Sulforaphane attenuates 5-FU–induced submandibular gland injury via SIRT1/Nrf2/HO-1 pathway

Abstract

AimThis study evaluated whether sulforaphane (SFN) prevents 5-fluorouracil (5-FU)-induced submandibular gland injury and investigated the involvement of the sirtuin 1 (SIRT1)/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling axis.MethodsForty male Wistar rats were divided into four groups (n = 10). Controls received vehicles only. The SFN group received oral SFN (20 mg/kg/day) for 14 days. The 5-FU group received intraperitoneal 5-FU (35 mg/kg/day) on days 10–14. The combination group received SFN for 14 days with 5-FU co-administered on days 10–14. Submandibular glands were collected for histology, biochemical assays, and gene expression analysis. Oxidative stress markers (malondialdehyde [MDA], superoxide dismutase [SOD], catalase [CAT], reduced glutathione [GSH]), inflammatory mediators (nuclear factor kappa B [NF-κB], tumor necrosis factor alpha [TNF-α], interleukin-6 [IL-6], interleukin-1 beta [IL-1β]), apoptotic regulators (B-cell lymphoma 2 [Bcl-2]-associated X protein [Bax], Bcl-2, caspase-3), and SIRT1/HO-1 protein levels were measured. Nrf2 messenger ribonucleic acid (mRNA) expression was quantified using qRT- polymerase chain reaction (PCR).Results5-FU significantly increased MDA, pro-inflammatory cytokines, NF-κB, Bax, and caspase-3 levels while reducing SOD, CAT, GSH, Bcl-2, SIRT1, Nrf2, and HO-1 (p<0.0001). Histology showed acinar destruction, edema, and intercellular widening. SFN administration before and during 5-FU exposure reversed these changes by restoring redox balance, suppressing inflammation and apoptosis, activating the SIRT1/Nrf2/HO-1 pathway, and largely preserving glandular architecture.ConclusionSFN mitigates 5-FU-induced submandibular gland injury by activating SIRT1/Nrf2/HO-1 signaling and suppressing oxidative stress, inflammation, and apoptosis, supporting its potential as an adjunctive therapy for chemotherapy-associated salivary gland dysfunction.

Keywords

Introduction

Oral mucositis represents a frequent and debilitating complication of radiotherapy and chemotherapy for head and neck cancers, especially following treatment with DNA synthesis inhibitors. Its clinical severity and progression are influenced by a complex interplay of patient-specific factors, treatment regimens, and the concurrent use of chemo- and radiotherapy, ultimately compromising oral health and patient quality of life.¹
5-Fluorouracil (5-FU), a cornerstone fluoropyrimidine chemotherapeutic agent, is widely employed against head and neck malignancies.² Its primary mechanism involves the inhibition of thymidylate synthase (TYMS), leading to a critical depletion of thymidine monophosphate (dTMP) and subsequent disruption of DNA replication.³ This action not only triggers genomic instability and programmed cell death but also induces significant oxidative stress.⁴ Clinically, 5-FU administration is associated with salivary gland dysfunction, including altered salivary composition, reduced flow rate (xerostomia), and elevated oxidative damage within the submandibular and sublingual glands, effects whose precise mechanisms remain partially elucidated.⁵
Sulforaphane (SFN), a bioactive isothiocyanate abundant in cruciferous vegetables like broccoli and Brussels sprouts, is recognized for its multifaceted health-promoting properties, including anti-cancer, anti-inflammatory, and cytoprotective activities.⁶ A key mediator of its effects is the potent activation of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), a master regulator of cellular antioxidant responses.⁷
Given this rationale, the present study was designed to investigate the potential of sulforaphane to confer cytoprotection against 5-FU-induced injury in the submandibular gland. Using a male albino rat model, we aimed to elucidate whether SFN can mitigate 5-FU-associated glandular damage. To our knowledge, this is the first study to explore SFN's protective role in this specific context. We further hypothesized that its mechanism involves the modulation of the sirtuin 1 (SIRT1)/Nrf2/heme oxygenase-1(HO-1) signaling axis, thereby reducing oxidative stress and subsequent cell death pathways, including apoptosis and inflammation.

Materials and Methods

AnimalsForty male Wistar albino rats (170–210 g body weight, 5–9 weeks of age) were utilized in the study. The animals were group-housed (five rats per cage) in standard polypropylene cages under controlled environmental conditions, including a temperature range of 21–25 °C and a standardized 12-hour light/dark cycle. Throughout the acclimatization and experimental periods, rats were provided ad libitum access to a standard laboratory pellet diet and filtered water.Study Design and Tissues SamplingThe experiment utilized a randomized design with four distinct treatment groups, each containing ten rats. The dosing protocols and their scientific justifications were as follows:
Group 1 (Control): Animals received only the experimental vehicles. For 14 days, they were orally administered salad oil (the vehicle for sulforaphane). From the 10th to the 14th day, they additionally received intravenous injections of saline (the vehicle for 5-fluorouracil).
Group 2 (SFN-only): Rats were orally administered SFN at a dose of 20 mg per kilogram of body weight (mg/kg), once daily for 14 consecutive days. This dosage is consistent with established protocols shown to be effective in mitigating drug-induced intestinal injury.⁸ The SFN (Sigma, USA) was dissolved in salad oil for oral gavage.
Group 3 (5-FU-only): This group received intraperitoneal injections of 5-FU at 35 mg/kg/day from the 10th to the 14th day of the study. This dose and administration schedule align with prior preclinical models investigating 5-FU-induced toxicity.⁹ The 5-FU (Sigma Co., USA) was prepared as a 50 mg/mL solution in 1 N ammonium hydroxide.
Group 4 (SFN + 5-FU): Animals underwent a combined treatment regimen. They were pre-treated with oral SFN (20 mg/kg/day) for the first 9 days. Subsequently, from the 10th to the 14th day, they received both the ongoing daily SFN and the daily intraperitoneal injections of 5-FU (35 mg/kg/day). This design allows for the assessment of SFN's potential protective effect against 5-FU toxicity. The selected doses for both 5-FU and SFN were determined based on evidence from previous scientific literature and were further validated by preliminary experiments conducted prior to this study.
At the conclusion of the study, euthanasia was performed under deep anesthesia, achieved via an intraperitoneal injection of urethane hydrochloride at a standardized dose of 1 gram per kilogram of body weight. Immediately following, the submandibular salivary glands were surgically removed. Each pair of glands was processed using a bilateral division protocol to enable both microscopic and molecular analyses. One gland from each animal was allocated for histological examination. This tissue sample was fixed in a 10% formalin solution and subsequently prepared for microscopic evaluation through paraffin embedding and sectioning. The corresponding contralateral gland was processed for biochemical assays. After being weighed and rinsed with chilled saline, the tissue was homogenized on ice using a glass homogenizer. The homogenization was performed in a cold phosphate buffer solution (0.01 M concentration, pH 7.4) at a standardized tissue-to-buffer ratio of 1 gram to 5 milliliters. The homogenate was then centrifuged at 5000 revolutions per minute for a duration of 15 minutes to separate cellular debris. The resulting supernatant, containing the soluble cellular components, was divided into aliquots in microcentrifuge tubes and stored at -80 °C to ensure stability of the biochemical parameters until analysis.Analysis of Oxidative Stress and Antioxidant Markers in Submandibular TissuesThe oxidative stability and antioxidant capacity within the salivary gland tissue were evaluated using a series of colorimetric assays. Commercial assay kits (Biodiagnostic, Egypt) were employed, with all procedures strictly following the manufacturer's established protocols. The following specific biomarkers were quantified: The activity levels of key antioxidant enzymes, including superoxide dismutase (SOD), and catalase (CAT), were measured to assess the tissue's intrinsic antioxidant defense capability. The extent of lipid peroxidation, a direct indicator of oxidative damage, was determined by measuring the concentration of malondialdehyde (MDA), a stable end-product of this process. Additionally, the concentration of reduced glutathione (GSH), a major non-enzymatic cellular antioxidant, was assayed.Histological Preparation and Light Microscopy AnalysisSubmandibular salivary gland (SMG) tissue was collected immediately after euthanasia. Samples were immersion-fixed in 10% neutral buffered formalin, processed through a standard graded ethanol dehydration series, and embedded in paraffin wax. Serial sections were cut at a thickness of 4 µm. For staining, sections were first deparaffinized in xylene and then rehydrated through a descending ethanol gradient (100% to 75%), followed by rinsing in tap water. Nuclear staining was performed using Gill II hematoxylin (Leica) for one minute, after which slides were rinsed under running tap water for three minutes. Cytoplasmic counterstaining was achieved using Eosin Y solution (Leica) for five minutes. After staining, sections were dehydrated through an ascending ethanol series (95% to 100%), cleared in xylene, and permanently mounted with a xylene-based mounting medium. Prepared slides were examined under a light microscope.Quantification of SIRT1, HO 1, Inflammatory, and Apoptotic Markers in Salivary Gland TissueThe concentrations of SIRT1, HO 1, nuclear factor kappa B (NF κB), interleukin-6 (IL 6), tumor necrosis factor alpha (TNF α), interleukin-1 beta (IL 1β), B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), Bcl-2 and caspase 3 in submandibular salivary gland tissue were quantified using commercial enzyme linked immunosorbent assay (ELISA) kits (Catalog Nos.: MBS2600246, MBS764989, EEL138, ERA31RB, KRC3011, BMS630, MBS2512405, E02B0033 and MBS261814, respectively). All assays were performed strictly according to the manufacturers’ protocols.Gene Expression Analysis of Nrf2 via Quantitative Real-Time Polymerase Chain Reaction (PCR)Total RNA was extracted from approximately 30 mg of submandibular salivary gland tissue using a TRIzol reagent–based protocol adapted from Badawy et al.¹⁰ RNA purity and concentration were determined spectrophotometrically by measuring the A260/A280 ratio. Subsequently, complementary DNA (cDNA) was synthesized from the purified RNA using a commercial high capacity reverse transcription kit. Quantitative real time PCR (qRT PCR) was performed to assess the expression of Nrf2, employing sequence specific primers designed according to the manufacturer’s guidelines. The primers used were: Nrf2 (NM_031789.2), Forward 5’-TTGTAGATGACCATGAGTCGC-3’ and Reverse 5’-TGTCCTGCTGTATGCTGCTT-3’; and the housekeeping gene β actin (NM_031144.3), Forward 5’-TACAACCTTCTTGCAGCTCCT-3’ and Reverse 5’-CCTTCTGACCCATACCCACC-3’. Relative messenger ribonucleic acid (mRNA) expression levels were calculated based on standard qRT PCR analytical methods.Ethical ApprovalThe study was approved by the Ethics Committee of Saveetha Dental College (Date: 10.04.2026, Decision No: SRB/SDC/ENDO-2301/25/444).Statistical AnalysisData are shown as mean ± SD. Group comparisons used one-way ANOVA, followed by Tukey's honestly significant difference (HSD) post hoc test for pairwise analyses. Statistical significance was set at p < 0.05. All analyses were performed with GraphPad Prism 8.3.0.Reporting GuidelinesThis study is reported in accordance with the ARRIVE guidelines for animal research.

Results

Antioxidant Effect of SFN Against 5 FU–Induced Submandibular Oxidative DamageAdministration of 5 FU resulted in a significant elevation of the oxidative stress marker MDA (p < 0.0001), accompanied by a marked reduction in the activities of the antioxidant enzymes SOD, CAT, and GSH when compared with the control group. Conversely, co treatment with SFN alleviated the oxidative insult induced by 5 FU, as demonstrated by the substantial decrease in MDA levels and the restoration of GSH content along with SOD and CAT activities (Figure 1A–C). These findings further support the well established antioxidant potential of SFN in counteracting 5 FU–associated oxidative stress within the salivary glands.Histological ResultsFigure 2 illustrates the morphology of the submandibular gland in the different experimental groups. In both the control and SFN only treated groups, the submandibular gland sections exhibited normal histological architecture, showing intact acini and ducts (Figure 2A–D). In contrast, the 5 FU–treated group demonstrated markedly disordered glandular morphology, with destroyed acini, noticeable spacing between them, and pronounced oedema (Figure 2E–H). However, the combined treatment group showed clear histological improvement, with acini and ducts appearing more preserved, although mild localized oedema was still observed around some ducts (Figure 2I, J). These findings collectively support the protective role of SFN against 5 FU–induced submandibular gland injury.SFN Activates SIRT1 and Stimulates the Nrf2/HO 1 Pathway to Suppress OxidationAs shown in Figure 3A–C, intraperitoneal administration of 5 FU resulted in a significant reduction in SIRT1 protein levels in submandibular gland homogenates, as determined by ELISA. Moreover, 5 FU enhanced oxidation activity, as evidenced by the marked downregulation of Nrf2 gene expression and decreased HO 1 protein levels compared with normal tissues. In contrast, rats in the SFN + 5 FU group displayed a substantial elevation in SIRT1 levels along with pronounced suppression of oxidative stress, demonstrated by enhanced activation of the Nrf2/HO 1 pathway relative to the 5 FU group. Collectively, these findings suggest that SFN exerts an upregulatory effect on the SIRT1/Nrf2/HO 1 axis, thereby mitigating oxidative alterations in the submandibular gland.SFN Halts 5 FU–Induced Submandibular Inflammation and ApoptosisAdministration of 5 FU induced significant submandibular gland inflammation, as evidenced by the marked elevation of NF κB levels measured by ELISA, accompanied by increased cytokines TNF α, IL 6, and IL 1β (Figure 3D–G). In addition, 5 FU triggered submandibular apoptosis, shown by elevated pro apoptotic Bax and caspase 3 levels, along with a reduction in the anti apoptotic protein Bcl 2 compared with normal animals (Figure 3I–J). Interestingly, SFN exhibited strong anti inflammatory and anti apoptotic effects in the submandibular gland against 5 FU toxicity, demonstrated by a significant reduction in inflammatory markers and modulation of both apoptotic and anti apoptotic proteins. Hence, the antioxidant, anti inflammatory, and anti apoptotic properties of SFN collectively explain its protective role in preserving normal submandibular gland morphology.

Discussion

Salivary gland injury results from a disruption in the balance among oxidative stress, inflammatory responses, and cellular death. The chemotherapeutic agent 5-fluorouracil (5-FU) induces this imbalance. Additionally, most chemotherapy drugs present significant risks to the structural and functional integrity of the salivary glands.¹¹
The transcription factor Nrf2 plays a pivotal role in orchestrating the antioxidant response. During oxidative stress, Nrf2 dissociates from its cytoplasmic inhibitor Kelch-like ECH-associated protein 1 (Keap1) and translocates to the nucleus, where it induces the transcription of a wide array of cytoprotective genes essential for restoring cellular redox homeostasis.¹² Previous research has shown that 5 FU disrupts this protective mechanism by suppressing the Nrf2/HO 1 pathway.¹³ Consistent with these findings, the present results demonstrate that 5 FU downregulates Nrf2 expression, decreases antioxidant enzyme activity, and increases MDA levels.
In contrast, SFN acts as a potent Nrf2 activator. It enhances the expression of Nrf2 and its downstream antioxidant enzymes, thereby reducing oxidative stress and lipid peroxidation. These findings are in agreement with the work of Ibrahim,¹⁴ who reported that SFN exerts organoprotective effects through Nrf2 mediated increases in total antioxidant capacity and catalase levels, accompanied by reduced MDA production. Mechanistically, SFN modifies specific cysteine residues on Keap1, disrupting the Keap1–Nrf2 interaction and promoting Nrf2 nuclear translocation. This activation leads to upregulation of detoxifying and antioxidant enzymes, reinforcing cellular defenses against oxidative stress and injury.¹⁵ Furthermore, the upregulatory effect of SFN on Nrf2 observed in the present study may also be attributed to its activation of SIRT1. SIRT1 stimulation is known to enhance Nrf2 activation, providing an additional mechanism through which SFN supports antioxidant signaling. This interpretation aligns with previous findings by Zhang and Wu,¹⁶ who demonstrated that SIRT1 positively regulates Nrf2, thereby amplifying cellular resilience to oxidative damage.
The results of this study confirm that 5-fluorouracil (5-FU) treatment induces a pronounced inflammatory state in the submandibular gland, characterized by significantly elevated levels of the mediators NF-κB, TNF-α, IL-1β, and IL-6. This finding aligns with existing literature, such as the work by Bomfin et al.,¹⁷ which established that 5-FU exacerbates salivary gland inflammation by stimulating pro-inflammatory cytokine release and recruiting inflammatory cells. Conversely, treatment with SFN effectively attenuated the rise in these inflammatory markers, a result consistent with the earlier findings of Heiss et al.¹⁸ The anti-inflammatory mechanism of SFN is closely linked to its activation of the Nrf2 pathway, which physiologically antagonizes the NF-κB signaling cascade. This counter-regulation occurs through multiple mechanisms: Nrf2 activation upregulates antioxidant and detoxifying enzymes, thereby diminishing the oxidative stress that normally triggers NF-κB activity. Furthermore.
Research has established that 5-FU is an inducer of programmed cell death, or apoptosis.¹⁹ This is confirmed by the present study's results, which demonstrate that 5-FU administration significantly increases the levels of the pro-apoptotic proteins caspase-3 and Bax while decreasing the anti-apoptotic protein Bcl-2 within submandibular gland tissue. This shift in the balance of apoptotic regulators indicates a potent activation of the cell death pathway. Treatment with SFN effectively countered this process, normalizing the expression of these key apoptotic markers and thereby halting 5-FU-induced cell death, a finding consistent with the earlier work of Wang et al.²⁰ The protective, anti-apoptotic action of SFN is primarily attributable to its potent antioxidant and anti-inflammatory properties, which mitigate the upstream cellular stresses that trigger apoptosis.

Limitations

This study has several limitations. Findings from male Wistar rats may not translate to humans or capture sex-specific responses. The 14-day single-dose regimen does not reflect chronic chemotherapy or dose-response relationships. Lack of specific inhibitors or gene knockouts limits causal confirmation of SIRT1/Nrf2/HO-1 activation. Salivary flow was not measured, so functional recovery is uncertain. Prophylactic SFN administration leaves the therapeutic efficacy after established injury unknown.

Conclusion

This study reveals that SFN effectively protects the submandibular gland from 5-FU-induced damage. The protective mechanism is driven by SFN's activation of the SIRT1/Nrf2 signaling pathway, which enhances cellular antioxidant defenses and reduces oxidative stress. Furthermore, SFN mitigates inflammation and apoptosis by inhibiting NF-κB and modulating apoptotic protein expression. Therefore, SFN may exert protective effects in an experimental model of submandibular gland injury.
However, its clinical applicability requires further investigation in human studies.

Declarations

Abbreviations

References

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