Effects of different concentrations of brusatol on silicosis fibrosis in mice

Background Silicosis is a diffuse fibrosis of the lungs caused by long-term inhalation of free silicon dioxide (SiO₂). It has a complex pathogenesis and lacks effective treatment. Brusatol (Bru) has a variety of biological activities, and its role in silicosis fibrosis is unclear yet.

Objective To investigate the effects of different concentrations of Bru on SiO₂-induced silicosis fibrosis in mice.

Methods Thirty male C57BL/6J mice were randomly divided into five groups: a control group, a silica group, and three Bru intervention groups with low, medium, and high doses (1, 2, and 4 mg·kg⁻¹), with 6 mice in each group. Except the control group, the remaining groups were established as SiO₂-induced silicosis mouse models by using a single tracheal infusion of 50 μL 60 mg·mL⁻¹ SiO₂ suspension. The control group was dosed with equal amount of saline. The Bru intervention groups were injected intraperitoneally with Bru for 5 consecutive days and then injected every other day. After 28 d of exposure, the mice were executed and lung tissues were collected. The lung coefficient of the mice was measured, and the pathological changes of the lung tissues were observed after hematoxylin-eosin (HE) and Masson staining. The levels of apoptotic protein Cleaved-caspase 3, fibrosis-related protein α-smooth muscle actin (α-SMA), type I collagen (Col-I), autophagy-associated protein Beclin1, microtubule-associated protein 1 light chain 3 (LC3), Sequestosome 1 (p62/SQSTM1), Kelch like ECH-associated protein-1 (Keap1), and nuclear factor erythroid 2 related factor 2 (Nrf2) were detected by Western blot. The mRNA levels of Caspase 3, α-SMA, and Col-I were measured by realtime fluorescence-based quantitative PCR.

Results Compared with the control group, the lung coefficient of mice in the silica group was significantly increased (P < 0.01); the lung tissues of the silicosis mice showed damaged alveolar walls, along with infiltration of inflammatory cells, fibrous nodules, and collagen deposition; furthermore, the protein and mRNA levels of Cleaved-caspase 3, α-SMA, and Col-I were significantly increased (P < 0.01); the expression levels of Beclin1, LC3-II/I, p62, and Nrf2 were increased, while that of Keap1 was decreased (P < 0.05). The interventions with low and medium doses of Bru reduced lung coefficient (P < 0.05) and protected against pathological damage and collagen deposition in the lung tissues of the silicosis mice; the protein and mRNA expression levels of Cleaved-caspase 3, α-SMA, and Col-I were significantly decreased in the low and medium dose groups (P < 0.05, P < 0.01), the expression levels of Beclin1, LC3-II/I, p62, and Nrf2 were also decreased (P < 0.05, P < 0.01), and the expression level of Keap1 was increased in the medium dose group (P < 0.05). However, compared with the silica group, the differences in lung coefficient, pathological damage, and protein and mRNA expression levels of Cleaved-caspase 3, α-SMA, and Col-I in the Bru high dose group were not statistically significant (P > 0.05). In addition, the high dose of Bru decreased Beclin1, LC3-II/I, and Nrf2 expression levels (P < 0.01), did not change p62 protein expression level (P > 0.05), while increased Keap1 protein level (P < 0.01).

Conclusion Low and medium doses of Bru might regulate autophagy through the Keap1-Nrf2 pathway, ameliorate autophagic degradation impairment, reduce pulmonary coefficient, attenuate apoptosis, and delay the progression of fibrosis in SiO₂-induced silicosis mice.