Objective: The current study aimed to evaluate the possible protective effects of N-acetylcysteine (NAC) against Indum-tin oxide (ITO) nanoparticle (Nano-ITO) -induced pulmonary alveolar proteinosis (PAP) in rats, especially via modulation of nuclear factor kappa B (NF-κB) signaling. Methods: In October 2019, 50 adult male Sprague-Dawley rats were randomly allocated into five groups (10 rats each) as follows: blank control group, saline control group, NAC control group (200 mg/kg), Nano-ITO group (receiving a repeated intratracheal dose of 6 mg/kg Nano-ITO) and NAC intervention group (pre-treated intraperitoneally with 200 mg/kg NAC 1.5 h before the administration of an intratracheal dose of 6 mg/kg Nano-ITO). The rats were exposed twice a week for 12 weeks. Rats were then euthanized under anesthesia, and their lungs were removed for histopathological and immunohistochemical analysis. The comparison of indicators reflecting oxidative stress and pulmonary inflammation among groups was conducted using one-way analysis of variance (ANOVA) and Bonferroni's test. The effect of NAC on Nano-ITO induced NF-κB signaling pathway in rats was analyzed. Results: Histopathological examination of Nano-ITO exposed rats revealed diffuse alveolar damage, including PAP, cholesterol crystals, alveolar fibrosis, pulmonary fibrosis, and alveolar emphysema. Immunohistochemical results of Nano-ITO exposed rats showed strong positive for nuclear factor κB p65 (NF-κB p65) and nuclear factor Kappa B inhibitory factor kinase (IKK-β) and weak positive for nuclear factor κB inhibitory protein α (IκB-α) in the nuclei of bronchiolar and alveolar epithelial cells. Compared with blank control group, saline control group and NAC control group, the level of total protein (TP) in bronchoalveolar lavage fluid of rats in Nano-ITO group was significantly increased (P<0.05), and the activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA) content and total antioxidant capacity (T-AOC) were significantly increased (P<0.05), the levels of proinflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) were significantly increased (P<0.05), and the levels of NF-κB p65, IKK-β, inducible nitric oxide synthase (iNOS) and reactive oxygen species (ROS) in lung tissue were significantly increased (P<0.05). Compared with Nano-ITO group, the levels of TP, T-AOC, MDA and TNF-α in bronchoalveolar lavage fluid of rats in NAC intervention group were significantly decreased (P<0.05), and the levels of NF-κB p65 and ROS in lung tissue were significantly decreased (P<0.05). Western blot results showed that compared with the control groups, the protein expressions of NF-κB p65 and IKK-β in the lung tissue of Nano-ITO group were increased, while the protein expression of IκB-α was decreased (P<0.05). Compared with Nano-ITO group, the protein expressions of NF-κB p65 and IKK-β in lung tissue of rats in NAC intervention group were decreased, while the protein expression of IκB-α was increased (P<0.05) . Conclusion: The study demonstrated that Nano-ITO might induce pulmonary toxicity through the activation of NF-κB signaling pathway, and NAC could antagonize the pulmonary toxicity of Nano-ITO by inhibiting the NF-κB signaling pathway.
目的： 评估N-乙酰半胱氨酸（NAC）通过调节核因子-κB（NF-κB）信号通路对氧化铟锡纳米颗粒（Nano-ITO）诱导的大鼠肺泡蛋白沉积症（PAP）的可能保护作用。 方法： 于2019年10月，将50只成年雄性SD大鼠随机分为5组（每组10只），分别为空白对照组、生理盐水对照组、NAC对照组（200 mg/kg，腹腔注射）、Nano-ITO组（6 mg/kg Nano-ITO，非暴露式气管灌注）和NAC干预组（大鼠腹腔注射20 mg/kg NAC，1.5 h后气管内灌注6 mg/kg Nano-ITO）。每周染毒2次，共染毒12周。大鼠麻醉下实施处死，取出肺脏进行组织病理学和免疫组化分析。各组间反映氧化应激和肺部炎症的指标比较采用单因素方差分析（ANOVA）和Bonferroni的事后检验进行，并分析NAC对Nano-ITO诱导的大鼠NF-κB信号通路的影响。 结果： Nano-ITO暴露大鼠的组织病理学检查显示弥漫性肺泡损伤，包括PAP、胆固醇晶体、肺泡纤维化、肺纤维化和肺泡肺气肿。Nano-ITO暴露大鼠的免疫组织化学结果显示，在细支气管和肺泡上皮细胞的细胞核中，核因子κB p65（NF-κB p65）和核因子Kappa B抑制物激酶（IKK-β）呈强阳性，核因子κB抑制蛋白α（IκB-α）呈弱阳性。与空白对照组、生理盐水对照组和NAC对照组比较，Nano-ITO组大鼠支气管肺泡灌洗液中总蛋白（TP）水平明显升高（P<0.05），乳酸脱氢酶（LDH）、超氧化物歧化酶（SOD）活力、丙二醛（MDA）含量以及总抗氧化能力（T-AOC）明显增加（P<0.05），促炎细胞因子白细胞介素1β（IL-1β）、白细胞介素6（IL-6）、肿瘤坏死因子α（TNF-α）的水平明显升高（P<0.05），大鼠肺组织中NF-κB p65、IKK-β、诱导型一氧化氮合成酶（iNOS）和活性氧（ROS）水平明显升高（P<0.05）。与Nano-ITO组比较，NAC干预组大鼠支气管肺泡灌洗液中TP水平、T-AOC、MDA含量和TNF-α水平明显降低（P<0.05），大鼠肺组织中NF-κB p65和ROS水平均明显降低（P<0.05）。Western blot结果显示，与各对照组比较，Nano-ITO组大鼠肺组织中NF-κB p65和IKK-β的蛋白表达增加，IκB-α的蛋白表达降低（P<0.05）；与Nano-ITO组比较，NAC干预组大鼠肺组织NF-κB p65和IKK-β的蛋白表达降低，IκB-α的蛋白表达升高（P<0.05）。 结论： Nano-ITO可能通过激活NF-κB信号通路对肺脏产生毒性作用，且NAC可能通过抑制NF-κB信号通路来拮抗Nano-ITO的肺毒性。.
Keywords: Metal nanoparticles; N-acetylcysteine; NF-κB signaling pathway; Pulmonary alveolar proteinosis; Rats; lIndium-tin oxide nanoparticles.