目的 探究砒霜（三氧化二砷）的生物转化产物亚砷酸钠（sodium arsenite，SA）与N-乙酰半胱氨酸（N-acetylcysteine，NAC）联合用药的抗肺纤维化作用。方法 小鼠L929成纤维细胞给予SA或NAC干预24 h后，测定细胞活力；通过考察不同比例的SA与NAC对L929细胞活力的影响，确定联合用药比例，采用划痕实验和Transwell实验检测细胞迁移能力；采用免疫荧光法检测SA与NAC联合给药对转化生长因子-β1（transforming growth factor-β1，TGF-β1）诱导的L929细胞α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）、I型胶原蛋白α1（collagen type I α1，COL1A1）表达和活性氧（reactive oxygen species，ROS）水平的影响。建立博来霉素诱导的小鼠特发性肺纤维化模型，给予SA或NAC干预28 d，观察小鼠生存情况和体质量变化；采用苏木素-伊红（hematoxylin-eosin，HE）和Masson染色观察肺组织病理变化；检测支气管肺泡灌洗液（bronchoalveolar lavage fluid，BALF）中总蛋白含量、TGF-β1和肺组织羟脯氨酸（hydroxyproline，Hyp）水平；采用免疫荧光法检测肺组织α-SMA、COL1A1、CD86和CD206表达。结果 SA与NAC联合用药的协同效应与二者比例有关，当SA∶NAC＝2∶1时，可显著抑制L929细胞活力与迁移（P＜0.001），且优于单药组（P＜0.01、0.001）；SA与NAC（2∶1）联合给药显著下调TGF-β1诱导的L929细胞中α-SMA、COL1A1表达（P＜0.001），并降低ROS水平（P＜0.01）。体内实验结果显示，与模型组比较，SA与NAC（2∶1）联合给药可以延长小鼠的生存周期，增加体质量，减轻肺组织损伤和胶原沉积，降低BALF中总蛋白含量、TGF-β1及肺组织Hyp水平（P＜0.01、0.001），降低肺组织α-SMA、COL1A1表达（P＜0.001），抑制肺组织巨噬细胞M2型极化（P＜0.001），且对主要脏器无明显毒性作用。结论 亚砷酸钠与NAC联合用药具有协同抗肺纤维化效果，为砷剂联合巯基供体治疗特发性肺纤维化提供了实验依据。

Objective To investigate the antifibrotic effect of combined treatment with sodium arsenite (SA, biotransformation product of arsenic trioxide) and N-acetylcysteine (NAC) on pulmonary fibrosis. Methods L929 fibroblasts were treated with SA or NAC for 24 h before measuring cell viability. The combination drug ratio was determined by examining the effects of different ratios of SA and NAC on viability of L929 cells, and the cell migration ability was detected using scratch assay and Transwell assay. Immunofluorescence assay was used to detect the effects of combined administration of SA and NAC on the expressions of α-smooth muscle actin (α-SMA), collagen type I α1 (COL1A1), and reactive oxygen species (ROS) level in transforming growth factor-β1 (TGF-β1)-induced L929 cells. A mouse model of idiopathic pulmonary fibrosis was established induced by bleomycin, after intervention with SA or NAC for 28 d, the survival and body weight changes of mice were observed. Hematoxylin-eosin (HE) and Masson staining were used to observe pathological changes in lung tissue. The total protein content, TGF-β1 level in bronchoalveolar lavage fluid (BALF), and hydroxyproline (Hyp) level in lung tissue were detected. Immunofluorescence assay was used to detect the expressions of α-SMA, COL1A1, CD86 and CD206 in lung tissue. Results The synergistic effect of SA and NAC combination therapy was related to their ratio. When SA∶NAC = 2∶1, the combination could significantly inhibit viability and migration of L929 cells (P < 0.001), and was superior to the monotherapy group (P < 0.01, 0.001). The combined administration of SA and NAC (2∶1) significantly down-regulated the expressions of α-SMA and COL1A1 in L929 cells induced by TGF-β1 (P < 0.001), and reduced ROS level (P < 0.01). The in vivo experimental results showed that compared with model group, the combined administration of SA and NAC (2∶1) could prolong the survival cycle of mice, increase body weight, reduce lung tissue damage and collagen deposition, decrease the total protein content, TGF-β1 level in BALF and Hyp level in liver tissue (P < 0.01, 0.001), reduce the expression of α-SMA and COL1A1 in lung tissue (P < 0.001), inhibit M2 polarization of lung tissue macrophages (P < 0.001), and had no significant toxic effects on major organs. Conclusion The combination of SA and NAC has a synergistic anti-pulmonary fibrosis effect, providing experimental evidence for the treatment of idiopathic pulmonary fibrosis with arsenic agents combined with thiol donors.

R285.5

浙江省自然科学基金资助项目（LY23H280010）；浙江中医药大学校级科研基金资助项目（2023JKZKTS23）