关键词: 多房棘球绦虫, 干扰素诱导的跨膜蛋白3, 叉头框P3蛋白, 蛋白互作关系

Abstract:

Objective To investigate the interaction between interferon-inducible transmembrane protein 3 (IFITM3) and forkhead box P3 (Foxp3) in mice during Echinococcus multilocularis infections. Methods E. multilocularis protoscolices were obtained from gerbil liver tissues, and a C57BL/6J mouse model of E. multilocularis infection was established via intraperitoneal injection with E. multilocularis protoscolices. Mice were euthanized 15, 30, 60 and 90 days post-infection, and mouse liver tissues were collected, with uninfected mice as controls. Pathological changes of mouse liver tissues were observed at different time points using hematoxylin-eosin (HE) staining, Masson staining and immunohistochemistry, and the relative expression of IFITM3 and Foxp3 proteins was determined in liver tissues using Western blotting. HEK-293T cells were transfected with IFITM3-ShRNA interference plasmids (ShIFITM3-1, ShIFITM3-2, ShIFITM3-3), and the optimal knockdown fragments were screened for dose-dependent experiments (transfected at 0, 0.5, 1.0, 1.5, 2.0 μg) to further detect changes in Foxp3 expression. The IFITM3-Foxp3 molecular docking was analyzed using the AutoDock software, and the tag plasmid HA-FITTM3 and Flag-Foxp3a were transfected into HEK-293T cells. Total cellular proteins were extracted for immunoprecipitation to verify the interaction between FITTM3 and Foxp3a. Results HE staining revealed disordered arrangements of liver tissues and gradually aggravated infiltration of inflammatory cells in liver tissues of mice infected with E. multilocularis. Masson staining displayed no significant blue staining areas in the control group, and a gradual increase in the blue staining area in the infection group, with (22.12 ± 3.09)%, (28.57 ± 2.1)%, (41.20 ± 2.01)%, and (58.30 ± 2.21)% proportions 15, 30, 60, and 90 days post-infection, respectively (F = 135.60, P < 0.01). Immunohistochemical staining showed that the positive expression rate of IFITM3 increased from (20.18 ± 7.25)% in the control group to (56.22 ± 4.49)% at 90 days post-infection (F = 25.40, P < 0.05), and the positive expression rate of Foxp3 increased from (2.12 ± 1.49)% in the control group to (59.10 ± 4.45)% at 90 days post-infection (F = 106.30, P < 0.05). Western blotting assay showed that the relative expression level of IFITM3 decreased to 0.27 ± 0.04 at 15 days post-infection, and then increased and reached 1.01 ± 0.05 at 90 days post-infection (F = 52.37, P < 0.05), and the relative expression level of Foxp3 gradually increased over time, from 0.03 ± 0.01 in the control group to 0.97 ± 0.03 at 90 days post-infection (F = 143.20, P < 0.05). The IFITM3 expression positively correlated with Foxp3 expression (R² = 0.687 3, P < 0.05). Transfection with ShIFITM3-1, ShIFITM3-2, and ShIFITM3-3 plasmids all resulted in a decrease in the relative expression level of IFITM3 (0.32 ± 0.02, 0.23 ± 0.05, 0.53 ± 0.09), with ShIFITM3-2 showing the highest knockdown efficiency (F = 37.05, P < 0.01), and the relative expression of Foxp3 gradually decreased with ShIFITM3-2 transfection at 0 (1.10 ± 0.14), 0.5 (0.62 ± 0.05), 1.0 (0.52 ± 0.04), 1.5 (0.49 ± 0.01), and 2.0 μg (0.33 ± 0.05), respectively (F = 42.06, P < 0.01). Molecular docking showed that the docking score between IFITM3 and Foxp3 was -312 kcal/mol, indicating a good affinity, and immunoprecipitation indicated an interaction between IFITM3 and Foxp3. Conclusion There is an interaction between IFITM3 and Foxp3 in mice during E. multilocularis infections, and IFITM3 and Foxp3 expression is positively correlated with the severity of liver damages.

Key words: Echinococcus multilocularis, Interferon-induced transmembrane protein 3, Forkhead box P3, Protein-protein interaction