Alpha-Mangostin as a New Therapeutic Candidate for Concanavalin A-Induced Autoimmune Hepatitis: Impact on the SIRT1/Nrf2 and NF-κB Crosstalk

Abstract

Alpha-mangostin (α-MN) is a xanthone obtained from Garcinia mangostana that has diverse anti-oxidative and anti-inflammatory potentials. However, its pharmacological activity against autoimmune hepatitis (AIH) has not been investigated before. Concanavalin A (Con A) was injected into mice to induce AIH and two doses of α-MN were tested for their protective effects against Con A-induced AIH. The results demonstrated the potent hepatoprotective activity of α-MN evidenced by a remarkable decrease of serum indices of the hepatic injury and amendment of the histological lesions. α-MN significantly attenuated the level and immuno-expression of myeloperoxidase (MPO) indicating a decrease in the neutrophil infiltration into the liver. Additionally, the recruitment of the CD4+ T cell was suppressed in the α-MN pre-treated animals. α-MN showed a potent ability to repress the Con A-induced oxidative stress evident by the reduced levels of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and protein carbonyl (PC), as well as the enhanced levels of antioxidants as the reduced glutathione (GSH), superoxide dismutase (SOD), and total antioxidant capacity (TAC). The ELISA, RT-PCR, and IHC analyses revealed that α-MN enhanced the sirtuin1/nuclear factor erythroid 2 related factor-2 (SIRT1/Nrf2) signaling and its downstream cascade genes concurrently with the inhibition of the nuclear factor kappa B (NF-κB) and the inflammatory cytokines (tumor necrosis factor-alpha and interleukine-6) signaling. Taken together, these results inferred that the hepatoprotective activity of α-MN could prevent Con A-induced AIH through the modulation of the SIRT1/Nrf2/NF-κB signaling. Hence, α-MN may be considered as a promising candidate for AIH therapy.

Keywords: Garcinia mangostana; SIRT1/Nrf2/NF-κB signaling; autoimmune hepatitis; concanavalin A; xanthones; α-mangostin.

Figure 1

Xanthone nucleus and the α-MN structure.

Figure 2

¹H (600 MHz) and ¹³C (150 MHz) NMR spectra of α-MN in CDCl₃.

Figure 3

Alpha-mangostin (α-MN) conferred protection against concanavalin-A (Con A)-induced hepatitis: improvement of biochemical and histopathological indices of the hepatic injury. (I) (A): Alanine aminotransferase (ALT); (B): Aspartate aminotransferase (AST); (C): Alkaline phosphatase (ALP); (D): Gamma-glutamyl transferase (γ-GT); (E): Lactate dehydrogenase (LDH); (F): Albumin. (II) Hepatic sections stained with H&E (×400); (A): Control and (B): α-MN groups displayed normal hepatic architecture with the hepatocytes arranged in regular plates separated by the blood sinusoids (S) and radiating from around the CV (central vein); (C): Con A group showed marked hepatocytes trabeculation, hydropic degeneration, and apoptotic changes (arrow heads), diffuse intense sinusoidal congestion (S) and several inflammatory cell infiltrations (arrows); (D): α-MN 25 + Con A group showed alternating areas of multiacinar necrosis with the same pathological changes with areas of a normal hepatic structure; (E): α-MN 50 + Con A group showed a marked improvement with still hepatocytes having a vacculated cytoplasm and a less marked apoptotic nuclei and inflammatory cell infiltration; (F): Scores of the histopathological hepatic lesion. Data are the mean ± SE. (n = 8); *** p < 0.001; ** p < 0.01; * p < 0.05 vs. control group; ^### p < 0.001; ^## p < 0.01, ^# p < 0.05 vs. Con A group (one-way ANOVA).

Figure 4

Alpha-mangostin (α-MN) decreased concanavalin A (Con A)-induced infiltration of the neutrophiles and the CD4+ T cells into the liver. (I) MPO immuno-expression and level; (II) Immuno-expression of CD4+ in the hepatic specimen. The immuno-staining of MPO and CD4+ of the control and α-MN groups was minimal while that of Con A was high; α-MN pre-treated groups showed a remarkable lowered level of immunostaining for both MPO and CD4+ (×200). Data are the mean ± SE. (n = 8); *** p < 0.001; ** p < 0.01; * p < 0.05 vs. control group; ^### p < 0.001; ^## p < 0.01, ^# p < 0.05 vs. Con A group (one-way ANOVA).

Figure 5

Alpha-mangostin (α-MN) ameliorated concanavalin-A (Con A)-induced oxidative stress and enhanced antioxidants in the hepatic tissue. (A) Malondialdehyde (MDA); (B) Protein carbonyl (PC); (C) 4-Hydroxynonenal (4-HNE); (D) Total antioxidant capacity (TAC); (E) Reduced glutathione (GSH); (F) Superoxide dismutase (SOD). Data are the mean ± SE. (n = 8); *** p < 0.001; ** p < 0.01; * p < 0.05 vs. control group; ^### p < 0.001; ^## p < 0.01, ^# p < 0.05 vs. Con A group (one-way ANOVA).

Figure 6

Alpha-mangostin (α-MN) counteracted concanavalin-A (Con A)-induced suppression in the SIRT1/Nrf2/HO-1 signaling. (A–C) mRNA and protein expression of SIRT1 showing an enhanced protein immuno-expression of SIRT1 in the α-MN pretreated groups; (D–G) mRNA and protein expression of Nrf2, HO-1, GClc, GCLm, NQO1; (H) Nrf2 binding activity; (I) HO-1 level. Data are the mean ± SE. (n = 8); *** p < 0.001; ** p < 0.01; * p < 0.05 vs. control group; ^### p < 0.001; ^## p < 0.01, ^# p < 0.05 vs. Con A group (one-way ANOVA).

Figure 7

Alpha-mangostin (α-MN) inhibited concanavalin-A (Con A)-induced activation of the NF-ĸB signaling in the hepatic tissue. (A) mRNA expression NF-ĸB p65; (B) Level of NF-ĸB; (C) % of the NF-ĸB immuno-positive cells; (D) Immuno-expression of NF-ĸB where control and α-MN groups exhibited minimal NF-ĸB immuno-staining, Con A group showed intense NF-ĸB immuno-staining; Both groups of α-MN + Con A exhibited low immuno-staining for NF-ĸB. Data are the mean ± SE. (n = 8). ** p < 0.01, *** p < 0.001 vs. control group; ^## p < 0.01, ^### p < 0.001 vs. Con A group (one-way ANOVA).

Figure 8

Alpha-mangostin (α-MN) decreased concanavalin-A (Con A)-induced enhancement of the TNF-α/IL-6 signaling in the hepatic tissue. (A) mRNA expression of TNF-α; (B) Immuno-expression of TNF-α (×400); (C) Level of TNF-α; (D) mRNA expression of IL-6; (E) Immuno-expression of IL-6 (×400); (F) Level of IL-6. The immuno-expression of TNF-α and IL-6 was minimal in α-MN pretreated groups compared to intense staining in the Con A group. Data are the mean ± SE. (n = 8). ** p < 0.01, *** p < 0.001 vs. control group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. Con A group (one-way ANOVA).

Figure 9

Schematic diagram showing the possible molecular mechanisms of the hepatoprotective potential of alpha-mangostin (α-MN) against concanavalin-A (Con A)-induced hepatitis.