Hesperidin‐Loaded Chitosan Nanoparticles Restore Hepatic Homeostasis by Targeting Nrf2/ <scp>HO</scp> ‐1 and <scp>NF</scp> ‐ <scp>κB</scp> /p53 Signaling in Malathion‐Exposed Rats

The present study investigated the hepatoprotective effects of hesperidin (HES) encapsulated in chitosan nanoparticles (HES-CNPs) against Malathion (MAL)-induced liver toxicity in rats. Ninety male Wistar rats were randomly divided into six groups: a control group, groups treated with HES (100 mg/kg BW) or HES-CNPs (100 mg/kg BW), a MAL-exposed group (27 mg/kg BW), and two combination groups receiving MAL (27 mg/kg BW) together with either HES or HES-CNPs (100 mg/kg BW) for four consecutive weeks. MAL administration induced profound biochemical and molecular alterations in rats. It significantly reduced serum total protein and its fractions, while elevating hepatic enzyme activities, bilirubin, cholesterol, and triglycerides (TGs). Oxidative stress was evident by decreased activities of antioxidant enzymes (CAT, SOD, GPx), reduced glutathione levels, and increased malondialdehyde and reactive oxygen species (ROS). At the molecular level, MAL exposure upregulated proapoptotic genes (p53, Bax, caspase-3, caspase-9) and inflammatory markers (TNF-α, NF-κB), while down-regulating the antiapoptotic gene Bcl-2 and the cytoprotective gene Nrf2. Exposure to MAL significantly increased hepatic DNA oxidative damage, as indicated by elevated 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels and DNA fragmentation. Treatment with HES-CNPs provided significantly greater protection than crude HES (p < 0.05). These hepatoprotective effects were evidenced by normalization of liver function biomarkers and lipid profile, restoration of antioxidant enzyme activities and glutathione levels, reduction of lipid peroxidation, suppression of proinflammatory and proapoptotic markers, upregulation of Nrf2 and Bcl-2, attenuation of DNA oxidative damage (8-OHdG) and fragmentation, and marked improvement of hepatic histoarchitectural and ultrastructural integrity. HES demonstrated strong binding affinity toward key proteins involved in oxidative stress, apoptosis, and inflammatory pathways, as revealed by in silico studies. In conclusion, HES-CNPs demonstrated improved hepatoprotection against MAL toxicity through the activation of the Nrf2/HO-1 antioxidant pathway and the inhibition of NF-κB/p53-mediated inflammation and apoptosis.