Dietary α-sitosterol mitigates titanium dioxide nanoparticle-induced hemato-immunotoxicity, oxidative stress, and gene expression dysregulation in Oreochromis niloticus

Introduction Titanium dioxide nanoparticles (TDNPs) are widely used in food industries, agricultural and consumer products, and diagnostic purposes, leading to their potential release into aquatic environments and associated physiological risks to non-target aquatic organisms, particularly fish. α-Sitosterol (STL), a phytosterol, acts by enhancing antioxidant defenses and modulating inflammatory signaling pathways. Hence, this study investigated whether dietary STL can protect Nile tilapia ( Oreochromis niloticus ) from TDNPs-induced toxicity during a 60-day exposure. Methods In this study, 300 Nile tilapia were allocated into four groups. The control group received a basal diet, the STL group was fed a diet supplemented with 80 mg STL/kg, the TDNPs group was exposed to 10 mg/L of TDNPs in water, and the TDNPs + STL group was exposed to TDNPs and fed the STL-supplemented diet. Results Dietary STL supplementation markedly improved growth performance, with increases of 33%–60% in final body weight, weight gain, and daily growth rate, and a 29% reduction in feed conversion ratio compared to TDNPs-exposed fish. STL supplementation also restored hematological parameters altered by TDNPs exposure, including significant recovery of red blood cells, hemoglobin, packed cell volume, and white blood cells, thereby reversing macrocytic normochromic anemia and leukopenia. Furthermore, STL significantly decreased elevated serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, urea, and creatinine levels induced by TDNPs, and normalized lipid profiles by reducing total cholesterol, triglycerides, and low-density lipoprotein cholesterol, while elevating high-density lipoprotein cholesterol. STL-fed fish also exhibited significant reductions in stress biomarkers (glucose and cortisol) and enhanced innate immune responses, as evidenced by higher lysozyme, complement 3, nitric oxide, nitro blue tetrazolium, and phagocytic activity. Antioxidant status was strengthened through increased superoxide dismutase, catalase, and glutathione peroxidase activities and reduced malondialdehyde levels. At the molecular level, STL supplementation downregulated endoplasmic reticulum stress-related genes ( chop, jnk, xbp-1 , and perk ), while upregulating autophagy-related genes ( beclin-1 and lc3-ii ) and downregulating mtor and p62 . Histological analysis confirmed STL’s protective effects, showing marked recovery of intestinal, hepatic, renal, and splenic structures. Conclusion These findings demonstrated that STL confers multi-level protection against TDNPs-induced oxidative, metabolic, and cellular stress, highlighting its potential as a functional dietary supplement for mitigating nanotoxicity in aquaculture.