GENE EXPRESSION ANALYSIS AND HEPATOPROTECTIVE ACTIVITY OF METFORMIN-LOADED CHITOSAN NANOPARTICLES AGAINST METHOTREXATE-INDUCED LIVER TOXICITY IN VIVO Authors: Nayyer Shamim, Muhammad Adil Rasheed, Muhammad Ovais Omer, Wasim Shehzad Journal: Journal of Animal and Plant Sciences (JAPS) ISSN: 1018-7081 (Print), 2309-8694 (Online) Volume: 37 Issue: 1 Year: 2027 DOI: https://doi.org/10.36899/JAPS.2027.1.0004 URL: https://doi.org/https://doi.org/10.36899/JAPS.2027.1.0004 Publisher: Pakistan Agricultural Scientists Forum Abstract:

The adverse hepatotoxic effect is a great barrier for both drug development and pharmacotherapy because of its complex pathogenesis and unpredictable outcome. This research aimed to explore the hepatic toxicity and gene expression studies after the oral methotrexate administration in Wistar rats and the hepatoprotective efficacy of the metformin-loaded chitosan nanoparticles system. The nanoparticles (NPs) were synthesized by means of the ionic gelation technique with the use of the following instrumental approaches: Fourier Transform Infrared Spectroscopy, Scanning Electron Microscopy, polydispersity index, size, zeta potential analysis, drug entrapment efficiency, and in vitro drug release test. Metformin-loaded chitosan NPs exhibited a distinctive spectral peak of 3447 cm⁻¹, spherical shape, low PDI = 0.30, size of 213 nm, zeta potential of +28.1 mV, and drug entrapment efficiency of 97.34%. A total of 48 male Wistar albino rats (3–4 months old; 220 ± 25 g) were randomly allocated into eight groups (n = 6 per group) under a Completely Randomized Design (CRD) for hepatotoxicity evaluation based on biochemical, histopathological, and gene expression studies. The methotrexate treatment (Group B) caused a severe hepatotoxic and hematological effect with the increased ALT, AST, ALP, total bilirubin serum concentrations and decreased total protein, albumin, TLC, hemoglobin, RBC, HCT, and platelets count compared to the normal control (p ≤ 0.001). These alterations were markedly attenuated by metformin-loaded chitosan nanoparticles (Met-CSNPs), particularly at 75 mg/kg/day (Group H), which produced the most favorable biochemical and hematological profile among the nanoparticle-treated groups. Histopathological examination revealed Met-CSNP treatment progressively reduced hepatic injury, with the 75 mg/kg/day dose showing the greatest tissue preservation and no detectable fibrosis. Gene-expression analysis demonstrated significant suppression of hepatic TIMP1 and TGF-β expression relative to the MTX toxic-control group, with a graded dose-response across the 25, 50, and 75 mg/kg/day Met-CSNP doses. The 75 mg/kg/day formulation produced the greatest reduction in TGF-β expression (0.51-fold relative to the normal control) and the strongest overall suppression of TIMP1 among the nanoparticle-treated groups. Overall, Met-CSNPs demonstrated dose-dependent hepatoprotective and anti-fibro genic effects against MTX-induced hepatic injury, with the 75 mg/kg/day formulation showing the most pronounced protective response.

Keywords: Chitosan nanoparticles, Nanoparticle drug delivery, Hepatic protection, Toxicity attenuation.