Dong R, Liu M, Huang X X, Liu Z, Jiang D Y, Xiao H J, Dai H P
Department of Respiratory and Critical Care Medicine, Beijing Chaoyang Hospital, Capital Medical University, Beijing Key Laboratory of Respiratory and Pulmonary Circulation Disorders, Beijing Institute of Respiratory Medicine, Beijing 100020, China.
Zhonghua Yi Xue Za Zhi. 2017 Jun 13;97(22):1740-1744. doi: 10.3760/cma.j.issn.0376-2491.2017.22.014.
To evaluate the role of water-soluble C(60) fullerenes in mice model of lung injury and fibrosis that induced by bleomycin. A total of 20 healthy C57BL/6J mice were randomly divided into normal control group, bleomycin group, high dose C(60) group, low dose C(60) group, each group with 5 mice. Mice were induced pulmonary fibrosis by intratracheal injection of bleomycin except the normal control group, which was induced by saline instead. In low dose C(60) group and high dose C(60) group, 1 mg·kg(-1)·d(-1) and 10 mg·kg(-1)·d(-1) water-soluble C(60) fullerenes was injected into mice intraperitoneally every day, which began from one day before intratracheal instillation of bleomycin until the end of observation. Saline was given to mice in the same way in normal control and bleomycin group. This study investigated the variation of weight and survival rate of mice for 14 d. HE-staining and Masson's trichrome staining were used to assess the severity of fibrosis according to the method proposed by Ashcroft at 14th day. Total lung collagen content was determined by hydroxyproline assay. The changes of transforming growth factor-β(1) (TGF-β(1)) and tumor necrosis factor α (TNF-α) in plasma, bronchial alveolar lavage fluid (BALF) and lung tissue were measured by enzyme-linked immunosorbent assay (ELISA). And, the amount of reactive oxygen species (ROS) was tested by 2, 7-dichlorofuorescin diacetate (DCFH-DA), and determined by the ratio of fluorescence intensity and protein content (OD/μg). C(60) can protect mice that injured by bleomycin from weight loss. According the method proposed by Ashcroft et al.HE and Masson's trichrome staining showed that collagen deposition in lung tissue were markedly attenuated in C(60) (1 mg·kg(-1)·d(-1) and 10 mg·kg(-1)·d(-1)) treated mice compared with bleomycin model mice[(4.08±0.52), (3.00±0.41) vs (6.75±0.75) points, both <0.01]. In low dose C(60) group and high dose C(60) group, the content of hydroxyproline in lung tissue were significantly lower than that in bleomycin group[(0.36±0.06), (0.35±0.08) vs (0.55±0.16) μg/mg, both <0.05]. The level of TGF-β(1) in BALF and lung tissue were also decreased in mice treated with C(60) (10 mg·kg(-1)·d(-1)) compared with bleomycin model mice, but the difference had no statistical significance[(9.38±5.32) vs (23.60±8.96) pg/ml, (2.89±0.35) vs (6.44±2.95) pg/mg, both >0.05]. Also, in high dose C(60) group, the content of TNF-α in plasma, BALF and lung tissue were significantly lower than those in bleomycin group[(4.56±0.73) vs (7.21±2.26) pg/ml, (34.58±23.30) vs (151.00±27.34) pg/ml, (22.99±5.83) vs (122.90±22.04) pg/mg, all <0.05]. In addition, Compared with bleomycin group, ROS in lung tissue was significantly decreased after treatment with C(60) (10 mg·kg(-1)·d(-1))[(19.68±0.91) vs (22.92±1.71) OD/μg, <0.05]. Water-soluble C(60) fullerenes reduce the severity of pulmonary fibrosis induced by bleomycin in mice.
评估水溶性C60富勒烯在博来霉素诱导的小鼠肺损伤和纤维化模型中的作用。将20只健康的C57BL/6J小鼠随机分为正常对照组、博来霉素组、高剂量C60组、低剂量C60组，每组5只。除正常对照组经气管内注射生理盐水诱导外，其余小鼠均经气管内注射博来霉素诱导肺纤维化。在低剂量C60组和高剂量C60组中，从气管内滴注博来霉素前一天开始，每天给小鼠腹腔注射1 mg·kg-1·d-1和10 mg·kg-1·d-1的水溶性C60富勒烯，直至观察结束。正常对照组和博来霉素组小鼠以同样方式给予生理盐水。本研究观察了14天内小鼠体重和存活率的变化。在第14天，按照Ashcroft提出的方法，采用苏木精-伊红（HE）染色和Masson三色染色评估纤维化严重程度。通过羟脯氨酸测定法测定肺组织总胶原含量。采用酶联免疫吸附测定（ELISA）法检测血浆、支气管肺泡灌洗液（BALF）和肺组织中转化生长因子-β1（TGF-β1）和肿瘤坏死因子α（TNF-α）的变化。并且，采用2,7-二氯荧光素二乙酸酯（DCFH-DA）检测活性氧（ROS）的量，并通过荧光强度与蛋白质含量之比（OD/μg）进行测定。C60可以保护博来霉素损伤的小鼠免于体重减轻。根据Ashcroft等人提出的方法，HE染色和Masson三色染色显示，与博来霉素模型小鼠相比，C60（1 mg·kg-1·d-1和10 mg·kg-1·d-1）处理的小鼠肺组织中的胶原沉积明显减轻[（4.08±0.52），（3.00±0.41）对（6.75±0.75）分，均<0.01]。在低剂量C60组和高剂量C60组中，肺组织中羟脯氨酸含量显著低于博来霉素组[（0.36±0.06），（0.35±0.08）对（0.55±0.16）μg/mg，均<0.05]。与博来霉素模型小鼠相比，C60（10 mg·kg-1·d-1）处理的小鼠BALF和肺组织中TGF-β1水平也降低，但差异无统计学意义[（9.38±5.32）对（23.60±8.96）pg/ml，（2.89±0.35）对（6.44±2.95）pg/mg，均>0.05]。此外，在高剂量C60组中，血浆、BALF和肺组织中TNF-α含量显著低于博来霉素组[（4.56±0.73）对（7.21±2.26）pg/ml，（34.58±23.30）对（151.00±27.34）pg/ml，（22.99±5.83）对（122.90±22.04）pg/mg，均<0.05]。此外，与博来霉素组相比，C60（10 mg·kg-1·d-1）处理后肺组织中的ROS显著降低[（19.68±0.91）对（22.92±1.71）OD/μg，<0.05]。水溶性C60富勒烯可减轻博来霉素诱导的小鼠肺纤维化严重程度。
