Rutin-Loaded Nanostructured Lipid Carriers Attenuate Doxorubicin-Induced Nephrotoxicity and Modulate Epigenetic Regulation and MyD88/STAT3 Signaling

Figure 1. Characterization of the developed RUT-NLCs. RUT-loaded nanostructured lipid carriers (RUT-NLCs) were prepared using the hot high-shear homogenization method and characterized for (A) particle size, (B) zeta potential, (C) morphology by scanning electron microscopy (SEM), and (D) in vitro drug release profile.

Figure 2. Effects of RUT-NLCs on Renal Function, Renal Injury Biomarkers, and Histopathological Alterations. Serum levels of (A) urea and (B) creatinine. Relative mRNA expression of (C) kidney injury molecule-1 (KIM-1), (D) neutrophil gelatinase-associated lipocalin (NGAL/Lcn2), and (E) fibroblast growth factor-23 (FGF-23). Data are presented as mean ± SD (n = 6). Different lowercase letters indicate statistically significant differences among groups (p < 0.05). (F) Representative hematoxylin and eosin (H&E)-stained sections of renal cortex from the experimental groups: (a,b) Ctrl; (c,d) RUT; (e,f) RUT-NLCs; (g–j) DOX; (k,l) DOX-RUT; and (m,n) DOX-RUT-NLCs. Panels (a,c,e,g,i,k,m) were acquired at ×200 magnification (scale bar = 100 µm). Boxed regions in panels (a,c,e,g,i,k,m) indicate areas shown at higher magnification in the corresponding panels (b,d,f,h,j,l,n) at ×400 magnification (scale bar = 50 µm). Representative histological features and histopathological alterations include normal proximal convoluted tubules (pt), distal convoluted tubules (dt), intact glomeruli (asterisk), congested glomeruli (square), hemorrhage (white arrows), inflammatory cell infiltration (blue arrow),…

Figure 3. Effects of RUT-NLCs on Renal Oxidative Stress in DOX-Treated Rats. Renal levels of (A) malondialdehyde (MDA), (B) total antioxidant capacity (TAC), and (C) glutathione peroxidase (GPx) activity in the experimental groups. Data are presented as mean ± SD (n = 6). Different lowercase letters indicate statistically significant differences among groups (p < 0.05).

Figure 4. Effects of RUT-NLCs on Renal Inflammation and MyD88/STAT3 Signaling in DOX-Treated Rats. Relative mRNA expression of (A) interleukin-1β (IL-1β), (B) interleukin-6 (IL-6), (C) tumor necrosis factor-α (TNF-α), (D) nuclear factor-κB (NF-κB), (E) myeloid differentiation primary response 88 (MyD88), (F) suppressor of cytokine signaling 3 (SOCS3), and (G) signal transducer and activator of transcription 3 (STAT3) in the experimental groups. Data are presented as mean ± SD (n = 6). Different lowercase letters indicate statistically significant differences among groups (p < 0.05). (H) Representative immunohistochemical staining of NF-κB in renal cortical sections from the experimental groups (×400 magnification; scale bar = 50 μm). Positive immunoreactivity is indicated by brown nuclear and cytoplasmic staining (arrows). (I) Quantitative analysis of NF-κB immunoreactivity expressed as the percentage area of positive staining. Data are presented as mean ± SD (n = 6). Different lowercase letters indicate statistically significant differences among groups (p < 0.05). Ctrl, control; RUT, rutin; RUT-NLCs, rutin-loaded nanostructured lipid carriers; DOX, doxorubicin; DOX-RUT, doxorubicin +…

Figure 5. Effect of RUT-NLCs on renal DNA methylation and histone modifications in DOX-treated rats. Renal mRNA expression of (A) DNMT1, (B) DNMT3a, (C) DNMT3b, (D) TET1, (E) TET2, (F) TET3, and (G) Klotho in the experimental groups. (H) Representative Western blots showing the protein expression of H3K4me3, H3K9me3, H3K27me3, and total H3. Quantitative analysis of renal protein expression of (I) H3K4me3, (J) H3K9me3, and (K) H3K27me3. Data are presented as mean ± SD (n = 6). Bars with different letters are significantly different (p < 0.05).

Figure 6. Effect of RUT-NLCs on renal TNF-α, DNMT1, and KIM-1 protein expression in DOX-treated rats. (A) Representative Western blots showing the protein expression of TNF-α, DNMT1, KIM-1, and β-actin in the experimental groups. Quantitative analysis of renal protein expression of (B) TNF-α, (C) DNMT1, and (D) KIM-1. Data are presented as mean ± SD (n = 6). Bars with different letters indicate statistically significant differences (p < 0.05).

Figure 7. Effect of RUT-NLCs on renal DNA damage in DOX-treated rats. (A) Representative fluorescence micrographs of comet assay images from the experimental groups. (B) Quantitative analysis of comet assay parameters, including percentage of tailed DNA, percentage of DNA in the tail, and tail moment. Data are presented as mean ± SD (n = 6). Bars with different letters indicate statistically significant differences (p < 0.05).

Figure 8. Molecular docking of RUT with STAT3 and TNF-α. (A) Three-dimensional representation of the predicted binding mode of RUT within the STAT3 binding pocket. Amino acid residues are shown in yellow, and RUT is shown in purple. (B) Three-dimensional representation of the predicted binding mode of RUT within the TNF-α binding pocket. Amino acid residues are shown in yellow, and RUT is shown in turquoise.