Fisetin attenuates cyclophosphamide-induced cystitis in rats via activation of the AMPK/SIRT1/Nrf2 pathway

Authors

Abstract

AimThis study evaluated the uroprotective effect of fisetin against cyclophosphamide (CYP)-induced hemorrhagic cystitis and explored its underlying mechanisms.
MethodsRats were divided into four groups: Control, Fisetin-only, CYP, and CYP + fisetin. The CYP + fisetin group received oral fisetin (10 mg/kg/day) alongside a single CYP injection. Bladder tissues were analyzed histologically and biochemically.
ResultsFisetin co-treatment significantly mitigated CYP-induced histological damage. It activated the adenosine monophosphate-activated protein kinase (AMPK)/SIRT1 pathway, which upregulated the Nrf2/HO-1 antioxidant axis, reducing lipid peroxidation and restoring superoxide dismutase activity. Concurrently, fisetin inhibited NF-κB, lowering inflammatory cytokines (TNF-α, IL-6, IL-1β), and suppressed apoptosis by reducing caspase-3.
ConclusionThe findings demonstrate that fisetin protects against cystitis by co-activating AMPK/SIRT1, which enhances antioxidant defenses via Nrf2 and attenuates inflammation and apoptosis via NF-κB inhibition, positioning it as a promising therapeutic candidate.

Keywords

Introduction

CYP belongs to the oxazaphosphorine class of alkylating agents. As a potent antineoplastic medication, it is commonly used to treat malignancies, including breast cancer, lymphoma, and leukemia. It also serves as an effective immunosuppressive therapy for autoimmune disorders. The drug exerts its cellular toxicity primarily by alkylating guanine bases in DNA. This action inhibits the uncoiling and separation of DNA double strands, thereby blocking cell division and ultimately leading to apoptosis. While cyclophosphamide affects both actively cycling and quiescent cells, it demonstrates greater toxicity toward rapidly dividing cells.1,2
The urinary bladder is a hollow, elastic muscular organ responsible for storing urine and periodically expelling it. Its epithelial lining has a relatively slow turnover rate of three to six months, compared to more rapidly renewing tissues like the epidermis and intestinal epithelium, which replace themselves every 1.5 to 30 days.3,4 Cystitis, or inflammation of the urinary bladder, can result from various factors, including infection, certain medications, and radiation therapy.5
The primary mechanism behind ifosfamide-induced cystitis is the direct exposure of the bladder lining to acrolein, a toxic metabolite produced when the drug breaks down.6 This metabolite is filtered by the kidneys and becomes concentrated in the urine. Upon contact with the urothelium, acrolein triggers significant cell death (apoptosis and necrosis), resulting in ulceration, swelling, and inflammation.7 This damage is further driven by the release of proinflammatory cytokines, such as TNF-α and IL-1β. Additionally, acrolein promotes the generation of reactive oxygen species and nitric oxide, which combine to form a potent oxidant called peroxynitrite. This compound causes oxidative damage through lipid peroxidation, exacerbating injury to the bladder tissue.8
Plant-based bioactive compounds show promise in modulating key biological pathways associated with chronic illnesses such as inflammatory disorders, cancer, and metabolic and degenerative diseases.9 One such compound, fisetin (3,3’,4’,7-tetrahydroxyflavone), is a lipophilic polyphenol predominantly present in strawberries, as well as in a variety of other produce, including apples, grapes, onions, cucumbers, and leafy greens like spinach.10 Research indicates that fisetin exhibits a wide spectrum of therapeutic properties, encompassing antioxidant, anti-inflammatory, antimicrobial, bone-protective, anti-diabetic, and anti-cancer activities.11 The antioxidant properties of fisetin are likely connected to its specific chemical structure and its capacity to influence key cellular signaling mechanisms.12
This study aimed to assess the protective effect of orally administered fisetin against cyclophosphamide (CP)-induced hemorrhagic cystitis in male albino rats and to investigate the underlying mechanisms involved.

Materials and Methods

Experimental AnimalsAdult male Wistar rats, with body weights ranging from 210 to 220 grams, were housed under standard laboratory conditions. They were maintained at a temperature of 22 ± 3 °C with a 12-hour light/dark cycle, provided a standard diet and water ad libitum, and allowed a one-week acclimatization period prior to the commencement of experimental procedures.
Experimental Design and Sample CollectionAdult male Wistar rats were randomly assigned to one of four experimental groups (n = 10 per group). The treatment protocol was as follows: Group 1 (normal control) received a 1% dimethyl sulfoxide (DMSO) solution orally by gavage for 10 days. Group 2 (fisetin control) was orally administered fisetin (10 mg/kg/day), dissolved in 1% DMSO, for 10 consecutive days. The fisetin used was a crystalline powder sourced from Naturewill Biotechnology Co., Ltd. (China). Group 3 (cyclophosphamide [CYP]) received saline for 10 days and a single intraperitoneal (i.p.) injection of 150 mg/kg cyclophosphamide (CYP; Baxter Oncology GmbH, Germany) on the eighth day. Group 4 (Fisetin + CYP) was given fisetin (10 mg/kg/day) orally for 10 days, plus a single i.p. injection of CYP (150 mg/kg) on day 8. The experimental timeline and the doses of fisetin and CYP were selected based on previous literature.13,14
Upon completion of the experiment, all animals were euthanized. The urinary bladders were promptly excised and divided for analysis. One portion was immediately frozen in liquid nitrogen and stored at -80 °C for subsequent biochemical assay preparation. For homogenate preparation, bladder tissues were homogenized in a tenfold volume of ice-cold sodium-potassium phosphate buffer (0.01 M, pH 7.4) containing 1.15% KCl. The resulting homogenates were centrifuged at 600 g for 10 minutes at 4 °C and stored at -80 °C until analysis. The remaining bladder tissue was preserved in 10% neutral buffered formalin for histopathological examination.
Histopathological ExaminationFollowing euthanasia, bladder tissue samples were fixed in 10% neutral buffered formalin. The tissues were subsequently dehydrated through a graded ethanol series, cleared, and embedded in paraffin wax. Sections 5 μm thick were cut from the paraffin blocks using a microtome. These sections were then stained with hematoxylin and eosin (H&E) for examination under a light microscope.
ELISA Assay for Urinary adenosine monophosphate-activated protein kinase (AMPK), SIRT1, Oxidative Stress Markers, Inflammatory Markers, and Apoptotic Markers
Levels of specific proteins in bladder homogenates were quantified using commercially available enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturers' protocols. The analytes measured included metabolic regulators AMPK (Cat# DYC3197; detection range: 625.0–40000 pg/mL) and SIRT1 (NBP2-80302; range: 0.156–10 ng/mL). Markers of the Nrf2 pathway were assessed using kits for Nrf2 (Cat# NBP3-08161; range: 15.63–1000 pg/mL) and heme oxygenase-1 (HO-1; Cat# CSB-E08267r; range: 0.312–20 ng/mL). Oxidative stress was evaluated by measuring malondialdehyde (MDA; Cat# MBS268427; range: 0.156–10 nmol/mL) and superoxide dismutase (SOD; Cat# MBS036924; range: 12.5–400 U/mL). Inflammatory cytokines, tumor necrosis factor-alpha (TNF-α; Cat# NBP1-92681; range: 39.1–2500 pg/mL), interleukin-1 beta (IL-1β; Cat# CSB-E08055r; range: 31.25–2000 pg/mL), and interleukin-6 (IL-6; Cat# CSB-E04640r; range: 0.312–20 pg/mL) were analyzed. Apoptosis was assessed via caspase-3 levels (Cat# CSB-E08857r; range: 0.312–20 ng/mL).
Real-Time Quantitative- Polymerase Chain Reaction (qPCR) Analysis for NFKBTotal RNA was extracted from frozen bladder tissue samples using TRIzol reagent (Invitrogen, Carlsbad, CA) and subsequently reverse-transcribed into complementary DNA (cDNA) with Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Madison, WI). Real-time PCR amplification was conducted in a final volume of 10 µL using SYBR Green PCR Master Mix (Applied Biosystems) on an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA), with thermal cycling conditions set to 95 °C for 10 minutes, followed by 40 cycles of 95 °C for 15 seconds and 60 °C for 1 minute. Amplification utilized specific primers for NF-kB (forward: 5′-TTACGGGAGATGTGAAGATG-3′; reverse: 5′-ATGATGGCTAAGTGTAGGAC-3′; accession NM_001276711.2) and for the internal control gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (forward: 5′-ACCAACTGCTTAGCCCCCC-3′; reverse: 5′-GCATGTCAGATCCACAACGG-3′; accession NM_017008.4). Relative NF-kB gene expression was calculated via the comparative threshold cycle (2^(-ΔΔCt)) method,15 with all values normalized to GAPDH expression.
Ethical ApprovalThis study was approved by the Ethics Committee of Umm Al-Qura University, Makkah, Saudi Arabia (Date: 2025-03-18, No: HAPO-02-K-012-2025-12-3140).
Statistical AnalysisStatistical analysis was performed using GraphPad Prism 8 software (GraphPad Software, La Jolla, CA, USA). Data are presented as the mean ± standard deviation (SD). To determine statistical significance between experimental groups, a one-way analysis of variance (ANOVA) was conducted, followed by Bonferroni’s post hoc test for multiple comparisons. Differences with a probability value (p) of less than 0.05 were considered statistically significant.
Reporting GuidelinesThis study was conducted and reported in accordance with the ARRIVE guidelines.

Results

Upregulatory Effect of Fisetin on AMPK/SIRT1 Signaling Pathway in Urinary Bladder TissuesAccording to the results shown in Figure 1A and B, CYP treatment induced a significant (p < 0.05) decline in the protein levels of AMPK and SIRT1 in the urinary bladder supernatant of rats, as measured by ELISA, compared to normal controls. Conversely, co-administration of fisetin with CYP markedly elevated the protein levels of these markers (AMPK, SIRT1) relative to the CYP-only group. These findings confirm the positive effect of fisetin in activating the SIRT1 pathway.
Impact of Fisetin on CYP-Induced Urinary Bladder Oxidative Stress and Nrf2 SignalingAs shown in Figure 1C and D, CYP administration induced a marked elevation in malondialdehyde (MDA), a marker of lipid peroxidation, alongside a decrease in the level of the antioxidant enzyme superoxide dismutase (SOD) in urinary bladder tissue compared to normal animals. In contrast, co-treatment with fisetin attenuated this CYP-induced oxidative stress, significantly reducing MDA levels and elevating SOD activity relative to the CYP-only group. The antioxidant effect of fisetin can be attributed to its upregulation of the Nrf2 signaling pathway, as demonstrated by increased protein levels of Nrf2 and its downstream target HO-1 in the urinary bladder homogenate of the CYP + fisetin group compared to rats treated with CYP alone (Figure 1E, F).

Figure 1. ELISA level of (A) AMPK, (B) SIRT1, (C) MDA, (D) SOD, (E) Nrf2, (F) HO-1 in urinary bladder tissue in different groups. All results are presented as M ± SD. †p this means significant to the control group, %p this means significant to the CYP group.
Effect of Fisetin on Urinary Bladder Histology Hematoxylin and eosin examination of urinary bladder sections from all groups of our work revealed clear morphological variations. The control and fisetin-only groups showed a normal structural integrity, with healthy transitional epithelium, lamina propria, and intact muscular wall (Figure 2A-D). Conversely, sections from the CYP-treated group exhibited pronounced pathological changes, including severe edema and capillary congestion within the lamina propria. Additionally, clusters of leukocytes were dispersed throughout the lamina propria and between muscle fibers (Figure 2E-J). In animals receiving both CYP and fisetin, these alterations were markedly reduced, with only mild capillary congestion and minimal leukocytic infiltration observed in the lamina propria (Figure 2K, L). These findings indicate a protective role of fisetin in maintaining urinary bladder morphology under CYP-induced injury.

Figure 2. (A-D) Microscopic pictures of HE-stained sections of UB showing normal transitional epithelial lining, normal lamina propria, and muscular layer in the control group & fisetin groups. (E-J) UB from CYP group showing marked edema (*) and congested capillaries (red arrow) in lamina propria with scattered leukocytic cells aggregation (thick arrow) in lamina propria/between muscle fibers in some sections. (K, L) UB from CYP + fisetin group showing mildly congested capillaries (red arrow) with few leukocytic cells infiltration (thick arrow) in the lamina propria. X: 100 scale bar 100 µm & X: 400 bar 50 µm.
Anti-Inflammatory and Anti-Apoptotic Effects of Fisetin Against CYC-Induced Urinary Bladder InjuryTo investigate CYP-induced inflammatory and apoptotic damage in the urinary bladder, we measured the expression of key markers. The mRNA expression of the inflammatory mediator NF-κB was assessed by PCR. Furthermore, protein levels of the proinflammatory cytokines TNF-α, IL-6, and IL-1β in the bladder supernatant were quantified by ELISA. Apoptotic changes were evaluated by measuring the protein level of the executioner caspase, caspase-3.
We found that CYP administration induced severe bladder inflammation and epithelial cell apoptosis, evidenced by a significant (p < 0.05) elevation in all the aforementioned markers compared to the control group (Figure 3A-E). Notably, co-treatment with fisetin and CYP produced a marked reduction in these inflammatory indicators, both at the gene expression level (NF-κB) and the protein level (cytokines), alongside a drastic decrease in caspase-3. These results elucidate the protective effect of fisetin against CYP-induced cystitis, which is mediated through its anti-inflammatory and anti-apoptotic properties.

Figure 3. (A) Relative gene expression of NF-κB, (B-D) Protein level of TNF-α, IL-6, IL-1β. (E) apoptotic caspase-3 in different groups. All data expressed as M ± SD. †p vs normal rats, % vs CYP treated group.

Discussion

Conventional chemotherapeutic agents such as ifosfamide and cyclophosphamide (oxazaphosphorines) frequently cause serious adverse effects, notably hemorrhagic cystitis (HC). This toxicity is primarily associated with their metabolism, which generates reactive compounds such as acrolein and chloroacetaldehyde.¹⁴ Acrolein, the main cytotoxic metabolite, damages the bladder epithelium and penetrates deeper tissue layers.
This study aimed to evaluate the protective effect of fisetin against CYP-induced HC and to elucidate its underlying mechanisms. The findings indicate that fisetin significantly protects the urinary bladder epithelium, as confirmed histologically. This effect is mediated by upregulation of SIRT1 signaling, which activates the Nrf2 antioxidant pathway and suppresses NF-κB–mediated inflammation. Consequently, oxidative stress, inflammatory cytokine production, and epithelial apoptosis are reduced, resulting in improved bladder tissue morphology.
AMPK serves as a master cellular energy sensor, integral to maintaining metabolic homeostasis.16 Concurrently, SIRT1 functions as an nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylase, playing a critical role in countering oxidative stress, mitigating inflammation, and modulating apoptotic pathways.17 A growing body of evidence underscores a significant, synergistic interaction between SIRT1 and AMPK in protection against CYP-induced toxicity. In experimental models, their co-activation has been demonstrated to alleviate oxidative damage, suppress pro-inflammatory cytokine production, and enhance autophagic clearance, collectively conferring a potent cytoprotective effect.18 Our findings align with this established protective axis. We observed a significant decrease in both SIRT1 and AMPK protein levels in the CYP-treated group. In contrast, co-administration with fisetin markedly elevated their expression. This corroborates the earlier work of Rizk et al.,19 who reported that fisetin mitigated testicular damage by upregulating phosphorylated AMPK and SIRT1. The mechanism underlying fisetin's positive effect on SIRT1 involves its function as a SIRT1-activating compound (STAC). Fisetin is understood to bind to an allosteric site on the SIRT1 enzyme complex, inducing a conformational change that increases the enzyme's affinity for its NAD⁺ and protein substrates, thereby amplifying its catalytic rate.
The exact pathogenesis of CYP-induced hemorrhagic cystitis remains incompletely understood; however, oxidative stress is recognized as a pivotal contributing factor.20 This oxidative insult is primarily attributed to acrolein, a key hepatic metabolite of CYP that concentrates in the bladder lumen, where it promotes the excessive generation of reactive oxygen species (ROS).21 Consistent with this mechanism, our study demonstrated that a single intraperitoneal injection of CYP induced significant oxidative damage in the bladder tissue. This was evidenced by a marked increase in lipid peroxidation (measured as MDA) and a concomitant decrease in the activity of the antioxidant enzyme SOD. These biochemical alterations were associated with a downregulation of the protective Nrf2 signaling pathway. Conversely, co-treatment with fisetin effectively mitigated this CYP-induced oxidative injury. Fisetin administration significantly reduced MDA levels and activated the Nrf2/HO-1 axis, leading to a recovery in the secretion of antioxidant enzymes compared to rats treated with CYP alone. These findings align with the work of Jabbar and Alshawi,22 who reported that fisetin counteracted CYP-related myelosuppression by reducing serum MDA and elevating levels of the antioxidants SOD1 and GSH. The mechanism by which fisetin upregulates Nrf2 is multifaceted. It can be partially attributed to fisetin's established ability to activate SIRT1, which in turn promotes Nrf2 activity. Furthermore, fisetin directly targets the kelch-like ECH-associated protein 1 (KEAP1)/Nrf2 interaction. Under basal conditions, Nrf2 is sequestered in the cytoplasm by its repressor protein, KEAP1, and targeted for degradation. Fisetin acts as an activator that disrupts this binding, stabilizing Nrf2 and enabling its nuclear translocation. Once in the nucleus, Nrf2 binds to the antioxidant response element (ARE), initiating the transcription of a broad network of cytoprotective genes, including HO-1 and various antioxidant enzymes.
CYP-induced cystitis is characterized by a well-established cascade of oxidative stress and inflammation, leading to distinct histological damage. The present study confirmed hallmark pathological features, including epithelial denudation, hemorrhage, edema, and inflammatory cell infiltration. This damage is driven by elevated levels of pro-inflammatory mediators such as TNF-α, IL-6, and IL-1β, which further amplify the inflammatory response.23 Accordingly, our results demonstrated a significant rise in key inflammatory markers (NF-κB, TNF-α, IL-6, IL-1β) compared to control animals. Apoptosis is another central mechanism of CYP-induced bladder injury,24 reflected in our findings by an increase in the apoptotic marker caspase-3. Importantly, co-treatment with fisetin (CYP + fisetin) markedly attenuated these inflammatory and apoptotic markers, aligning with the protective effects reported by Althunibat et al.25

Limitations

While the results of this study demonstrate a promising uroprotective effect for fisetin, several limitations must be acknowledged. Primarily, the use of an acute rodent model may not fully replicate the complex pathophysiology of cyclophosphamide-induced cystitis in human patients, limiting direct clinical translation. The mechanistic insights, though significant, remain somewhat descriptive; the absence of interventional experiments using specific pathway inhibitors (e.g., for SIRT1 or AMPK) precludes definitive causal conclusions about the observed protection. Furthermore, the study design lacked a positive control group treated with a standard agent like mesna, which would have allowed for a direct comparative assessment of efficacy. Other constraints include the use of a single fisetin dose without a dose-response analysis, the absence of pharmacokinetic data, and a focus on short-term biochemical and histological markers without evaluating long-term functional recovery or potential fibrotic changes. These limitations highlight important avenues for future research to further validate and refine the therapeutic potential of fisetin.

Conclusion

In summary, fisetin exerts a potent uroprotective effect by simultaneously modulating the AMPK/SIRT1/Nrf2 and AMPK/SIRT1/NF-κB cascades. This dual action effectively mitigates the core pathological triad of CYP-induced cystitis—oxidative stress, inflammation, and apoptosis—positioning fisetin as a promising therapeutic candidate for managing this debilitating chemotherapy complication.

Declarations

Abbreviations

AMPK: adenosine monophosphate-activated protein kinase
ANOVA: analysis of variance
ARE: antioxidant response element
ARRIVE: Animal Research: Reporting of In Vivo Experiments
CYP: cyclophosphamide
DNA: deoxyribonucleic acid
DMSO: dimethyl sulfoxide
ELISA: enzyme-linked immunosorbent assay
GAPDH: glyceraldehyde-3-phosphate dehydrogenase
HC: hemorrhagic cystitis
HO-1: heme oxygenase-1
IL-1β: interleukin-1 beta
IL-6: interleukin-6
i.p.: intraperitoneal
KEAP1: kelch-like ECH-associated protein 1
MDA: malondialdehyde
M-MLV: moloney murine leukemia virus
mRNA: messenger ribonucleic acid
NAD⁺: nicotinamide adenine dinucleotide
NF-κB: nuclear factor kappa B
nm: nanometer
Nrf2: nuclear factor erythroid 2–related factor 2
PCR: polymerase chain reaction
qPCR: quantitative polymerase chain reaction
RNA: ribonucleic acid
ROS: reactive oxygen species
SD: standard deviation
SIRT1: sirtuin 1
SOD: superoxide dismutase
STAC: sirtuin-activating compound
TNF-α: tumor necrosis factor alpha

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About This Article

Received:

January 20, 2026

Accepted:

April 24, 2026

Published Online:

May 24, 2026