Liposomal Piceatannol May Reduce Methotrexate Kidney Damage in Rats by Regulating Antioxidant, Inflammation, and Cell Death Pathways

Methotrexate (MTX; 50 mg/5 mL injectable solution) was obtained from EIPICO Pharmaceutical Company, Tenth of Ramadan City, Egypt. Piceatannol (PIC, ≥98%), cholesterol, and L-α-phosphatidylcholine (L-PC) were purchased from Sigma-Aldrich, Cairo, Egypt, and dissolved in DMSO (Sigma-Aldrich, Cairo, Egypt) immediately before use to ensure complete solubilization. Piceatannol-loaded liposomal nanoparticles (PIC-LNPs) were prepared as described below. The MTX dose was selected according to [20], while the PIC dose was within the previously reported safe and effective range [21].

PIC-LNPs were prepared by the thin-film hydration method (Figure 1). L-PC (70 mg; 0.09 mmol) and cholesterol (30 mg; 0.078 mmol) were dissolved in 10 mL chloroform: methanol (2:1, v/v) in a round-bottom flask to obtain a clear lipid solution (10 mg/mL). Organic solvents were removed under reduced pressure to form a thin, uniform lipid film. The film was hydrated with 5 mL phosphate-buffered saline (PBS; pH 7.4; VWR Chemicals, Radnor, PA, USA) containing 1 mg PIC (approximately 5 μmol), yielding a final PIC concentration of 200 μg/mL. The suspension was incubated at 37 °C with gentle stirring for 30 min to allow bilayer swelling and drug incorporation. Multilamellar vesicles were then probe-sonicated (SONICS Vibra-Cell, ultrasonic processor, Sonics & Materials, Inc., Newtown, CT, USA) in an ice bath using 10 s pulses at 30% amplitude, intermittently for 10 min. Unencapsulated PIC was removed by centrifugation at 15,000× g for 30 min. The supernatant containing PIC-LNPs was characterized for particle size, polydispersity index (PDI), and zeta potential using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). Particle size was measured in distilled water, and zeta potential was measured in triplicate. Vesicle morphology and integrity were examined by transmission electron microscopy (TEM; JEM-2100, JEOL Ltd., Tokyo, Japan) at 160 kV.

Encapsulation efficiency (EE%) and drug loading (DL%) were determined by measuring free (non-encapsulated) PIC in the supernatant. The PIC content was quantified by UV–Vis spectrophotometry (UV–Vis spectrophotometer, model UV-1800, Shimadzu Corporation, Kyoto, Japan) at 260 nm. Encapsulated PIC was calculated by subtracting the free PIC from the total amount added. EE% and DL% were calculated as: Drug loading (%) = [Encapsulated PIC/Total liposome mass] × 100 Encapsulation efficiency (%) = [Encapsulated PIC/Total PIC added] × 100

FTIR analysis was used to evaluate functional groups and potential interactions between PIC, lipid components, and PIC-LNPs. Samples were scanned in the range 4000–400 cm⁻¹. Characteristic peaks were compared to confirm successful incorporation of PIC into the lipid matrix.

The release behaviour of PIC from PIC-LNPs was evaluated in phosphate-buffered saline (PBS, pH 7.4) maintained at 37 °C with mild shaking. At defined time points, samples of the release medium were collected and immediately replaced with fresh PBS. The amount of PIC liberated at each interval was quantified by UV–Vis spectrophotometry at 260 nm, and the percentage of drug released was calculated and plotted over time.

Sixty male albino Sprague–Dawley rats (200–300 g) were obtained from the “Medical Experimental Research Center (MERC), Faculty of Medicine, Mansoura University, Mansoura, Egypt.” Animals were clinically inspected, housed in clean plastic cages, and maintained under controlled conditions (25 °C, 45% relative humidity, 12 h light/dark cycle) with free access to standard chow and water. Rats were acclimatized for two weeks before experimentation. All procedures complied with OECD Guideline 420 [22] and were conducted in accordance with internationally accepted standards for animal care and use and aligned with “ARRIVE (Animal Research: Reporting of In Vivo Experiments)” guidelines [23]. The study protocol was approved by the “Research Ethics Committee, Faculty of Veterinary Medicine, Mansoura University, Egypt, approval code MU-ACUC; VM.R.26.03.272.”

Rats were randomly assigned to six groups (n = 10/group):(1)Control: received vehicle only.(2)PIC: intraperitoneal (i.p.) PIC (20 mg/kg) once daily for 7 days.(3)PIC-LNPs: i.p. PIC-LNPs (20 mg/kg equivalent PIC) once daily for 7 days.(4)MTX: single i.p. MTX dose (20 mg/kg) on day 1.(5)PIC + MTX: MTX as above, followed by i.p. PIC (20 mg/kg) once daily for 5 days.(6)PIC-LNPs + MTX: MTX as above, followed by i.p. PIC-LNPs (20 mg/kg equivalent PIC) once daily for 5 days.

This design allowed comparison of free versus liposomal PIC in the context of MTX-induced renal injury. Dose/treatment schedules were adapted from earlier studies and refined through preliminary work to achieve reliable MTX-mediated renal injury, thereby enabling evaluation of the mitigating effects of PIC and PIC-LNPs [3,24,25].

Rats were fasted for 10 h before sacrifice. Anesthesia was induced and maintained with isoflurane, then euthanasia was completed using 5% isoflurane in oxygen for 5 min; the absence of cardiac and respiratory activity confirmed death. In each group, seven rats were used for biochemical and molecular analyses, and three for histopathological and ultrastructural studies. Blood was collected from the retro-orbital plexus, allowed to clot, and centrifuged at 3000× g for 10 min. Serum aliquots were stored at −80 °C. Kidneys were removed, rinsed in ice-cold 1.15% KCl, and sectioned; fragments designated for biochemical and molecular work were stored at −20 °C, while others were fixed in 10% neutral buffered formalin for light microscopy and TEM. For biochemical assays, tissue was homogenized in 50 mmol/L Tris–HCl (pH 7.4), centrifuged at 10,000× g for 15 min at 4 °C, and the clarified supernatants were preserved at −20 °C pending analysis.

All Kidney function assessment assays were purchased from BioDiagnostic, Cairo, Egypt. Serum creatinine was quantified using a kinetic colorimetric assay based on the Jaffé reaction, with absorbance readings at 495 nm. An enzymatic urease measured serum urea—Berthelot colorimetric method with absorbance recorded at 530–560 nm. Serum uric acid was determined using an enzymatic uricase-peroxidase colorimetric assay, with absorbance measured at 510 nm, according to standard clinical chemistry procedures described in [26].

Renal antioxidant status was assessed by measuring reduced glutathione (GSH) and the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) using validated colorimetric assay kits (Table S1) according to the manufacturers’ instructions [27]. Oxidative damage was evaluated by determining renal reactive oxygen species (ROS), malondialdehyde (MDA), protein carbonyls (PC), and 8-hydroxy-2′-deoxyguanosine (8-OHdG), whereas nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) levels were quantified using specific ELISA kits (Table S1) [28,29,30].

To evaluate renal inflammation, nuclear NF-κB p65 DNA-binding activity was measured as follows: renal cortical tissue was homogenized in ice-cold lysis buffer, and nuclear proteins were isolated using the Nuclear Extraction Kit (ab113474; Abcam, Cambridge, UK) strictly according to the manufacturer’s instructions. Briefly, cytoplasmic and nuclear fractions were separated by sequential low- and high-speed centrifugation steps as prescribed by the kit protocol, yielding a purified nuclear extract. Nuclear NF-κB p65 DNA-binding activity was subsequently quantified using the NF-κB p65 Transcription Factor Assay Kit (ab133112; Abcam, Cambridge, UK). Fifty micrograms of nuclear extract per well were loaded onto the pre-coated 96-well plate containing an immobilized consensus oligonucleotide (5′-GGGACTTTCC-3′), which selectively captures activated, DNA-binding p65. Absorbance was measured at 450 nm, and results were normalized to total nuclear protein content as determined by the BCA assay. The cytokines TNF-α and IL-1β were measured in total tissue homogenate supernatants using rat-specific ELISA kits, and Toll-like receptor 4 (TLR4) levels were determined with a dedicated ELISA kit for rat kidney tissue (). Nitrosative stress was assessed by quantifying total nitric oxide (NO) metabolites with a microplate-based colorimetric assay (). All measurements were carried out according to the manufacturer’s protocols. Table S1 Table S1

Apoptosis indices in renal tissue were assessed by measuring Bax, Bcl-2, and caspase-3 protein levels. These markers were quantified using rat-specific ELISA kits applied to supernatants from kidney homogenates (). Assays were conducted strictly according to the manufacturers’ protocols, and the resulting values were normalised to each sample’s total protein content. Table S1

Activation of mitogen-activated protein kinase (MAPK) signalling was investigated by determining phosphorylated ERK1/2, p38 MAPK, and JNK in kidney homogenates. Before analysis, total protein concentrations in the supernatants were established by a BCA assay to allow appropriate normalisation. Phosphorylated forms of ERK1/2, p38, and JNK were then quantified using SimpleStep ELISA kits following the supplier’s instructions (). Each sample was analysed in duplicate, and absorbance at 450 nm was used to interpolate protein concentrations from standard curves. Table S1

Total RNA was extracted from renal samples using QIAzol Lysis Reagent (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. After phase separation with chloroform, RNA was precipitated using isopropanol, washed with ethanol (both from Sigma-Aldrich, St. Louis, MO, USA), air-dried, and finally dissolved in DNase/RNase-free water. RNA yield and purity were evaluated spectrophotometrically, and equal amounts of RNA were reverse-transcribed into cDNA using the iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA). Quantitative real-time PCR was carried out on a Rotor-Gene Q system (Qiagen, Hilden, Germany) with iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) and the gene-specific primers listed in Table 1. Relative transcript levels were calculated by the 2^−ΔΔCt method, using β-actin as the internal control [31].

All histopathology-related reagents were purchased from Sigma-Aldrich, St. Louis, MO, USA, except where otherwise specified. Kidney specimens were immersed in 10% neutral buffered formalin (fixative: tissue ≈ 20:; Piochem, Cairo, Egypt) for three days, then processed through graded alcohols, cleared in xylene, and embedded in paraffin wax. Paraffin blocks were sectioned on a rotary microtome, and the sections were rehydrated and stained with hematoxylin and eosin for examination by light microscopy. Tubular and glomerular alterations, inflammation, and hemorrhage were semi-quantitatively scored (0–3) according to AASLD-based criteria (Table 2). For each rat (3 per group), three sections were prepared, and four non-overlapping fields per section (400×) were examined (12 fields/rat); mean scores were calculated per animal.

All related reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA). For ultrastructural evaluation, kidney pieces were initially fixed in 2.5% glutaraldehyde prepared in 0.1 M phosphate buffer (pH 7.4) at 4 °C for 24 h, rinsed in the same buffer, and then post-fixed in 1% osmium tetroxide for 2 h. Specimens were subsequently dehydrated through ascending ethanol concentrations, passed through acetone, and embedded in epoxy resin. Ultrathin sections (approximately 60–70 nm) were cut, collected on copper grids, and contrasted with uranyl acetate followed by lead citrate before examination in a JEM-2100 transmission electron microscope (JEOL Ltd., Tokyo, Japan) operating at 160 kV.

Paraffin-embedded renal sections were deparaffinised in xylene, rehydrated through graded ethanols, and subjected to heat-induced antigen retrieval in 0.01 M citrate buffer (pH 6.0). Endogenous peroxidase activity was quenched with 3% H₂O₂ in methanol, and non-specific binding was minimised by pre-incubation with 5% bovine serum albumin (all from Sigma-Aldrich, St. Louis, MO, USA). Sections were then incubated overnight at 4 °C with rabbit anti-Nrf2 and anti-NF-κB p65 primary antibodies (Abcam, Cambridge, UK; 1:200). After PBS washing, a biotinylated goat anti-rabbit secondary antibody, followed by streptavidin–HRP, was applied sequentially according to the supplier’s recommendations. Immunoreactivity was visualized with DAB and counterstained with Mayer’s hematoxylin, then dehydrated, cleared, and mounted with DPX. Stained sections were examined at 400× magnification. Five random fields per section were photographed, and the percentage of positively stained area was quantified using Fiji (ImageJ, version 1.54g; National Institutes of Health, Bethesda, MD, USA).

The investigators responsible for drug administration and sample collection were aware of the group allocation. Histopathological and immunohistochemical evaluations were performed by an experienced pathologist who was blinded to the treatment groups; kidney sections were coded before scoring and image analysis. Biochemical, ELISA, and RT-qPCR assays were carried out according to predefined protocols using coded samples, and the operator was not informed of group identity during measurement. Statistical analyses were conducted on datasets labeled only with group codes, and the treatment allocation was revealed only after completion of the primary analyses.

Before analysis, datasets were examined for normal distribution and equality of variances using the Shapiro–Wilk and Levene tests, respectively. Group comparisons were then performed using one-way analysis of variance (ANOVA) in SAS (version 2012; PROC ANOVA). When a significant overall effect was detected, Tukey’s honestly significant difference test was applied for pairwise post hoc comparisons. Data are presented as mean ± standard error (SE), and differences were regarded as statistically significant when p < 0.05. All graphical summaries were created with GraphPad Prism (version 9.5.1, GraphPad Software, San Diego, CA, USA).

Figure 2A shows PIC-LNPs with clear spherical shapes and no aggregation. This confirms their structural integrity. Particle size measurements (Figure 2B) ranged from 75 to 100 nm. The results indicate uniform and consistent distribution. DLS analysis (Figure 2C) indicated that PIC-LNPs possess an average hydrodynamic diameter of 128 nm and a low PDI of 0.157, confirming their consistent and narrowly distributed particle sizes. The zeta potential of PIC-LNPs was measured at −37 mV (Figure 2D), suggesting excellent colloidal stability.

The PIC-LNPs showed high encapsulation efficiency (~90%) and drug loading (~5.5%), demonstrating effective incorporation of Piceatannol into the lipid matrix. These values confirm the formulation’s ability to deliver the drug efficiently while maintaining stability.

The FTIR spectra (Figure 3A) reveal clear interactions between Piceatannol (PIC) and the liposomal matrix. PIC shows characteristic O–H stretching around 3290 cm⁻¹, C–H stretching near 2920 cm⁻¹, and C=O stretching at 1650 cm⁻¹. In the PIC-loaded liposomes (PIC-LNPs), these peaks are slightly shifted and broadened, indicating successful incorporation of PIC into the lipid bilayers without forming new chemical bonds. The blank liposomes show only typical lipid peaks, confirming that the observed changes in PIC-LNPs arise from drug encapsulation.

The release profile of Piceatannol from the liposomal nanoparticles (PIC-LNPs) shows a gradual and sustained pattern over 24 h (Figure 3B). Around 12% of the drug was released within the first hour, followed by a steady increase to nearly 80–95% by 24 h. This sustained release highlights the liposomal matrix’s ability to deliver drugs in a controlled manner over an extended period.

The administration of MTX resulted in a marked deterioration of renal function, as evidenced by significant elevations in serum urea by 76.24% (Figure 4A), creatinine by 141.56% (Figure 4B), and uric acid levels by 278.29% (Figure 4C) compared with the normal control group. Treatment with PIC, in both its crude and nanoliposomal formulations, significantly reduced the levels of these renal biomarkers. Notably, the nanoliposomal PIC formulation demonstrated greater efficacy than the crude form. However, no significant differences were detected between the MTX-exposed group and the MTX + PIC-LNPs group regarding serum urea and creatinine levels.

MTX administration led to a clear downregulation of the renal Nrf2/HO-1 axis, as reflected by significant decreases in Nrf2 protein and transcript levels by nearly 55.60% (Figure 5A,B) and in HO-1 protein and transcript levels by 68.53% (Figure 5C,D). In contrast, co-treatment with PIC, particularly in its liposomal form, counteracted this suppression. In the MTX + PIC-LNPs group, Nrf2 expression at both the protein and gene levels was restored to values that did not differ from the control group. In parallel, MTX markedly depleted renal GSH content by 39.00% (Figure 5E) and significantly reduced the activities of SOD by 59.55% (Figure 5F), CAT by 44.84% (Figure 5G), and GPx by 47.32% (Figure 5H). Both PIC preparations improved these antioxidant parameters, with the PIC-LNPs regimen producing the most pronounced recovery. Notably, GSH, CAT, and GPx values in MTX + PIC-LNPs rats were comparable to those of the normal controls.

Consistent with impaired antioxidant status, MTX exposure substantially increased indices of oxidative damage, including renal ROS by 158.01% (Figure 6A), MDA by 201.68% (Figure 6B), protein carbonyls by 237.93% (PC; Figure 6C), and the DNA oxidation marker 8-OHdG by 241.96% (Figure 6D), relative to the negative control group. Co-administration of PIC in either form significantly attenuated these changes, with the nanoliposomal formulation again providing superior protection. In the MTX + PIC-LNPs group, MDA levels were reduced to values indistinguishable from those of the control rats. Nitrosative stress followed a similar pattern: renal NO (Figure 6E) was significantly elevated after MTX treatment by 117.75%, and this increase was effectively blunted only in the PIC-LNPs group. In contrast, crude PIC did not significantly modify NO levels compared with MTX alone.

In MTX-treated rats, the renal TLR4/NF-κB pathway was markedly activated, as indicated by significant elevations in TLR4 protein and gene expression by 134.73% (Figure 7A,B) and in NF-κB protein and gene expression by 132.62% (Figure 7C,D) relative to controls. Co-treatment with PIC attenuated this activation, with the liposomal formulation exerting a more robust effect than the crude compound. Of note, nuclear NF-κB p65 was measured in purified nuclear fractions isolated from renal cortical homogenates, thereby specifically reflecting activated, nuclear-translocated p65 rather than total cellular p65 protein. The NF-κB levels in the MTX + crude PIC group did not differ significantly from those in MTX-only animals at either the protein or mRNA level, underscoring the limited impact of the non-encapsulated form on this pathway.

MTX exposure also resulted in a significant rise in renal pro-inflammatory cytokines TNF-α by 150.51% (Figure 7E), IL-1β by 223.36% (Figure 7F), and IL-6 by 169.99% (Figure 7G) compared with the negative control group. Co-administration of PIC-LNPs with MTX significantly lowered these cytokine levels and outperformed crude PIC. For TNF-α and IL-6, values in the MTX + crude PIC group remained comparable to those in the MTX-only group, whereas PIC-LNPs produced a clear anti-inflammatory effect.

MTX challenge activated the renal MAPK cascade. Relative renal protein expression analysis demonstrated that phosphorylated ERK1/2 increased by 156.94% (p-ERK1/2; Figure 8A), phosphorylated JNK increased by 126.92% (p-JNK; Figure 8B), and phosphorylated p38 increased by 239.53% (p-p38; Figure 8C) compared with the normal control group, confirming marked activation of MAPK phosphorylation. In parallel, the downstream transcription factors c-Fos and c-Jun were significantly elevated by 245.67% (Figure 8D) and 202.55% (Figure 8E), respectively. Co-treatment with PIC-LNPs significantly dampened this activation. In the MTX + PIC-LNPs group, phosphorylation levels of ERK1/2, JNK, and p38 were markedly reduced compared with the MTX group, accompanied by suppression of downstream c-Fos and c-Jun expression. In rats receiving MTX + PIC-LNPs, c-Fos expression was normalized to values that were not significantly different from those of controls. By contrast, p-JNK and p-p38 levels in the MTX + crude PIC group remained similar to those of the MTX-only group, indicating that the crude formulation failed to restrain these MAPKs effectively.

MTX exposure markedly enhanced renal apoptosis, as evidenced by significant increases in the pro-apoptotic proteins Bax (77.05%; Figure 8F) and caspase-3 (169.20%; Figure 8G) compared with the control group. Co-administration of PIC-LNPs substantially reduced the levels of both markers, whereas crude PIC had a more modest effect. In fact, Bax concentrations in the MTX + crude PIC group did not differ significantly from those in MTX-only rats. Conversely, MTX significantly decreased the anti-apoptotic protein Bcl-2 by 56.86% (Figure 8H). Treatment with PIC-LNPs effectively restored Bcl-2 to near-normal levels, with no significant difference versus the control group, highlighting a more favorable Bax/Bcl-2 balance in the liposomal PIC-treated animals.

Light microscopic examination of kidney sections from the negative control, PIC-LNPs, and PIC groups showed preserved renal architecture, with normal-appearing glomeruli and intact proximal and distal tubules (Figure 9A–C). In contrast, MTX-treated rats exhibited extensive structural damage, including glomerular atrophy, marked vascular congestion, and prominent periglomerular lymphocytic infiltration (Figure 9D). Co-treatment with either crude PIC or PIC-LNPs mitigated these lesions. Renal sections from the MTX + PIC and MTX + PIC-LNPs groups showed largely preserved glomerular and tubular structures with only mild congestion (Figure 9E,F). Semi-quantitative scoring confirmed a significant increase in renal injury in the MTX group compared with controls and antioxidant-only groups, partial improvement with crude PIC, and the greatest reduction in damage scores in the PIC-LNPs–treated animals (Figure 9G).

Electron microscopy of proximal tubular epithelial cells from control, PIC-LNPs, and PIC groups revealed normal ultrastructure, characterized by an intact basement membrane, dense apical brush border, rounded euchromatic nuclei, and abundant elongated mitochondria (Figure 10A–C). MTX exposure produced severe ultrastructural alterations, including shrunken, heterochromatic nuclei, extensive basal cytoplasmic vacuolation, swollen and fragmented mitochondria, and shortened, irregular apical microvilli (Figure 10D). Co-treatment with PIC-LNPs or crude PIC markedly improved these features. In the MTX + PIC and MTX + PIC-LNPs groups, proximal tubular cells showed largely restored mitochondrial morphology, euchromatic nuclei, and reformed brush borders, with substantially fewer vacuoles compared with the MTX-only group (Figure 10E,F).

In kidneys from control animals, Nrf2 immunoreactivity was intense and diffusely distributed in cortical tubular epithelial cells, consistent with an active endogenous antioxidant defense (Figure 11A–C). MTX treatment markedly diminished Nrf2 staining, which became weak and patchy within the tubular epithelium (Figure 11D). PIC co-treatment partially restored Nrf2 positivity (Figure 11E), whereas PIC-LNPs produced strong and widespread staining throughout the cortical tubules (Figure 11F). Quantitative analysis confirmed a significant MTX-induced reduction in Nrf2 immunostaining intensity and a graded restoration with PIC and PIC-LNPs, with the highest values observed in the MTX + PIC-LNPs group (Figure 11G).

NF-κB expression was barely detectable in renal cortical tubules of control rats, indicating a basal, non-activated inflammatory status (Figure 12A–C). MTX exposure elicited a marked increase in NF-κB immunostaining, particularly in proximal tubular epithelial cells surrounding the glomeruli, with intense brown nuclear and cytoplasmic labeling (Figure 12D). Treatment with PIC or PIC-LNPs clearly attenuated this response. In the MTX + PIC and MTX + PIC-LNPs groups, NF-κB staining in cortical tubules was weak and approached control levels, with the most pronounced reduction observed in the nanoparticle-treated animals (Figure 12E,F). Densitometric analysis corroborated these observations: MTX significantly elevated NF-κB immunostaining, and both PIC formulations reduced this increase, with PIC-LNPs achieving the greatest normalization (Figure 12G).