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. 2025 Jan;43(1):62-73.
doi: 10.1007/s11419-024-00697-x. Epub 2024 Aug 8.

Silymarin ameliorates diazinon-induced subacute nephrotoxicity in rats via the Keap1-Nrf2/heme oxygenase-1 signaling pathway

Eman Mohamed Fath  1 , Hatem H Bakery  1 , Ragab M El-Shawarby  1 , Mohamed E S Abosalem  1 , Samar S Ibrahim  1 , Nesrine Ebrahim  2   3   4 , Ahmed Medhat Hegazy  5
Affiliations

Silymarin ameliorates diazinon-induced subacute nephrotoxicity in rats via the Keap1-Nrf2/heme oxygenase-1 signaling pathway

Eman Mohamed Fath et al. Forensic Toxicol. 2025 Jan.

Abstract

Purpose: The goal of the current study was to clarify the potential molecular mechanism underlying the protective effects of silymarin (SIL) administration against diazinon-induced subacute nephrotoxicity, with a special emphasis on the role of the Kelch-like-associated protein-1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-heme oxygenase-1 (HO-1) signaling pathway in minimizing the oxidative stress induced by diazinon (DZN).

Methods: Five equal groups of thirty adult male Wistar rats were created at random. Group 1 (G1) was maintained under typical control conditions and administered saline intragastrically (I/G) once daily for 4 weeks; G2 was administered olive oil I/G for 4 weeks; G3 was I/G administered silymarin daily for 4 weeks; G4 was I/G administered diazinon daily for 4 weeks. G5 was I/G administered silymarin daily 1 h before the I/G administration of the diazinon for 4 weeks. Blood samples were collected at the end of the experiment for the determination of complete blood cell count, and kidney function tests. Kidney specimens were collected for the evaluation of the oxidative markers, mRNA gene expression, protein markers, and histopathological examination.

Results: SIL reduced the renal dysfunction caused by DZN by restoring urea and creatinine levels, as well as oxidative indicators. Although the expression of Keap-1 was also elevated, overexpression of Nrf2 also enhanced the expression of HO-1, a crucial target enzyme of Nrf2.

Conclusions: SIL is hypothesized to potentially aid in the prevention and management of nephrotoxicity caused by DZN.

Keywords: Diazinon; Heme oxygenase-1; Keap1; NF-κB; Nephrotoxicity; Silymarin.

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Conflict of interest statement

Declarations. Conflict of interest: The authors declare no competing interests. Ethical approval: Regulations established by the Institutional Animal Ethics Committee of Benha University in Egypt were followed when conducting the current investigation under protocol approval number BUFVTM 03–04-21. Consent to participate: Acceptance of participation, each individual participant in this study provided both oral and written informed permission.

Figures

Fig. 1
Fig. 1
Western blots with densitometric analysis of the Nrf2, keap-1, HO-1, and NF-κB proteins in kidney tissue homogenates from rats treated with olive oil, silymarin, diazinon, silymarin plus diazinon, and the control at 4 weeks after treatment (n = 6)
Fig. 2
Fig. 2
Photomicrograph of the kidneys of the experimental rats. A–C Normal control rats (G1), olive oil-treated rats (G2) and rats treated with silymarin (G3). The kidney showed a normal glomerulus (G) and Bowmen’s capsule, distal tubules (DTs) and proximal tubules (PTs). D Intoxicated rats (G4) induced by diazinon showed dilatation of Bowman’s space and degenerated glomeruli (DG) and inflammatory cell infiltration (arrow), as well as glomerular and peritubular vascular congestion and swelling of renal tubular epithelial cells (curved arrow). E Intoxicated rats treated with diazinon and then treated with silymarin (G5) had normal glomeruli (G) and tubules (PTs & DTs). H&E, ×200; the scale bar represents 25 μm
Fig. 3
Fig. 3
Photomicrographs of kidney sections stained with Masson’s trichrome. A–C Normal control rats (G1), olive oil-treated rats (G2) and rats treated with silymarin (G3). The kidney showed minimal collagen fibers among the glomerular capillaries (arrow) and surrounding the renal corpuscles and tubules (arrow). D The kidneys of diazinon-intoxicated rats (G4) showed the accumulation of collagen fibers among the glomerular capillaries (arrow) and surrounding the renal corpuscles and tubules (arrow). E Diazinon-intoxicated rats treated with silymarin (G5) showed minimal collagen fibers among the glomerular capillaries (arrow). F Histogram representing the mean area percentage of collagen fiber deposition in all the experimental groups. The scale bar represents 25 μm
Fig. 4
Fig. 4
Tumor necrosis factor alpha (TNF-α) immunohistochemical staining of kidney sections from experimental rats. A–C Normal control rats (G1), olive oil-treated rats (G2) and rats treated with silymarin (G3) showed minimal changes in glomerular epithelial cells. D Diazinon-intoxicated rats (G4) showed a strong positive cytoplasmic reaction in glomerular epithelial cells (arrow). E After the rats were treated with diazinon and then with silymarin (G5), minimal positive cytoplasmic reactions were detected in the glomerular epithelial cells (arrow). F Histogram showing the mean area percentage of TNF-α-immunoreactive cells in all the experimental groups (anti-TNF-α, ×400). The scale bar represents 25 μm
Fig. 5
Fig. 5
Cyclooxygenase 2 (COX2) immunohistochemical staining of kidney sections from experimental rats. A–C Normal control rats (G1), olive oil-treated rats (G2) and rats treated with silymarin (G3) showed minimal changes in glomerular epithelial cells. D Diazinon-intoxicated rats (G4) showed a strong positive cytoplasmic reaction in glomerular epithelial cells (arrow). E After the rats were treated with diazinon and then with silymarin (G5), minimal positive cytoplasmic reactions were detected in the glomerular epithelial cells (arrow). F Histogram representing the mean area percentage of COX2-immunoreactive cells in all the experimental groups (anti-COX2, × 400). The scale bar represents 25 μm
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