Impairment of thymocyte function via induction of apoptosis by areca nut extract

Figures & data

Figure 1. Administration of ANE impaired thymocyte IL-2 production. Thymocytes (5 × 10⁶ cells/ml) isolated from different groups treated with or without ANE in vivo were cultured ex vivo and stimulated with PMA + ionomycin for 24 h. (a) Metabolic activity of the thymic cells was measured by MTT assay. (b) Concentrations of IL-2 in culture supernatants were measured by ELISA. Data are expressed as mean (±SE) of quadruplicate cultures. Results are representative of three independent experiments. *Value significantly different compared to VH group at p < 0.05.

Table 1. The in vivo effect of ANE on body weight and on thymus weight and cellularity.

			ANE
	NA	VH	1 mg/kg	5 mg/kg	25 mg/kg
Body weight (g)
Day 1	16.53 ± 0.25	16.15 ± 0.27	16.19 ± 0.18	16.33 ± 0.18	16.25 ± 0.14
Day 6	20.19 ± 0.22	19.42 ± 0.30	19.04 ± 0.27	19.29 ± 0.29	17.20 ± 0.22*
Thymus
Weight (mg)	65.33 ± 3.05	61.75 ± 2.25	62.05 ± 3.73	54.84 ± 2.29*	37.18 ± 1.72*
Total number (10^8)	2.06 ± 0.11	1.97 ± 0.13	1.96 ± 0.11	1.74 ± 0.08*	0.99 ± 0.07*
Cell viability (%)	91.00 ± 0.77	90.20 ± 0.86	89.00 ± 0.71	83.00 ± 0.71*	77.40 ± 0.51*
Cellularity^a (% of total cells)
CD4^+CD8^−	10.99 ± 0.92	10.62 ± 0.46	9.97 ± 0.35	10.50 ± 0.374	17.53 ± 0.82*
CD8^+CD4^−	4.44 ± 0.23	4.88 ± 0.28	4.25 ± 0.23	4.83 ± 0.23	7.19 ± 0.55*
CD4^+CD8^+	81.58 ± 1.03	81.40 ± 0.81	82.90 ± 0.64	81.84 ± 0.72	71.34 ± 1.52*
CD4^−CD8^−	3.25 ± 0.19	3.65 ± 0.13	3.41 ± 0.06	3.32 ± 0.23	4.68 ± 0.19*
Cells/thymus^b (×10^7)
CD4^+CD8^−	2.27 ± 0.19	2.09 ± 0.09	1.95 ± 0.07	1.82 ± 0.07*	1.74 ± 0.08*
CD8^+CD4^−	0.91 ± 0.05	0.96 ± 0.05	0.83 ± 0.05	0.84 ± 0.04	0.71 ± 0.05*
CD4^+CD8^+	16.81 ± 0.21	16.04 ± 0.16	16.25 ± 0.13	14.24 ± 0.13*	7.06 ± 0.15*
CD4^−CD8^−	0.62 ± 0.05	0.61 ± 0.05	0.56 ± 0.04	0.49 ± 0.04	0.39 ± 0.03*

*Value significantly different compared to VH group at p < 0.05.

^aValues represent percentage of different thymocyte population measured by a flow cytometer and analyzed using Flowjo 8.0 software.

^bPercent values and total number of thymus were used to calculate the total number of each cell population in the thymus.

Figure 2. ANE directly suppressed IL-2 production by thymocytes. Thymocytes (5 × 10⁶ cells/ml) were left untreated (NA) or treated with ANE (1–40 µg/ml) for 30 min and then stimulated with PMA + ionomycin for 24  h. (a) Metabolic activity of the thymic cells was measured by MTT assay. (b) Production of IL-2 as measured by ELISA. Data are expressed as mean (±SE) of quadruplicate cultures. Results are representative of three independent experiments. *Value significantly different compared to VH group at p < 0.05.

Figure 3. ANE-induced thymocyte apoptosis. Thymocytes were left untreated (NA) or treated with ANE (1–40 µg/ml) for 12 h, and then fixed with ethanol. The single cell fluorescence of 5000 cells per sample was then measured by flow cytometry. (a) Data presented as a proportion of apoptotic cells, defined as sub-G₀/G₁-phase cells with hypodiploid DNA content. (b) Portion of TUNEL⁺ cells. (c) Representative histograms of TUNEL analyses on NA and ANE (20–40 μg/ml)-treated cells. Data are expressed as mean (±SE) of quadruplicate cultures. Results are representative of three independent experiments. *Value significantly different compared to VH group at p < 0.05.

Figure 4. Induction of apoptosis-inducing factor (AIF) and active caspase-3 by ANE. Thymocytes were left untreated (NA) or treated with ANE (10–40 μg/ml) for 3 h. The cells were then lysed and amounts of apoptosis-inducing factor (AIF) and the active form of caspase-3 in the cytosolic extracts were measured using Western blotting. The level of actin was used as a loading control. The data shown are representative of three independent experiments.