Amelioration of concanavalin A-induced autoimmune hepatitis by magnesium isoglycyrrhizinate through inhibition of CD4(+)CD25(-)CD69(+) subset proliferation

Abstract

Magnesium isoglycyrrhizinate (MGL) is a new stereoisomer of glycyrrhizic acid, which is clinically used as a hepatoprotective medicine with more potent effects and less side effects than glycyrrhizic acid. This study was designed to evaluate the protective effects and possible mechanism of MGL against concanavalin A (Con A)-induced autoimmune hepatitis. Hepatitis was induced by Con A in C57/6J mice with or without MGL administration; injury score and serum ALT were evaluated. The CD4(+) T-cells were isolated from splenocytes and challenged with Con A after coculturing with MGL. The injury score was significantly improved in MGL-treated mice after Con A challenging for 12 and 24 hours compared with those merely challenged with Con A. Similar trends were observed in the serum levels of ALT and AST. The most interesting result was that MGL administration significantly decreased the frequency of CD4(+)CD25(-)CD69(+) T-cells rather than CD4(+)CD25(+)CD69(+) T-cells in peripheral blood mononuclear cells, after Con A challenging 12 and 24 hours. Moreover, the serum ALT levels were markedly correlated with the frequency of CD4(+)CD25(-)CD69(+) cells, but only weakly correlated with CD4(+)CD25(+)CD69(+) cells in peripheral blood mononuclear cells. More importantly, MGL (5 mg/mL) almost completely eliminated the proliferation of the CD25(-)CD69(+) subset in primary CD4(+) T-cells after Con A challenge. Compared with merely Con A-challenged mice, those with MGL administration significantly demonstrated decreased NALP3, NLRP6, and caspase-3 expression, in which the NALP3 and caspase-3 downregulated in a dose-dependent manner. Our results indicate that MGL may have potential as a therapeutic agent in autoimmune hepatitis by ameliorating liver injury. Its molecular mechanism may be involved in inhibiting CD4(+)CD25(-)CD69(+) subset proliferation and downregulating inflammasome expression in liver tissue.

Keywords: adaptive immunity; autoimmune hepatitis; concanavalin A; drug therapy; mouse; regulatory T-cell.

Figure 1

Study design (in vivo). Abbreviations: MGL, magnesium isoglycyrrhizinate; ALT, alanine aminotransferase; AST, aspartate aminotransferase; H&E, hematoxylin and eosin; Con A, concanavalin A; PBMC, peripheral blood mononuclear cell.

Figure 2

Liver function tests and injury scoring. Notes: (A) Injury score of liver injury. (B) Serum ALT levels. (C) Serum AST levels. The data on the graphs indicate P values (treated group vs control group). Abbreviations: ALT, alanine aminotransferase; Con A, concanavalin A; AST, aspartate aminotransferase; MGL, magnesium isoglycyrrhizinate.

Figure 3

H&E staining of liver tissue. Notes: MGL administration significantly improved liver injury compared with merely challenging with Con A. The blue arrow indicates the infiltrating inflammatory cells in necrotic area (200×). Abbreviations: Con A, concanavalin A; MGL, magnesium isoglycyrrhizinate; H&E, hematoxylin and eosin. subsets and ALT levels before and after Con A challenging

Figure 4

The subsets of CD4⁺ T-cells in PBMCs. Notes: (A) Shows the frequency analysis of CD4⁺CD25⁻CD69⁺ T-cells, representing results from control mice. (B) The frequency of CD4⁺CD25⁻CD69⁺ T-cells in PBMCs, before and after Con A challenge for 6, 12, and 24 hours. (C) The frequency of CD4⁺CD25⁺CD69⁺ T-cells in PBMCs, before and after Con A challenge for 6, 12, and 24 hours. The data on the graphs indicate P values (treated group vs control group). Abbreviations: Con A, concanavalin A; MGL, magnesium isoglycyrrhizinate; PBMC, peripheral blood mononuclear cell; SSC, side scatter; FSC, forward scatter.

Figure 5

The subsets of CD4⁺ T-cells in splenocytes. Notes: (A) Shows the frequency analysis of CD4⁺CD25⁻CD69⁺ T-cells, representing results from control mice. (B) The frequency of CD4⁺CD25⁻CD69⁺ T-cells in splenocytes, before and after Con A challenge for 6, 12, and 24 hours. (C) The frequency of CD4⁺CD25⁺CD69⁺ T-cells in splenocytes, before and after Con A challenge for 6, 12, and 24 hours. The data on the graphs indicate P values (treated group vs control group). Abbreviations: Con A, concanavalin A; MGL, magnesium isoglycyrrhizinate; SSC, side scatter; FSC, forward scatter.

Figure 6

The apoptosis of T-cell subsets in thymocytes. Notes: (A) Shows the analyzing method of CD4⁺ T-cell apoptosis in thymus. (B) CD4⁺ T-cell apoptosis in thymocytes, before and after Con A challenge for 6, 12, and 24 hours. (C) CD8⁺ T-cell apoptosis in thymocytes, before and after Con A challenge for 6, 12, and 24 hours. The data on the graphs indicate P values (treated group vs control group). Abbreviations: Con A, concanavalin A; MGL, magnesium isoglycyrrhizinate; SSC, side scatter; FSC, forward scatter.

Figure 7

The proliferation of cells in the CD4⁺ T-cell subset before and after MGL administration and/or Con A challenging. Notes: The upper part of figure shows the study design in vitro. (A) The CD4⁺ T-cells were isolated from splenocytes using a specific CD4⁺ T-cells isolation kit and were then cultured in plates. (B) Con A challenging significantly increased the CD25⁻CD69⁺ subset of CD4⁺ T-cells, but the 5 mg/mL MGL almost completely eliminated proliferation of the CD25⁻CD69⁺ subset of cells after Con A challenge for 6, 12, and 24 hours. (C) Con A challenging significantly increased the CD25⁺CD69⁺ subset of CD4⁺ T-cells, but the 5 mg/mL MGL almost completely eliminated proliferation of the CD25⁺CD69⁺ subset of cells after Con A challenge for 6, 12, and 24 hours. The data on the graphs indicate P values (treated group vs control group). Abbreviations: Ab, antibody; Con A, concanavalin A; MGL, magnesium isoglycyrrhizinate; SSC, side scatter; FSC, forward scatter.

Figure 8

The inflammasome expression in liver tissue. Notes: After Con A challenge for 6, 12, and 24 hours, the NALP3, NLRP6, and caspase-3 expression increased. (A) Shows the results of the Western blotting analysis, and (B) shows the plots of pixel intensity. For simplicity, representative blots of actin and GAPDH are shown. MGL administration significantly downregulated inflammasome expression, in which caspase-3 decreased in a dose-dependent manner. Abbreviations: Con A, concanavalin A; MGL, magnesium isoglycyrrhizinate.

Figure 9

The possible target in the pathogensis of autoimmune hepatitis. Notes: The blue arrow indicates that the possible molecular mechanism of pathogenesis of autoimmune hepatitis. The red arrows indicate the possible targets of MGL’s proliferation inhibiting-capacity of the regulatory T-cell subsets. Based on these data, MGL inhibiting the expression of inflammasome may be a downstream event in CD4⁺ T-cell subsets regulation. Abbreviations: APC, antigen presenting cell; MGL, magnesium isoglycyrrhizinate; Con A, concanavalin A; Treg, T regulator cell.