Impact of Losartan Nanoparticles on Carbon Tetrachloride-induced Cerebellar Injury in Rats: Association with Alterations in RIPK1/RIPK3/MLKL Necroptosis Markers.

Carbon tetrachloride (CCL₄) is a lipophilic industrial solvent metabolized by cytochrome P450 enzymes. Its rapid diffusion across membranes facilitates widespread tissue distribution, including the brain, where it induces oxidative stress and alters the expression of necroptosis-related markers, including tumor necrosis factor-alpha (TNF-α). Losartan, an angiotensin II receptor antagonist, has demonstrated attenuation of cerebellar injury in many neurodegenerative diseases. This study evaluated the potentials of losartan and its nanoparticle formulation against CCL₄-induced cerebellar injury. Twenty-four adult male Sprague-Dawley rats were assigned into four groups: Control, CCL₄, Losartan, and Losartan potassium nanoparticles (LP-NPs)-treated groups. Oxidative stress parameters, quantitative Real time- PCR (qRT-PCR) for Tnf-α, receptor interacting protein kinase 1 (Ripk1), receptor interacting protein kinase 3 (Ripk3), and mixed lineage kinase domain-like protein (Mlkl), along with histological and immunohistochemical (IHC) assessments, were performed. CCL₄ exposure significantly elevated malondialdehyde (MDA) while reducing superoxide dismutase (SOD), reduced glutathione (GSH), and catalase. Molecular study shows upregulation of mRNA expression of Tnf-α, Ripk1, Ripk3, and Mlkl, which correlates with alterations in necroptosis-related marker expression. IHC showed marked increases in RIPK1, RIPK3, Glial Fibrillary Acidic Protein (GFAP), and Ionized calcium-binding adaptor molecule1 (Iba1) immunoreactivity, with a concomitant decrease in caspase-8. Histologically, CCL₄ induced severe cerebellar alterations, including reduced myelinated fiber area, Purkinje cell loss, and granular layer thinning. Both Losartan- and LP-NPs-treated groups significantly mitigated these biochemical, molecular, and histological changes, with LP-NPs exerting greater observed effects in histological parameters and selected IHC markers. Both treatments produced comparable reductions in qRT-PCR mRNA expressions of Tnf- α , Ripk1, and Ripk3. In conclusion, losartan, particularly in nanoparticle form, attenuates CCL₄-induced cerebellar toxicity and is associated with alteration in necroptosis-related markers and attenuation of GFAP and Iba1 immunoreactivity, accompanied by structural improvement. These findings highlighted LP-NPs as a prospective biochemical, molecular, and histological protective agent against CCL₄-induced cerebellar injury. Further studies are required to confirm the direct involvement of the RIPK1/RIPK3/MLKL necroptosis pathway.