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Full Research Article

Role of Quercetin against the Effect of Piroxicam on the Liver and Kidney in Pregnant Rats

F
Fawziah Alshabanat1,*
P
Promy Virk1
D
Dalia Fouad1,*
1Department of Zoology, College of Science, King Saud University, PO Box 22452, Riyadh 11495, Saudi Arabia.

ABSTRACT

Background: Piroxicam (PX) is an NSAID linked to hepato-renal toxicity. This study evaluated quercetin’s (QUE) protective role against PX-induced damage in pregnant rats.

Methods: Pregnant rats (n=40) were divided into four groups (13 days): Control, QUE (75 mg/kg), PX (7 mg/kg i.p.) and QUE+PX. Hepato-renal toxicity was assessed via hematological, biochemical, histopathological, immunohistochemical (caspase-3, TNF-α, IL-6), oxidative stress markers and DNA fragmentation.

Result: PX caused hematological disturbances, elevated liver enzymes (ALT, AST) and kidney markers (creatinine, urea), decreased albumin and tissue damage. Increased MDA and decreased GSH/SOD indicated oxidative stress. Elevated caspase-3, TNF-α and DNA fragmentation confirmed apoptosis. QUE supplementation significantly reversed these alterations. Quercetin effectively ameliorated PX-induced hepato-renal toxicity through its antioxidant and anti-apoptotic properties.

INTRODUCTION

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most prescribed medications worldwide, accounting for approximately 5% of all prescriptions, due to their well-established analgesic, anti-inflammatory and antipyretic properties. Beyond these effects, they may also play a protective role against certain serious conditions, including cardiovascular diseases and cancer (Panchal et al., 2023; Ignácio  et al., 2024; Montinari et al., 2019). However, their use is not without risk. Numerous studies have documented adverse effects associated with NSAIDs overdosing, affecting the cardiovascular, gastrointestinal, hepatic and renal systems (Zhang et al., 2023; Masubuchi et al., 2019). During pregnancy, NSAIDs can cross the placental barrier, potentially impacting both embryonic and neonatal development. The severity of these effects is influenced by the specific drug, dosage, duration of therapy and gestational period (Antonucci et al., 2012; Burdan et al., 2004). Notably, certain NSAIDs such as piroxicam (PX) are associated with a higher risk profile compared to others like ibuprofen or diclofenac (Plappert et al., 2019; Ghlichloo et al., 2023).
       
Piroxicam (PX), a member of the oxicam class, is widely prescribed for various inflammatory conditions, including rheumatoid arthritis, osteoarthritis and post-traumatic inflammation (Sahu, 2016; Abdeen et al., 2020). Its primary mechanism involves the non-selective inhibition of cyclooxygenase (COX-1 and COX-2) enzymes, thereby suppressing prostaglandin synthesis (Burdan, 2015). However, the same mechanism contributes to its toxicity profile. Given that the liver is the main site of drug metabolism, it is particularly susceptible to PX-induced injury (Orinya et al., 2016; Badawi, 2018). Similarly, the kidney, where prostaglandins play a key role in regulating renal blood flow, is also a target for PX toxicity, especially following acute or chronic exposure (Shaheen et al., 2019; Rahmani Del Bakhshayesh  et al., 2020). Accumulating evidence indicates that oxidative stress is a central mediator of PX-induced hepato-renal damage. This is characterized by lipid peroxidation (LPO), mitochondrial dysfunction, depletion of endogenous antioxidants, DNA oxidation and ultimately apoptosis (Ai-Rufaei et al., 2025; Abdeen et al., 2019). Therefore, strategies aimed at enhancing the body’s antioxidant capacity may represent a viable approach to mitigating PX-induced organ toxicity (Vivek et al., 2025).
       
Quercetin (QUE), a naturally occurring flavonol abundant in fruits and vegetables, has garnered considerable attention for its potent antioxidant, anti-inflammatory and anti-apoptotic properties (Qi et al., 2022; Ullah et al., 2020; Gungor et al., 2025). Its ability to scavenge free radicals and modulate key cellular pathways positions it as a promising candidate for protecting against chemical-induced tissue injury (Yang et al., 2020; Khan et al., 2025).
       
The main objective of current research was effect of quercetin in pregnant Wistar rats against piroxicam-induced toxicity in the liver and kidney by ameliorating the oxidative stress, DNA damage and apoptosis.

MATERIALS AND METHODS

Chemicals and reagents
 
Piroxicam (C15H13N3O4) was purchased from Bruschettini S.R.I. Company (USP, Genoa) and the exposure dose for the experiment was 7 mg/kg according to a previous study by Abdeen et al., (2020). Quercetin (>95% purity) was procured from Sigma-Aldrich Chemical Co., Saint Louis, MO, USA, (CAS No: 117-39-5). Assay kits for biochemical analyses were purchased from Cayman Chemical Company (Sieda) and Sigma-Aldrich (Rockwood, United States). The antibodies for caspase-3, TNF-α and IL-6 used for immunohistochemistry were purchased from Thermo Scientific (Waltham, Massachusetts, USA). DNA extraction kits were supplied by Qiagen (Hilden, Germany). All other chemicals used in the experiments were of high-quality analytical grade.
 
Animals and experimental design
 
Forty female Wistar rats weighing 180 to 200 grams were procured from the animal house facility at King Saud University in Riyadh, Saudi Arabia. The study was reviewed and approved by the Institutional Review Board (IRB) and ethical consent was provided with reference number (KSU-SE-24-23) through the Ethics Committee at King Saud University. The experimental work was conducted at the animal house of the central lab at King Saud University during the period from January to October 2025. All animals were housed in plastic crates under standard conditions with regular changes in light and dark periods, constant humidity and a temperature between 23 and 28oC. The rats had access to both a commercial pellet meal and tap water ad libitum.

After acclimatization, animals were randomly divided into four groups (n=10 each) and treated for 13 consecutive days as follows:
-   Group 1 (Control): Animals were orally administered normal saline for 13 days.
-   Group 2 (QUE): Animals were orally administered quercetin (75 mg/kg) dissolved in normal saline for 13 days (Molina et al., 2003).
-   Group 3 (PX): Animals were injected intraperitoneally (i.p.) with piroxicam (7 mg/kg body weight/day) for 13 days.
-   Group 4 (QUE+PX): Animals were orally administered quercetin (75 mg/kg) and injected intraperitoneally with piroxicam (7 mg/kg body weight/day) for 13 days.
    On the 19th day of pregnancy, animals were anesthetized with CO2  and sacrificed and embryos were extracted from each group (Karampour et al., 2014).
 
Sample collection and preparation
 
After decapitation, blood was immediately collected from the trunks of the rats. Blood samples were placed in EDTA tubes for hematological testing and in serum separator tubes for biochemical studies. Blood samples were also collected in sterile, closed plain tubes and placed at 25oC to coagulate. All tubes were then cooled and centrifuged for 15 minutes at 4oC at a speed of 3500 rpm. Serum samples were transferred into sterile Eppendorf tubes and frozen at -80oC for further analyses.
       
Liver and kidney tissues were excised immediately after collection and placed in ice-cold saline. A portion of each tissue was transferred immediately to 10% buffered formaldehyde for histopathological and immunohistochemical investigations. The remaining tissue samples were stored at -80oC for further analysis of oxidative stress markers, antioxidant defense enzymes and DNA damage assays.
 
Laboratory analyses
 
Hematological parameters
 
Hematological parameters, including red blood cells (RBC), hemoglobin (HGB), white blood cells (WBC) and platelets (PLT), were measured using a fully automated hematology analyzer (Beckman Coulter Ac. T 5diff, USA) following standard protocols and manufacturer’s instructions (Shuker et al., 2022).
 
Biochemical analysis of Liver and Kidney function
 
Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, urea and albumin were determined spectrophotometrically using commercial diagnostic kits according to the manufacturer’s guidelines (Fouad et al., 2019).
 
Histopathological examination
 
Liver and kidney specimens, fixed in 10% buffered formalde- hyde and embedded in paraffin, were sectioned at 5-6 μm thickness, stained with hematoxylin and eosin (H and E) and examined under a light microscope (Olympus CX23, Japan) for histopathological changes (Abdeen et al., 2020).
 
Immunohistochemical detection of caspase-3, TNF-α and IL-6
 
Tissue sections were incubated overnight at 4oC with primary monoclonal antibodies against caspase-3, TNF-α and IL-6. Following PBS washing, sections were incubated with HRP-conjugated secondary antibody (1:2500), developed with DAB and counterstained with hematoxylin. Positively stained cells were counted in three fields per sample (Potocnjak, 2016; Abdeen et al., 2019).
 
Oxidative stress markers and antioxidant enzymes
 
Liver and kidney tissue homogenates (5 mg) were assayed for malondialdehyde (MDA), reduced glutathione (GSH) and superoxide dismutase (SOD) using commercial assay kits following the manufacturer’s protocols (Abdeen et al., 2020).
 
DNA fragmentation assay
 
DNA was extracted from kidney tissue (25 mg) using a Qiagen DNeasy kit according to the manufacturer’s instructions. DNA integrity was assessed by 1.5% agarose gel electrophoresis, visualized under UV light at 300 nm and photographed (Shuker et al., 2022).
 
Statistical analysis
 
One-way ANOVA was performed using SPSS software (version 22). All P-values were analyzed as two-sided and values below 0.05 were regarded as statistically significant. The analysis was complemented with a least significant difference (LSD) post-hoc test to identify specific group differences. Results were expressed as mean ± standard deviation (SD) for continuous data and as percentages for categorical data.

RESULTS AND DISCUSSION

Hematological parameters
 
When compared to the control group, the PX-treated pregnant rats’ hemoglobin (HGB) and red blood cell counts were considerably lower, but their blood platelet (PLT) and white blood cell counts were higher (Fig 1). When compared to the PX group, the treated group (QUE+PX) showed a decrease in WBCs and PLT count, as well as an increase in RBCs and HGB levels.  Significant restoration against the hematological alterations was observed on quercetin supplementation. The ameliorative effect of quercetin against xenobiotic-induced hepato-renal dysfunction is well-documented, with recent studies confirming its ability to attenuate hematological and biochemical alterations caused by various toxins (Alanazi et al., 2025). This protective effect indicates quercetin’s ability to mitigate PX-induced hematotoxicity, aligning with Pasdar et al., (2020), who reported that quercetin supplementation improves hematological profile through its antioxidant and immunomodulatory properties.

Fig 1: (A) Effects of Quercetin on Piroxicam induces changes in level of WBC count (103/μL) in the control and experimental pregnant rats. (B) Effect of Quercetin on piroxicam induces changes in level of RBC count (106/μL) in the control and experiment pregnant rats. (C) Effect of Quercetin on piroxicam induces changes in level of hemoglobin (HGB) (g/dL) in the control and experimental pregnant rats. (D) Effect of quercetin on piroxicam induces changes in level of platelet count (103/μL) in the control and experimental pregnant rats.


 
Liver and kidney function
 
Biochemical analysis revealed that PX exposure significantly elevated serum ALT, AST, creatinine and urea levels, while reducing albumin concentration (Table 1, 2). These findings indicate substantial hepato-renal dysfunction, corroborating earlier reports by Abdeen et al., (2020) and Lina et al., (2017), who demonstrated similar patterns of liver enzyme elevation and renal impairment following PX administration. The reduction in albumin may be attributed to impaired hepatic synthetic function secondary to hepatocellular damage. Notably, quercetin supplementation markedly attenuated these alterations, supporting this, dietary quercetin supplementation has been shown to significantly elevate the activity of hepatic enzymes ALT and AST, indicating enhanced protein metabolism and improved physiological function (Raghuvaran et al., 2025). This hepatoprotective and nephroprotective effect is consistent with Jia et al., (2023), who documented quercetin’s ability to modulate liver enzyme activity in hepatocarcinogenesis models and with Qi et al., (2022), who highlighted its capacity to prevent lipid peroxidation in renal tissues.

Table 1: Effect of quercetin on piroxicam induced alterations in alanine transaminase (ALT) (U/L) and aspartate aminotransferase (AST) (U/L) in the different pregnant rats.



Table 2: Effect of quercetin on piroxicam induced changes in creatinine (U/L), urea (mg/dl) and albumin (g/dl) in the different pregnant rats.


 
Histopathological examination
 
Histological examination of liver tissues using HandE staining revealed normal hepatocytes and blood sinusoids in the control group (Fig 2A). Similar normal architecture was observed in the quercetin-treated group (Fig 2B). In contrast, liver sections from PX-treated rats showed marked pathological alterations including congested veins with hemorrhage, inflammatory cell infiltration, severe steatosis and edema (Fig 2C). However, the QUE+PX group exhibited notable improvement in liver architecture, with most hepatocytes appearing healthy and only sporadic infiltrative cells observed (Fig 2D). Kidney sections from control and QUE-treated rats showed normal glomeruli and tubules (Fig 3A, 3B). PX exposure resulted in significant renal damage characterized by interstitial hemorrhage, inflammatory cell aggregates and tubular casts (Fig 3C). Meanwhile, QUE+PX treatment markedly improved renal histology, demonstrating enhanced glomerular structure and reduced inflammatory infiltration (Fig 3D).

Fig 2: Histological examinations by hematoxylin and eosin staining demonstrating the effect of quercetin (QUE) on piroxicam (PX) induced liver damage in pregnant rats.



Fig 3: Histological examinations by hematoxylin and eosin staining demonstrating the effect of quercetin (QUE) on piroxicam (PX) induced kidney damage in pregnant rats’ Light micrographs of the kidney.


       
The histopathological alterations observed in PX-treated rats, including hepatic steatosis, inflammatory infiltration and renal tubular casts, confirm the biochemical evidence of hepato-renal damage and align with previous reports of piroxicam-induced tissue injury (Al-Hamdany  et al., 2023; Abdeen et al., 2020). The marked improvement in both hepatic and renal architecture following quercetin co-administration demonstrates its tissue-protective potential, consistent with Badawi (2018), who reported that antioxidants facilitate tissue repair by neutralizing free radicals and reducing inflammatory cell recruitment. Additionally, the reduction in inflammatory infiltrates and restoration of normal glomerular structure support quercetin’s well-documented anti-inflammatory properties (Qi et al., 2022; Ullah et al., 2020).
 
Immunohistochemical investigation
 
Immunohistochemical analysis demonstrated strong positive expression of caspase-3 and TNF-α in both liver and kidney tissues of PX-treated rats, indicating enhanced apoptosis and inflammation (Fig 4-7). These findings align with Abdeen et al., (2019), who reported that PX-induced oxidative stress triggers apoptotic pathways through caspase activation. Interestingly, IL-6 expression showed only minimal changes across all groups (Fig 8,9). Quercetin co-treatment markedly reduced caspase-3 and TNF-α expression, confirming its anti-apoptotic and anti-inflammatory properties. The moderate reduction in IL-6 immunoreactivity observed in the QUE+PX group further supports quercetin’s modulatory effect on inflam-matory mediators (Michala, 2022). Oxidative Stress Markers and Antioxidant Enzymes in Liver and Kidney Tissues.

Fig 4: Effects of quercetin on activated caspases-3 expression after piroxicam intoxication in the liver pregnant rats.



Fig 5: Effects of quercetin on activated caspases-3 expression after PX intoxication in the kidney of pregnant rats.



Fig 6: Immunohistochemical staining for TNF- expression in the liver of pregnant rat’s section obtained.



Fig 7: Immunohistochemical staining for TNF- expression in the kidney of pregnant rats section obtained.



Fig 8: Immunohistochemical staining for IL-6 expression in the liver of pregnant rat’s section obtained.



Fig 9: Immunohistochemical staining for IL-6 expression in the kidney of pregnant rat’s section obtained.


 
Oxidative stress markers
 
PX treatment significantly increased hepatic and renal MDA levels, indicative of enhanced lipid peroxidation, while concomitantly depleting GSH content and reducing SOD activity (Table 3,4). This redox imbalance confirms the role of oxidative stress in PX-mediated tissue injury, consistent with Ahmed et al., (2015) and Chen et al., (2023), who demonstrated that NSAIDs-induced mitochondrial dysfunction elevates ROS production and impairs antioxidant defenses. Quercetin supplementation effectively reversed these changes, restoring antioxidant enzyme activity and reducing lipid peroxidation. The ability of quercetin to scavenge free radicals and enhance endogenous antioxidant capacity has been well-documented (Ullah et al., 2020; Yang et al., 2020), explaining its protective efficacy in the present study. However, quercetin is known to inhibit key drug-metabolizing enzymes (e.g., CYP3A4) and efflux transporters (e.g., P-gp, MRP2), which may reduce the formation of toxic metabolites and enhance the bioavailability of co-administered drugs (Patel et al., 2022).

Table 3: Effect of quercetin on piroxicam induced alterations in malondialdehyde (MDA) levels (nmol/g fresh tissue) and reduced glutathione level (GSH) (nmol/g fresh tissue) in liver and kidney in the different pregnant rats.



Table 4: Effect of quercetin on piroxicam induced alterations in superoxide dismutase activity (SOD) (U/ml fresh tissue) in liver and kidney in the different pregnant rats.


 
DNA fragmentation
 
A major indicator of apoptosis is DNA fragmentation.  Agarose gel electrophoresis has been used to investigate the qualitative assessment of the integrity of the altered genomic DNA (Fig 10).  While PX treatment caused DNA fragmentation (lanes 3), the DNA isolated from control pregnant rats (lane 1) and QUE-treated animals (lane 2) showed high-quality DNA.  QUE+ PX-treated groups (lane 4) showed reduced DNA damage, nevertheless. These findings are consistent with Hosseini et al., (2021), who reported that quercetin protects against DNA damage through its antioxidant activity and ability to modulate apoptotic signaling pathways. The observed reduction in DNA fragmentation following quercetin co-treatment further supports its anti-apoptotic role, complementing the caspase-3 immunohistochemical findings.        

Fig 10: DNA fragmentation in control and experimental in the kidney of pregnant rats.

CONCLUSION

Taken together, treatment with PX showed detectable changes in blood analysis, serum biochemical parameters, antioxidants and DNA in both the liver and kidney of the animals, as well as noticeable histological alterations. The use of quercetin clearly mitigated the renal and hepatotoxicity. However, quercetin could be incorporated as a dietary supplement only under the approval of a healthcare professional in accordance with the daily recommended dose.

ACKNOWLEDGEMENT

The authors extend their appreciation to the “Ongoing Research Funding Program” (ORF-2026-965), King Saud University, Riyadh, Saudi Arabia.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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    Role of Quercetin against the Effect of Piroxicam on the Liver and Kidney in Pregnant Rats

    F
    Fawziah Alshabanat1,*
    P
    Promy Virk1
    D
    Dalia Fouad1,*
    1Department of Zoology, College of Science, King Saud University, PO Box 22452, Riyadh 11495, Saudi Arabia.

    ABSTRACT

    Background: Piroxicam (PX) is an NSAID linked to hepato-renal toxicity. This study evaluated quercetin’s (QUE) protective role against PX-induced damage in pregnant rats.

    Methods: Pregnant rats (n=40) were divided into four groups (13 days): Control, QUE (75 mg/kg), PX (7 mg/kg i.p.) and QUE+PX. Hepato-renal toxicity was assessed via hematological, biochemical, histopathological, immunohistochemical (caspase-3, TNF-α, IL-6), oxidative stress markers and DNA fragmentation.

    Result: PX caused hematological disturbances, elevated liver enzymes (ALT, AST) and kidney markers (creatinine, urea), decreased albumin and tissue damage. Increased MDA and decreased GSH/SOD indicated oxidative stress. Elevated caspase-3, TNF-α and DNA fragmentation confirmed apoptosis. QUE supplementation significantly reversed these alterations. Quercetin effectively ameliorated PX-induced hepato-renal toxicity through its antioxidant and anti-apoptotic properties.

    INTRODUCTION

    Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most prescribed medications worldwide, accounting for approximately 5% of all prescriptions, due to their well-established analgesic, anti-inflammatory and antipyretic properties. Beyond these effects, they may also play a protective role against certain serious conditions, including cardiovascular diseases and cancer (Panchal et al., 2023; Ignácio  et al., 2024; Montinari et al., 2019). However, their use is not without risk. Numerous studies have documented adverse effects associated with NSAIDs overdosing, affecting the cardiovascular, gastrointestinal, hepatic and renal systems (Zhang et al., 2023; Masubuchi et al., 2019). During pregnancy, NSAIDs can cross the placental barrier, potentially impacting both embryonic and neonatal development. The severity of these effects is influenced by the specific drug, dosage, duration of therapy and gestational period (Antonucci et al., 2012; Burdan et al., 2004). Notably, certain NSAIDs such as piroxicam (PX) are associated with a higher risk profile compared to others like ibuprofen or diclofenac (Plappert et al., 2019; Ghlichloo et al., 2023).
           
    Piroxicam (PX), a member of the oxicam class, is widely prescribed for various inflammatory conditions, including rheumatoid arthritis, osteoarthritis and post-traumatic inflammation (Sahu, 2016; Abdeen et al., 2020). Its primary mechanism involves the non-selective inhibition of cyclooxygenase (COX-1 and COX-2) enzymes, thereby suppressing prostaglandin synthesis (Burdan, 2015). However, the same mechanism contributes to its toxicity profile. Given that the liver is the main site of drug metabolism, it is particularly susceptible to PX-induced injury (Orinya et al., 2016; Badawi, 2018). Similarly, the kidney, where prostaglandins play a key role in regulating renal blood flow, is also a target for PX toxicity, especially following acute or chronic exposure (Shaheen et al., 2019; Rahmani Del Bakhshayesh  et al., 2020). Accumulating evidence indicates that oxidative stress is a central mediator of PX-induced hepato-renal damage. This is characterized by lipid peroxidation (LPO), mitochondrial dysfunction, depletion of endogenous antioxidants, DNA oxidation and ultimately apoptosis (Ai-Rufaei et al., 2025; Abdeen et al., 2019). Therefore, strategies aimed at enhancing the body’s antioxidant capacity may represent a viable approach to mitigating PX-induced organ toxicity (Vivek et al., 2025).
           
    Quercetin (QUE), a naturally occurring flavonol abundant in fruits and vegetables, has garnered considerable attention for its potent antioxidant, anti-inflammatory and anti-apoptotic properties (Qi et al., 2022; Ullah et al., 2020; Gungor et al., 2025). Its ability to scavenge free radicals and modulate key cellular pathways positions it as a promising candidate for protecting against chemical-induced tissue injury (Yang et al., 2020; Khan et al., 2025).
           
    The main objective of current research was effect of quercetin in pregnant Wistar rats against piroxicam-induced toxicity in the liver and kidney by ameliorating the oxidative stress, DNA damage and apoptosis.

    MATERIALS AND METHODS

    Chemicals and reagents
     
    Piroxicam (C15H13N3O4) was purchased from Bruschettini S.R.I. Company (USP, Genoa) and the exposure dose for the experiment was 7 mg/kg according to a previous study by Abdeen et al., (2020). Quercetin (>95% purity) was procured from Sigma-Aldrich Chemical Co., Saint Louis, MO, USA, (CAS No: 117-39-5). Assay kits for biochemical analyses were purchased from Cayman Chemical Company (Sieda) and Sigma-Aldrich (Rockwood, United States). The antibodies for caspase-3, TNF-α and IL-6 used for immunohistochemistry were purchased from Thermo Scientific (Waltham, Massachusetts, USA). DNA extraction kits were supplied by Qiagen (Hilden, Germany). All other chemicals used in the experiments were of high-quality analytical grade.
     
    Animals and experimental design
     
    Forty female Wistar rats weighing 180 to 200 grams were procured from the animal house facility at King Saud University in Riyadh, Saudi Arabia. The study was reviewed and approved by the Institutional Review Board (IRB) and ethical consent was provided with reference number (KSU-SE-24-23) through the Ethics Committee at King Saud University. The experimental work was conducted at the animal house of the central lab at King Saud University during the period from January to October 2025. All animals were housed in plastic crates under standard conditions with regular changes in light and dark periods, constant humidity and a temperature between 23 and 28oC. The rats had access to both a commercial pellet meal and tap water ad libitum.

    After acclimatization, animals were randomly divided into four groups (n=10 each) and treated for 13 consecutive days as follows:
    -   Group 1 (Control): Animals were orally administered normal saline for 13 days.
    -   Group 2 (QUE): Animals were orally administered quercetin (75 mg/kg) dissolved in normal saline for 13 days (Molina et al., 2003).
    -   Group 3 (PX): Animals were injected intraperitoneally (i.p.) with piroxicam (7 mg/kg body weight/day) for 13 days.
    -   Group 4 (QUE+PX): Animals were orally administered quercetin (75 mg/kg) and injected intraperitoneally with piroxicam (7 mg/kg body weight/day) for 13 days.
        On the 19th day of pregnancy, animals were anesthetized with CO2  and sacrificed and embryos were extracted from each group (Karampour et al., 2014).
     
    Sample collection and preparation
     
    After decapitation, blood was immediately collected from the trunks of the rats. Blood samples were placed in EDTA tubes for hematological testing and in serum separator tubes for biochemical studies. Blood samples were also collected in sterile, closed plain tubes and placed at 25oC to coagulate. All tubes were then cooled and centrifuged for 15 minutes at 4oC at a speed of 3500 rpm. Serum samples were transferred into sterile Eppendorf tubes and frozen at -80oC for further analyses.
           
    Liver and kidney tissues were excised immediately after collection and placed in ice-cold saline. A portion of each tissue was transferred immediately to 10% buffered formaldehyde for histopathological and immunohistochemical investigations. The remaining tissue samples were stored at -80oC for further analysis of oxidative stress markers, antioxidant defense enzymes and DNA damage assays.
     
    Laboratory analyses
     
    Hematological parameters
     
    Hematological parameters, including red blood cells (RBC), hemoglobin (HGB), white blood cells (WBC) and platelets (PLT), were measured using a fully automated hematology analyzer (Beckman Coulter Ac. T 5diff, USA) following standard protocols and manufacturer’s instructions (Shuker et al., 2022).
     
    Biochemical analysis of Liver and Kidney function
     
    Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, urea and albumin were determined spectrophotometrically using commercial diagnostic kits according to the manufacturer’s guidelines (Fouad et al., 2019).
     
    Histopathological examination
     
    Liver and kidney specimens, fixed in 10% buffered formalde- hyde and embedded in paraffin, were sectioned at 5-6 μm thickness, stained with hematoxylin and eosin (H and E) and examined under a light microscope (Olympus CX23, Japan) for histopathological changes (Abdeen et al., 2020).
     
    Immunohistochemical detection of caspase-3, TNF-α and IL-6
     
    Tissue sections were incubated overnight at 4oC with primary monoclonal antibodies against caspase-3, TNF-α and IL-6. Following PBS washing, sections were incubated with HRP-conjugated secondary antibody (1:2500), developed with DAB and counterstained with hematoxylin. Positively stained cells were counted in three fields per sample (Potocnjak, 2016; Abdeen et al., 2019).
     
    Oxidative stress markers and antioxidant enzymes
     
    Liver and kidney tissue homogenates (5 mg) were assayed for malondialdehyde (MDA), reduced glutathione (GSH) and superoxide dismutase (SOD) using commercial assay kits following the manufacturer’s protocols (Abdeen et al., 2020).
     
    DNA fragmentation assay
     
    DNA was extracted from kidney tissue (25 mg) using a Qiagen DNeasy kit according to the manufacturer’s instructions. DNA integrity was assessed by 1.5% agarose gel electrophoresis, visualized under UV light at 300 nm and photographed (Shuker et al., 2022).
     
    Statistical analysis
     
    One-way ANOVA was performed using SPSS software (version 22). All P-values were analyzed as two-sided and values below 0.05 were regarded as statistically significant. The analysis was complemented with a least significant difference (LSD) post-hoc test to identify specific group differences. Results were expressed as mean ± standard deviation (SD) for continuous data and as percentages for categorical data.

    RESULTS AND DISCUSSION

    Hematological parameters
     
    When compared to the control group, the PX-treated pregnant rats’ hemoglobin (HGB) and red blood cell counts were considerably lower, but their blood platelet (PLT) and white blood cell counts were higher (Fig 1). When compared to the PX group, the treated group (QUE+PX) showed a decrease in WBCs and PLT count, as well as an increase in RBCs and HGB levels.  Significant restoration against the hematological alterations was observed on quercetin supplementation. The ameliorative effect of quercetin against xenobiotic-induced hepato-renal dysfunction is well-documented, with recent studies confirming its ability to attenuate hematological and biochemical alterations caused by various toxins (Alanazi et al., 2025). This protective effect indicates quercetin’s ability to mitigate PX-induced hematotoxicity, aligning with Pasdar et al., (2020), who reported that quercetin supplementation improves hematological profile through its antioxidant and immunomodulatory properties.

    Fig 1: (A) Effects of Quercetin on Piroxicam induces changes in level of WBC count (103/μL) in the control and experimental pregnant rats. (B) Effect of Quercetin on piroxicam induces changes in level of RBC count (106/μL) in the control and experiment pregnant rats. (C) Effect of Quercetin on piroxicam induces changes in level of hemoglobin (HGB) (g/dL) in the control and experimental pregnant rats. (D) Effect of quercetin on piroxicam induces changes in level of platelet count (103/μL) in the control and experimental pregnant rats.


     
    Liver and kidney function
     
    Biochemical analysis revealed that PX exposure significantly elevated serum ALT, AST, creatinine and urea levels, while reducing albumin concentration (Table 1, 2). These findings indicate substantial hepato-renal dysfunction, corroborating earlier reports by Abdeen et al., (2020) and Lina et al., (2017), who demonstrated similar patterns of liver enzyme elevation and renal impairment following PX administration. The reduction in albumin may be attributed to impaired hepatic synthetic function secondary to hepatocellular damage. Notably, quercetin supplementation markedly attenuated these alterations, supporting this, dietary quercetin supplementation has been shown to significantly elevate the activity of hepatic enzymes ALT and AST, indicating enhanced protein metabolism and improved physiological function (Raghuvaran et al., 2025). This hepatoprotective and nephroprotective effect is consistent with Jia et al., (2023), who documented quercetin’s ability to modulate liver enzyme activity in hepatocarcinogenesis models and with Qi et al., (2022), who highlighted its capacity to prevent lipid peroxidation in renal tissues.

    Table 1: Effect of quercetin on piroxicam induced alterations in alanine transaminase (ALT) (U/L) and aspartate aminotransferase (AST) (U/L) in the different pregnant rats.



    Table 2: Effect of quercetin on piroxicam induced changes in creatinine (U/L), urea (mg/dl) and albumin (g/dl) in the different pregnant rats.


     
    Histopathological examination
     
    Histological examination of liver tissues using HandE staining revealed normal hepatocytes and blood sinusoids in the control group (Fig 2A). Similar normal architecture was observed in the quercetin-treated group (Fig 2B). In contrast, liver sections from PX-treated rats showed marked pathological alterations including congested veins with hemorrhage, inflammatory cell infiltration, severe steatosis and edema (Fig 2C). However, the QUE+PX group exhibited notable improvement in liver architecture, with most hepatocytes appearing healthy and only sporadic infiltrative cells observed (Fig 2D). Kidney sections from control and QUE-treated rats showed normal glomeruli and tubules (Fig 3A, 3B). PX exposure resulted in significant renal damage characterized by interstitial hemorrhage, inflammatory cell aggregates and tubular casts (Fig 3C). Meanwhile, QUE+PX treatment markedly improved renal histology, demonstrating enhanced glomerular structure and reduced inflammatory infiltration (Fig 3D).

    Fig 2: Histological examinations by hematoxylin and eosin staining demonstrating the effect of quercetin (QUE) on piroxicam (PX) induced liver damage in pregnant rats.



    Fig 3: Histological examinations by hematoxylin and eosin staining demonstrating the effect of quercetin (QUE) on piroxicam (PX) induced kidney damage in pregnant rats’ Light micrographs of the kidney.


           
    The histopathological alterations observed in PX-treated rats, including hepatic steatosis, inflammatory infiltration and renal tubular casts, confirm the biochemical evidence of hepato-renal damage and align with previous reports of piroxicam-induced tissue injury (Al-Hamdany  et al., 2023; Abdeen et al., 2020). The marked improvement in both hepatic and renal architecture following quercetin co-administration demonstrates its tissue-protective potential, consistent with Badawi (2018), who reported that antioxidants facilitate tissue repair by neutralizing free radicals and reducing inflammatory cell recruitment. Additionally, the reduction in inflammatory infiltrates and restoration of normal glomerular structure support quercetin’s well-documented anti-inflammatory properties (Qi et al., 2022; Ullah et al., 2020).
     
    Immunohistochemical investigation
     
    Immunohistochemical analysis demonstrated strong positive expression of caspase-3 and TNF-α in both liver and kidney tissues of PX-treated rats, indicating enhanced apoptosis and inflammation (Fig 4-7). These findings align with Abdeen et al., (2019), who reported that PX-induced oxidative stress triggers apoptotic pathways through caspase activation. Interestingly, IL-6 expression showed only minimal changes across all groups (Fig 8,9). Quercetin co-treatment markedly reduced caspase-3 and TNF-α expression, confirming its anti-apoptotic and anti-inflammatory properties. The moderate reduction in IL-6 immunoreactivity observed in the QUE+PX group further supports quercetin’s modulatory effect on inflam-matory mediators (Michala, 2022). Oxidative Stress Markers and Antioxidant Enzymes in Liver and Kidney Tissues.

    Fig 4: Effects of quercetin on activated caspases-3 expression after piroxicam intoxication in the liver pregnant rats.



    Fig 5: Effects of quercetin on activated caspases-3 expression after PX intoxication in the kidney of pregnant rats.



    Fig 6: Immunohistochemical staining for TNF- expression in the liver of pregnant rat’s section obtained.



    Fig 7: Immunohistochemical staining for TNF- expression in the kidney of pregnant rats section obtained.



    Fig 8: Immunohistochemical staining for IL-6 expression in the liver of pregnant rat’s section obtained.



    Fig 9: Immunohistochemical staining for IL-6 expression in the kidney of pregnant rat’s section obtained.


     
    Oxidative stress markers
     
    PX treatment significantly increased hepatic and renal MDA levels, indicative of enhanced lipid peroxidation, while concomitantly depleting GSH content and reducing SOD activity (Table 3,4). This redox imbalance confirms the role of oxidative stress in PX-mediated tissue injury, consistent with Ahmed et al., (2015) and Chen et al., (2023), who demonstrated that NSAIDs-induced mitochondrial dysfunction elevates ROS production and impairs antioxidant defenses. Quercetin supplementation effectively reversed these changes, restoring antioxidant enzyme activity and reducing lipid peroxidation. The ability of quercetin to scavenge free radicals and enhance endogenous antioxidant capacity has been well-documented (Ullah et al., 2020; Yang et al., 2020), explaining its protective efficacy in the present study. However, quercetin is known to inhibit key drug-metabolizing enzymes (e.g., CYP3A4) and efflux transporters (e.g., P-gp, MRP2), which may reduce the formation of toxic metabolites and enhance the bioavailability of co-administered drugs (Patel et al., 2022).

    Table 3: Effect of quercetin on piroxicam induced alterations in malondialdehyde (MDA) levels (nmol/g fresh tissue) and reduced glutathione level (GSH) (nmol/g fresh tissue) in liver and kidney in the different pregnant rats.



    Table 4: Effect of quercetin on piroxicam induced alterations in superoxide dismutase activity (SOD) (U/ml fresh tissue) in liver and kidney in the different pregnant rats.


     
    DNA fragmentation
     
    A major indicator of apoptosis is DNA fragmentation.  Agarose gel electrophoresis has been used to investigate the qualitative assessment of the integrity of the altered genomic DNA (Fig 10).  While PX treatment caused DNA fragmentation (lanes 3), the DNA isolated from control pregnant rats (lane 1) and QUE-treated animals (lane 2) showed high-quality DNA.  QUE+ PX-treated groups (lane 4) showed reduced DNA damage, nevertheless. These findings are consistent with Hosseini et al., (2021), who reported that quercetin protects against DNA damage through its antioxidant activity and ability to modulate apoptotic signaling pathways. The observed reduction in DNA fragmentation following quercetin co-treatment further supports its anti-apoptotic role, complementing the caspase-3 immunohistochemical findings.        

    Fig 10: DNA fragmentation in control and experimental in the kidney of pregnant rats.

    CONCLUSION

    Taken together, treatment with PX showed detectable changes in blood analysis, serum biochemical parameters, antioxidants and DNA in both the liver and kidney of the animals, as well as noticeable histological alterations. The use of quercetin clearly mitigated the renal and hepatotoxicity. However, quercetin could be incorporated as a dietary supplement only under the approval of a healthcare professional in accordance with the daily recommended dose.

    ACKNOWLEDGEMENT

    The authors extend their appreciation to the “Ongoing Research Funding Program” (ORF-2026-965), King Saud University, Riyadh, Saudi Arabia.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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