Abstract
Background:
Carbon tetrachloride (CCl₄) is a widely used hepatotoxin that induces liver injury through apoptosis and inflammation. Magnesium oxide (MgO) nanoparticles have shown potential hepatoprotective effects, which may depend on synthesis conditions such as pH.

Aim:
To investigate the hepatoprotective effects of biosynthesized MgO nanoparticles from Ornithogalum cuspidatum against CCl₄-induced liver injury in rats, and to compare the efficacy of nanoparticles synthesized at pH 5 and pH 9.

Methods:
MgO nanoparticles were biosynthesized at pH 5 and pH 9 and characterized for crystalline structure (XRD), elemental composition (EDS), and thermal stability (TGA). Male Wistar rats were divided into six groups: control negative, control positive (CCl₄), MgO pH 5, MgO pH 5 + CCl₄, MgO pH 9, and MgO pH 9 + CCl₄. Liver sections were evaluated for caspase-3 and TNF-α expression using immunohistochemistry. Staining was assessed semi-quantitatively based on intensity and percentage of positive cells, and a final immunoreactivity score was calculated as Intensity × % positive cells.

Results:
Characterization confirmed the successful synthesis and structural features of MgO nanoparticles. CCl₄ exposure increased caspase-3 and TNF-α immunoexpression, with higher scores observed in the CCl₄ group (caspase-3 = 12; TNF-α = 10) compared with the control group (score = 0 for both markers). MgO nanoparticles reduced caspase-3 and TNF-α expression in a pH-dependent manner. The pH 5 formulation showed moderate reduction (caspase-3 = 6; TNF-α = 6), while the pH 9 formulation showed greater reduction (caspase-3 = 4; TNF-α = 2), indicating a stronger protective trend.

Conclusion:
MgO nanoparticles mitigate apoptosis and inflammation in CCl₄-induced liver injury, with higher efficacy observed for nanoparticles synthesized at pH 9, highlighting the importance of synthesis conditions for optimizing hepatoprotective activity.

Key words: Carbon tetrachloride; Caspase-3; Immunohistochemistry; Magnesium oxide nanoparticles; TNF-α.