Effect of water-soluble C(60) fullerenes on pulmonary fibrosis induced by bleomycin in mice

Abstract

Objective: To evaluate the role of water-soluble C(60) fullerenes in mice model of lung injury and fibrosis that induced by bleomycin. Methods: 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). Results: 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 P<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 P<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 P>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 P<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, P<0.05]. Conclusion: Water-soluble C(60) fullerenes reduce the severity of pulmonary fibrosis induced by bleomycin in mice.