关键词: 日本血吸虫, 肝纤维化, NK细胞, 流式细胞术

Abstract:

Objective Using monoclonal antibodies to deplete or passively transfer natural killer (NK) cell, to explore the role of NK cells in schistosomiasis liver fibrosis. Methods The livers of 24 C57BL/6 healthy mice were collected to isolate NK cells, and the NK cells were activated with interleukin-15 (IL-15), IL-2 and IL-18. Forty C57BL/6 mice were randomly divided into five groups, namely monoclonal antibody group, IgG group, normal saline group, NK cell group and PBS group, with 8 mice in each group. Each mouse was infected with Schistosoma japonicum cercariae (20 ± 1) via an abdominal patch. The mice in the monoclonal antibody group, IgG group and normal saline group were injected with anti-NK1.1 monoclonal antibody (100 μg/mouse), IgG (100 μg/mouse), and normal saline through the tail vein at a dose of 200 μl/mouse each time, once a week, started one week before the infection. Four weeks after infection, the mice in the NK cell group and PBS group were transferred with activated NK cells (1 × 10⁵/mouse) and PBS through the tail vein weekly at dose of 200 μl/mouse each time. At six and eight weeks after infection, flow cytometry was used to detect changes in the proportion of NK cells in the liver of the mice. Masson staining was used to observe liver tissue lesions, and Real-time PCR was used to detect indicators of liver fibrosis, including mRNA relative transcription levels of Ⅰ-collagen (Ⅰ-C), Ⅲ-C, Ⅳ-C, fibronectin (FN), and laminin (LN). Results The proportion of liver NK cells at six weeks after infection in the monoclonal antibody group was (2.56 ± 0.47)%, which was lower than that in the IgG group (4.75 ± 0.62)% and normal saline group (5.35 ± 0.40)% (F = 31.59, P < 0.01). At 8 weeks post-infection, the proportion of liver NK cells were (2.65 ± 0.23)%, (3.43 ± 0.46)%, and (3.56 ± 0.46)% for the monoclonal antibody group, the IgG group and the normal saline group, respectively, and the difference was not statistically significant (F = 4.48, P > 0.05). The proportion of liver NK cells in the NK cell group was (5.58 ± 0.30)% and (3.73 ± 0.42)% for 6 and 8 weeks after infection, respectively. There was no statistically significant difference between 6 weeks (5.51 ± 0.27)% and 8 weeks (2.32 ± 0.40)% after infection for the PBS group (t = 0.25, 2.64, P > 0.05). The area of liver fibrosis in the monoclonal antibody group was (8.42 ± 1.30) × 10⁴ μm² at 6 weeks after infection, which was larger than that in the IgG group (6.40 ± 1.40) × 10⁴ μm² and normal saline group (6.73 ± 1.61) × 10⁴ μm² (F = 13.63, P < 0.01). The area of liver fibrosis for the monoclonal antibody group was (9.55 ± 1.55) × 10⁴ μm² at 8 weeks after infection, which was larger than that in the IgG group (6.11 ± 1.10) × 10⁴ μm² and normal saline group (6.62 ± 1.60) × 10⁴ μm² (F = 30.33, P < 0.01). The area of liver fibrosis in the NK cell group was (5.97 ± 0.96) × 10⁴ μm² at 6 weeks after infection, which was smaller than that in the PBS group (8.27 ± 1.62) × 10⁴ μm² (t = 4.85, P < 0.01). However, at 8 weeks after infection, the area of liver fibrosis for the NK cell group and the PBS group were (6.33 ± 0.98) × 10⁴ μm² and (6.97 ± 1.11) × 10⁴ μm², respectively. The difference was not statistically significant (t = 1.80, P > 0.05). The relative transcription levels of Ⅳ-C, FN, and LN mRNA in the monoclonal antibody group were 1.81 ± 0.47, 1.63 ± 0.38 and 1.41 ± 0.16, which are higher than that in the IgG group (1.00 ± 0.35, 0.81 ± 0.29, 0.79 ± 0.16) and the normal saline group (1.00 ± 0.12, 1.00 ± 0.10, 1.00 ± 0.14) (F = 8.30, 8.25, 14.40, P < 0.01). At 8 weeks after infection, the relative transcription level of Ⅳ-C mRNA in the monoclonal antibody group was 1.66 ± 0.21, which is higher than that in the IgG group (0.94 ± 0.26) and the saline group (1.00 ± 0.09) (F = 15.95, P < 0.01). At 6 weeks after infection, the relative transcription levels for Ⅰ-C, Ⅲ-C and LN mRNA in the NK cell group were 0.66 ± 0.12, 0.61 ± 0.06, 0.64 ± 0.09, respectively. The relative transcription levels were much higher compared with the PBS group (1.01% ± 0.14, 1.01 ± 0.14, 1.01 ± 0.16) (t = 3.36, 4.41, 3.59, P < 0.05). No statistical significant differences were observed at 8 weeks after infection for the NK cell group (0.82 ± 0.40, 0.93 ± 0.37, 0.73 ± 0.30) and the PBS group (1.04 ± 0.38, 1.02 ± 0.25, 1.06 ± 0.49) (t = 0.55, 0.27, 0.81, P > 0.05). Conclusion It was shown that after being infected with S. japonicum, NK cells from mouse liver might inhibit liver fibrosis in the early stage of its formation.

Key words: Schistosoma japonicum, Liver fibrosis, Natural killer cells, Flow cytometry