Data Sheet 1_Dietary α-sitosterol mitigates titanium dioxide nanoparticle-induced hemato-immunotoxicity, oxidative stress, and gene expression dysregulation in Oreochromis niloticus.pdf

IntroductionTitanium 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.
MethodsIn 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.
ResultsDietary 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.
ConclusionThese 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.

引言
二氧化钛纳米颗粒（Titanium dioxide nanoparticles, TDNPs）被广泛应用于食品工业、农业、消费品及诊断领域，其潜在释放进入水生环境后，会对非靶标水生生物尤其是鱼类产生相关生理风险。α-谷甾醇（α-Sitosterol, STL）作为一种植物甾醇，可通过增强抗氧化防御能力及调控炎症信号通路发挥作用。因此，本研究探讨了为期60天的暴露周期内，日粮添加STL是否能够保护尼罗罗非鱼（Oreochromis niloticus）免受TDNPs诱导的毒性损伤。

方法
本研究将300尾尼罗罗非鱼随机分为四组。对照组饲喂基础日粮，STL组饲喂添加80 mg/kg STL的日粮，TDNPs暴露组水体暴露于10 mg/L的TDNPs，TDNPs+STL共处理组则进行TDNPs水体暴露并饲喂添加STL的日粮。

结果
日粮添加STL可显著改善生长性能：与TDNPs暴露组鱼类相比，终末体重、增重率及日生长率提升33%~60%，饲料转化率降低29%。STL补充还可恢复TDNPs暴露导致的血液学参数异常，包括红细胞、血红蛋白、红细胞压积及白细胞水平的显著恢复，从而逆转大细胞正色素性贫血与白细胞减少症。此外，STL可显著降低TDNPs诱导升高的血清谷丙转氨酶、谷草转氨酶、碱性磷酸酶、尿素及肌酐水平，并通过降低总胆固醇、甘油三酯和低密度脂蛋白胆固醇，同时升高高密度脂蛋白胆固醇，使血脂谱恢复正常。饲喂STL的鱼类还表现出应激生物标志物（葡萄糖与皮质醇）水平显著降低，先天免疫应答增强：溶菌酶、补体3、一氧化氮、氮蓝四唑及吞噬活性均显著升高。抗氧化状态通过提升超氧化物歧化酶、过氧化氢酶及谷胱甘肽过氧化物酶活性，以及降低丙二醛水平得到强化。在分子层面，STL补充可下调内质网应激相关基因（chop、jnk、xbp-1及perk），同时上调自噬相关基因（beclin-1与lc3-ii），并下调mtor与p62。组织学分析证实了STL的保护作用，可见肠道、肝脏、肾脏及脾脏结构得到显著修复。

结论
本研究结果表明，STL可对TDNPs诱导的氧化应激、代谢应激及细胞应激提供多维度保护，凸显其作为功能性日粮添加剂在缓解水产养殖中纳米颗粒毒性的应用潜力。