulation of extra MMP secretion by the cells (
The results indicate that gliclazide has an anti-oxidant and anti-inflammatory effect, including leukocyte migration and influence on MDA, MPO, NFκB, IL-1β, TNF-α, COX2, MMP-2, iNOS, and P-selectin levels, improving OM (
Pharmacological modulation of the 5-FU-induced oral mucositis by Giclazide. DNA/RNA damage due to 5-fluoro-2′-deoxyuridine 5′-monophosphate (FdUMP) activates ROS, NFκB inducing the proinflammatory cytokines, such as IL-1β and TNF-α and cellular adhesion molecules, which promote inflammation and tissue damage in the oral mucosa. Gliclazide interferes with MDA, MPO, NFκB P50, NFκB P65, IL-1β, TNF-α, COX2, iNOS, MMP-2, GPx, and P-selectin, improving oral mucositis.
CAM, CXM, and AA devised the study design. CAM, CXM, RL, DC, RV, GB, GG, RA, AM, and AA participated in the interpretation of data, and drafted the manuscript. CAM, CXM, RL, DC, RV, GB, GG, RA, AM, and AA carried out the data collection, participated in the interpretation of data, and assisted in the writing the manuscript. CAM, CXM, RL, RV, GG, AM, and AA participated in the interpretation of data and drafted the manuscript. CAM and AA and performed all statistical analysis, participated in the interpretation of data, and assisted in the writing of the manuscript. All authors read and approved the final manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We would like to thank the laboratorial assistance from the Research Base of Pharmacology/UFRN/Brazil. We would also like to thank the Biosys group, Rio de Janeiro, Brazil.