Figure 1.
Asiatic acid induces apoptosis of activated T cells but not naïve T cells.
(A) The chemical structure of asiatic acid. (B–D) Lymph node-derived T cells isolated from BALB/c mice were incubated with medium or in the presence of Con A (5 µg/ml) or anti-CD3/anti-CD28 (1 µg/ml) or PMA (100 ng/ml)/ionomycin (1 µg/ml) for 24 h. Then cells were further incubated with or without various concentrations of asiatic acid for 24 h. Cell proliferation was determined by MTT assay (B). Cells apoptosis were determined by Annexin V/PI staining (C). Data are shown as means ± SEM of three independent experiments (D). *P<0.05, **P<0.01 vs. drug-untreated group.
Figure 2.
Asiatic acid results in caspases activation in activated T cells.
(A–B) Lymph node-derived T cells isolated from BALB/c mice were incubated in medium or in the presence of Con A (5 µg/ml) or anti-CD3/anti-CD28 (1 µg/ml) or PMA (100 ng/ml)/ionomycin (1 µg/ml) for 24 h. Then cells were further incubated with or without various concentrations of asiatic acid for 24 h. Protein levels of Bid, caspase-3, -9, -7, -8 and -12, and PARP were examined by Western blotting. (C) Enzymatic activities of caspase-3, -9, and -8 of Con A-activated T cells after asiatic acid treated for 12 h were determined. Data are shown as means ± SEM of three independent experiments. *P<0.05 vs. drug-untreated group.
Figure 3.
Asiatic acid triggers apoptosis of Con A-activated T cells through mitochondrial pathway.
Lymph node-derived T cells isolated from BALB/c mice were incubated in medium or in the presence of Con A (5 µg/ml) for 24 h. Then cells were further incubated with or without various concentrations of asiatic acid for 24 h. Cells were stained with JC-1 intensity of FL-2 was analyzed by flow cytometry to determine mitochondrial membrane potential. The cells located with low red fluorescence were considered as low mitochondrial transmembrane potential (A). Data are shown as means ± SEM of three independent experiments (B). *P<0.05, **P<0.01 vs. drug-untreated group. (C) The release level of cytochrome c from mitochondria was examined by Western blotting. The results shown are representative of three experiments.
Figure 4.
Fas-mediated death-receptor apoptotic pathway does not mainly contribute to asiatic acid-induced cell death of activated T cells.
(A) Lymph node-derived T cells isolated from BALB/c mice were incubated in medium or in the presence of Con A (5 µg/ml) for 24 h. Then cells were further incubated with or without various concentrations of asiatic acid for 24 h. Fas expression at the surface of T cells was evaluated by flow cytometry. (B, C) Lymph node-derived T cells isolated from MRLlpr/Lpr mice were incubated in medium or in the presence of Con A (5 µg/ml) for 24 h. Then cells were further incubated with or without various concentrations of asiatic acid for 24 h. Apoptosis and mitochondria membrane potential were evaluated by Annexin V-FITC/PI (B) and JC-1 staining (C), respectively. Data are shown as means ± SEM of three independent experiments. *P<0.05, **P<0.01 vs. drug-untreated group.
Figure 5.
Asiatic acid protects against Con A-induced T-cell-mediated acute fulminant hepatitis in mice. BALB/c mice were received an intravenous Con A injection (15 mg/kg body weight).
In the drug treatment group, asiatic acid (intragastric administration) and dexamethason (intramscular injection) were given twice at 8 h before and 1 h after Con A injection, respectively. (A, B) Time course of serum alanine transaminase (ALT) and aspartate transaminase (AST) release. Values are shown as the means ± SEM of three mice at each time point. *P<0.05 vs. Con A group at the same time point. (C, D) Dose-dependent inhibition on ALT and AST release. Values are shown as the means ± SEM of eight mice in each group. *P<0.05, **P<0.01 vs. Con A group. (E) Photomicrographs of representative mouse livers with H&E staining are shown (original magnification ×100).
Figure 6.
Asiatic acid inhibited inhibits TNF-α and IFN-γ levels in serum from Con A-induced hepatitis in mice.
Cytokine levels in serum were measured by ELISA. (A, B) Time course of TNF-α and IFN-γ released into serum. Values are shown as the means ± SEM of three mice at each time point. *P<0.05, **P<0.01 vs. Con A group at the same time point. (C, D) Dose-dependent inhibition on TNF-α and IFN-γ levels. Values are shown as the means ± SEM of eight mice in each group. *P<0.05, **P<0.01 vs. Con A group.
Figure 7.
Asiatic acid induces apoptosis of activated T cells in Con A-induced T-cell-mediated hepatitis.
BALB/c mice were received an intravenous Con A injection (15 mg/kg body weight). Asiatic acid (30 and 60 mg/kg) were intragastric given twice at 8 h before and 1 h after Con A injection, respectively. Splenic T cells were isolated at 2 h after Con A administration. After culture for 24 h, cells were co-stained with Annexin V/PI to determine apoptosis by flow cytometry (A). Values are shown as the means ± SEM of eight mice (B). *P<0.05, **P<0.01 vs. Con A group. (C) Paraffin-embedded liver tissue sections from vehicle- and asiatic acid-treated group were stained for TUNEL-FITC and CD4-PE. The nuclei were stained with DAPI. Original magnification, ×100.
Figure 8.
Overview of cell death pathways for asiatic acid-induced apoptosis in Con A-activated T cells.
Asiatic acid triggered apoptosis of Con A-activated T cells in a mitochondria-dependent manner indicated by the disruption of the mitochondrial transmembrane potential, release of cytochrome c from mitochondria to cytosol, caspase-9 and caspase-3 activation and cleavage of PARP. At the same time, following activation of T cells from MRLlpr/lpr mice with mutation of Fas demonstrated a similar susceptibility to asiatic acid-induced apoptosis compared with normal T cells. In addition, asiatic acid hardly influenced caspase-12 activation and the expression of GRP78 and CHOP. Taken together, these results suggest that Fas-mediated death-receptor apoptotic pathway and endoplasmic reticulum (ER) stress-mediated apoptotic pathway do not mainly contribute to asiatic acid-induced cell death.