Hesperetin and Isoliquiritigenin Attenuate the Progression of Diabetic Nephropathy via Inhibition of NF-κB/NLRP3 Pathways in Type 2 Diabetic Rats

Abstract

Glucose toxicity associated with oxidant and inflammatory triggers causes diabetic nephropathy (DN), thus necessitating the investigation of potential therapeutic interventions. This study investigated the nephroprotective activities of hesperetin (HSP) and isoliquiritigenin (ISL) in diabetic rats. Type 2 diabetes (T2D) was generated in Wistar rats by feeding them a high-fat diet (HFD, ad libitum) and injecting intraperitoneally with a single dose of streptozotocin (35 mg/kg). T2D rats were then administered orally HSP (50 and 100 mg/kg), ISL (10 and 20 mg/kg), and metformin (180 mg/kg) for 4 weeks along with the continuous feeding of HFD. The study assessed various renal function parameters, including kidney index, kidney inflammatory biomarkers, kidney oxidative stress biomarkers, fasting blood glucose, serum insulin, serum lipids, blood urea nitrogen, serum creatinine, urine protein, creatinine clearance, protein/creatinine ratio, renal histopathology, and relative gene expressions of kidney NLRP3 and NF-κB proteins, comparing these parameters with normal and diabetic control groups. The findings indicated that HSP and ISL attenuated the pathological progression of DN, as demonstrated through the normalization of various biochemical and gene expression biomarkers, indicating a marked anti-inflammatory and antioxidant effect, improved kidney histology, and mitigated renal dysfunction. These findings suggest that HSP and ISL exhibit nephroprotective effects via mechanisms including inhibition of NLRP3 and NF-κB activation, decrease in oxidative stress, and improvement of the blood glucose status of T2D rats.

1

Molecular docking of HSP and ISL on NLRP3 and NF-κB target proteins. For NLRP3, the binding energies of HSP, ISL, and MTF were −9.0, −8.3, and −6.3 kcal/mol, respectively; and for NF-κB, the binding energies of HSP, ISL, and MTF were −8.4, −7.6, and −6.4 kcal/mol, respectively. Interactions: green color, conventional hydrogen bond; light purple, Pi-alkyl; pink, Pi–Pi stacked.

2

Effect of HSP and ISL on BUN (A), serum creatinine (B), urine protein (C), creatinine clearance (D), and protein/creatinine ratio (E) in Type 2 diabetic rats. Data are shown as mean ± SEM for eight rats (^## p < 0.01 versus the NC group; *p < 0.05 and **p < 0.01 versus the DC group).

3

Effect of HSP and ISL on kidney MDA (A), GSH (B), SOD (C), and CAT (D) levels in Type 2 diabetic rats. Data are shown as mean ± SEM for eight rats (^## p < 0.01 versus the NC group; *p < 0.05 and **p < 0.01 versus the DC group).

4

Effect of HSP and ISL on kidney IL-1β (A), IL-6 (B), and TNF-α (C) levels in Type 2 diabetic rats. Data are shown as mean ± SEM for eight rats (^## p < 0.01 versus the NC group; *p < 0.05 and **p < 0.01 versus the DC group).

5

Effect of HSP and ISL on kidney NLRP3 (A), and NF-κB (B) gene expression in Type 2 diabetic rats. The relative mRNA expressions of NLRP3 and NF-κB in renal tissue were assessed by using qRT-PCR. Each qRT-PCR was performed in triplicate. Data are shown as mean ± SEM (^## p < 0.01 versus the NC group; *p < 0.05 and **p < 0.01 versus the DC group).

6

Effect of HSP and ISL on kidney histopathological changes in Type 2 diabetic rats. NC (image A) showed normal histopathology of renal tissues. No evidence of cellular disintegration, pyknosis, fibrosis, vacuolation, damaged proximal convoluted tubule (PCT), distal convoluted tubule (DCT), glomerulus, basement, podocytes, or hemorrhage is seen (green arrow). The DC (image B) group showed significantly damaged renal tissues. Significantly damaged glomerulus/mesangial cells (red arrow), basement membrane (blue arrow), and severe disintegration in PCT and DCT are seen (black arrow). Additionally, hemorrhage (white arrow), fibrotic changes (yellow arrow), pyknosis (purple arrow), and vacuolation (orange arrow) are also seen. Treatment with HSP, ISL, and MTF showed minimal (HSP-100 [image D] and MTF-180 [image G]), mild (HSP-50 [image C]), moderate (ISL-20 [image F]), or marked (ISL-10 [image E]) disintegration in PCT and DCT (black arrow) and vacuolation (orange arrow) and pyknosis (purple arrow). Notably, no damaged (HSP-50 [image C], HSP-100 [image D], and MTF-180 [image G]), mild (ISL-20 [image F]), or moderately damaged (ISL-10 [image E]) glomerulus, basement membrane, or podocytes (green arrow) are observed. No evidence of fibrosis is seen except for mild fibrosis (yellow arrow) in ISL-10 (image E).