Liposomal Piceatannol Mitigates Methotrexate-Induced Oxidative Renal Injury via Modulation of Nrf2/HO-1, TLR4/NF-κB, MAPK, and Apoptotic Pathways in Rats

Figures

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  Figure 1. Schematic representation of PIC-LNP preparation by thin-film hydration, sonication, and purification, followed by physicochemical characterization (particle size, zeta potential, TEM).

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  Figure 2. Physicochemical Characterization of PIC-LNPs: (A) TEM Micrograph Showing Predominantly Spherical Morphology, (B) Particle Size Distribution Histogram Indicating Most Particles Range Between 75 and 100 nm with Narrow Dispersion, (C) Zeta Size Distribution by Volume, and (D) Zeta Potential Profile.

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  Figure 3. (A) FTIR spectra of Piceatannol (PIC), PIC-loaded liposomal nanoparticles (PIC-LNPs), and blank liposomes (Blank LNPs), highlighting key functional group peaks and confirming successful drug incorporation. (B) Cumulative in vitro release of Piceatannol from PIC-LNPs over 24 h, showing gradual drug release with variable standard deviations at each time point.

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  Figure 4. Effect of Crude and Nanoliposomal PIC on Serum Renal Function Biomarkers in Methotrexate-Induced Nephrotoxicity: (A) Urea, (B) Creatinine, and (C) Uric Acid Compared with the Normal Control Group; Values are reported as mean ± SE. Marks “a” and “b” indicate statistically significant differences from the control and MTX-treated groups, respectively.

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  Figure 5. Effect of crude and nanoliposomal piceatannol on renal Nrf2/HO-1 signaling and antioxidant status in methotrexate-induced nephrotoxicity. (A,B) Nrf2 protein and mRNA expression; (C,D) HO-1 protein and mRNA expression; (E) GSH content; (F) SOD activity; (G) CAT activity; (H) GPx activity. Data are presented as mean ± SE. Letters “a” and “b” denote significant differences versus the control and MTX groups, respectively (p < 0.05).

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  Figure 6. Effect of crude and nanoliposomal piceatannol on renal oxidative and nitrosative stress markers in methotrexate-induced nephrotoxicity. (A) ROS; (B) MDA; (C) protein carbonyls (PC); (D) 8-OHdG; (E) nitric oxide (NO). Data are presented as mean ± SE. Letters “a” and “b” denote significant differences versus the control and MTX groups, respectively (p < 0.05).

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  Figure 7. Effect of crude and nanoliposomal piceatannol on renal TLR4/NF-κB signaling and pro-inflammatory cytokines levels in methotrexate-induced nephrotoxicity. (A,B) TLR4 protein and mRNA expression; (C,D) NF-κB protein and mRNA expression; (E) TNF-α; (F) IL-1β; (G) IL-6. Data are presented as mean ± SE. Letters “a” and “b” denote significant differences versus the control and MTX groups, respectively (p < 0.05).

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  Figure 8. Effect of crude and nanoliposomal piceatannol on renal MAPK signaling and apoptosis markers in methotrexate-induced nephrotoxicity. (A) p-ERK1/2; (B) p-JNK; (C) p-p38; (D) c-Fos transcript; (E) c-Jun transcript; (F) Bax; (G) caspase-3; (H) Bcl-2. Data are presented as mean ± SE. Letters “a” and “b” denote significant differences versus the control and MTX groups, respectively (p < 0.05).

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  Figure 9. Representative photomicrographs of renal histology in control and treated rats. (A) Control; (B) PIC-LNPs; (C) PIC; (D) MTX; (E) MTX + PIC; (F) MTX + PIC-LNPs. Photomicrographs of kidney sections from (A–C) groups show well-preserved renal architecture. The glomeruli (g) appear intact with normal Bowman’s spaces (S), and both proximal (PCT) and distal (DCT) convoluted tubules are clearly organized. The tubular epithelial cells (arrowheads) retain normal cytoplasmic features and distinct nuclei, indicating healthy tissue structure. In contrast, the (D) MTX group displays clear histopathological damage, including distortion of the glomerular tuft (g), vascular congestion (black arrow), and noticeable degeneration of the tubular epithelial cells. These changes are accompanied by interstitial edema “E” and lymphocytic infiltration around the glomeruli and renal tubules (yellow arrows). Meanwhile, the (E) MTX + PIC-LNPs and (F) MTX + PIC-LNPs treated groups show a marked improvement, with kidney architecture largely restored. The glomeruli (g) regain their normal structure, and only mild vacuolation is observed in the tubular epithelial cells (arrowheads). H&E, 400×; scale bar = 50…

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  Figure 10. Transmission electron micrographs of renal cortex in control and treated groups. (A) Control; (B) PIC; (C) PIC-LNPs; (D) MTX; (E) MTX + PIC; (F) MTX + PIC-LNPs. Nucleus (N), mitochondria (M), cytoplasmic vacuolation (V), and basement membrane (BM) are indicated.

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  Figure 11. Representative photomicrographs of Nrf2 immunolocalization in the rat kidney. (A–C) Control groups showing strong Nrf2 immunoreactivity in cortical tubules; (D) MTX group with weak Nrf2 staining (arrows); (E) MTX + PIC-LNPs and (F) MTX + PIC groups showing marked enhancement of Nrf2 immunostaining. Original magnification 400×. (G) Quantitative analysis of Nrf2 immunostaining intensity in renal cortex across experimental groups. Data are presented as mean ± SE. Letters “a” and “b” denote significant differences versus the control and MTX groups, respectively (p < 0.05).

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  Figure 12. Representative photomicrographs of NF-κB immunoreactivity in the rat kidney. (A–C) Control groups with minimal NF-κB staining; (D) MTX group showing strong NF-κB immunolocalization in proximal tubular epithelial cells around glomeruli (arrows); (E,F) MTX + PIC-LNPs and MTX + PIC groups exhibiting weak NF-κB staining in cortical tubules, approaching control patterns. Original magnification 400×. (G) Quantitative analysis of NF-κB immunostaining intensity in renal cortex across experimental groups. Data are presented as mean ± SE. Letters “a” and “b” denote significant differences versus the control and MTX groups, respectively (p < 0.05).