Effect of rapamycin nanoparticles in an animal model of primary biliary cholangitis

Abstract

Background: Primary biliary cholangitis (PBC) is a chronic autoimmune-mediated cholestatic liver disease. Nanoparticles encapsulating rapamycin (ImmTOR) suppress adaptive immune responses and induce the hepatic tolerogenic immune response.

Aim: To investigate the effects of ImmTOR in PBC mouse models.

Methods: PBC models were induced in C57BL/6 mice by two immunizations of 2-octynoic acid-coupled bovine serum albumin at two-week intervals, and polycytidylic acid every three days. The PBC mouse models were separated into the treatment group and the control group. The levels of alkaline phosphatase (ALP) and alanine aminotransferase in the mice were detected using an automatic biochemical analyzer. Liver and spleen mononuclear cells were analyzed by flow cytometry, and serum anti-mitochondrial antibodies (AMA) and the related cytokines were analyzed by enzyme-linked immunosorbent assay. Liver histopathology was examined by hematoxylin and eosin staining and scored.

Results: After treatment with ImmTOR, the ALP level was significantly decreased (189.60 U/L ± 27.25 U/L vs 156.00 U/L ± 17.21 U/L, P < 0.05), the level of AMA was reduced (1.28 ng/mL ± 0.27 ng/mL vs 0.56 ng/mL ± 0.07 ng/mL, P < 0.001) and the expression levels of interferon gamma and tumor necrosis factor α were significantly decreased (48.29 pg/mL ± 10.84 pg/mL vs 25.01 pg/mL ± 1.49 pg/mL, P < 0.0001) and (84.24 pg/mL ± 23.47 pg/mL vs 40.66 pg/mL ± 14.65 pg/mL, P < 0.001). The CD4+ T lymphocytes, CD8+ T lymphocytes and B lymphocytes in the liver were significantly reduced, with statistically significant differences (24.21% ± 6.55% vs 15.98% ± 3.03%, P < 0.05; 9.09% ± 1.91% vs 5.49% ± 1.00%, P < 0.001; 80.51% ± 2.96% vs 75.31% ± 4.34%, P < 0.05). The expression of CD8+ T lymphocytes and B lymphocytes in the ImmTOR treatment group also decreased (9.09% ± 1.91% vs 5.49% ± 1.00%, P < 0.001; 80.51% ± 2.96% vs 75.31% ± 4.34%, P < 0.05). The liver pathology of PBC mice in the treatment group showed reduced inflammation and a decreased total pathology score, and the difference in the scores was statistically significant (4.50 ± 2.88 vs 1.75 ± 1.28, P < 0.05).

Conclusion: ImmTOR can improve biochemistry and pathology of liver obvious by inhibiting the expression of CD8+ T cells and B cells, and reducing the titer of AMA.

Keywords: Anti-mitochondrial antibodies; Cytokine; Mouse model; Nanoparticles; Primary biliary cholangitis; Rapamycin.

Figure 1

Animal modeling and intervention protocol. CFA: Complete Freund's adjuvant; IFA: Incomplete Freund’s adjuvant; ImmTOR: Nanoparticles encapsulating rapamycin; Poly I: C: Polycytidylic acid; 2-OA-BSA: 2-octynoic acid-coupled bovine serum albumin.

Figure 2

Analysis of biochemical parameters in serum in the treatment and control groups. A: Nanoparticles encapsulating rapamycin treatment decreased alkaline phosphatase in the primary biliary cholangitis (PBC) mouse models (^aP < 0.05); B: Comparison of alanine aminotransferase in the treatment and control group PBC mouse models. ALP: Alkaline phosphatase; ALT: Alanine aminotransferase.

Figure 3

Nanoparticles encapsulating rapamycin treatment decreased serum anti-mitochondrial antibodies levels and inhibited the expression of cytokines. A: Levels of anti-mitochondrial antibodies at 4 weeks, 8 weeks and 12 weeks in primary biliary cholangitis (PBC) mice treated with nanoparticles encapsulating rapamycin and in mice in the control group; B: Comparative serum profiling of interferon gamma between control and treated PBC mice; C: Comparative serum levels of tumor necrosis factor α between the control and treatment group (^aP < 0.05). AMA: Anti-mitochondrial antibodies; IFN-γ: Interferon gamma; TNF-α: Tumor necrosis factor α.

Figure 4

Nanoparticles encapsulating rapamycin treatment decreased the lymphocyte expression in the liver and spleen. A: The percentage of CD4-positive T cells in the liver of the treatment group decreased compared with that in the control group; B: A comparison of the control and treatment groups showed that the percentage of CD8-positive T cells in the liver of the treatment group was decreased; C: The expression of B lymphocytes was decreased in the liver of the treatment group; D: There was no difference in CD4-positive T cells in the spleen between the two groups; E: The percentage of CD8-positive T lymphocytes in the spleen of the treatment group decreased compared with that in the control group; F: The expression of B lymphocytes in the spleen was decreased in the treatment group (^aP < 0.05).

Figure 5

Nanoparticles encapsulating rapamycin treatment improved liver pathology. A: Comparison of the hepatic histopathological score between the two groups (^aP < 0.05); B: Hepatic histopathology visualized using hematoxylin and eosin staining in control mice; C: Hepatic pathological manifestations of mice in the treatment group. HE: Hematoxylin and eosin.