Sesamin Induces MCL-1-Dependent Apoptosis in Activated T Cells and Ameliorates Experimental Atopic Dermatitis

Abstract

Sesamin, a natural lignan derived from Sesamum indicum, has been reported to possess anti-inflammatory and pro-apoptotic properties. However, its effect on T cell-mediated diseases and the underlying molecular mechanisms remain unclear. In this study, we demonstrate that sesamin selectively induces apoptosis in activated T cells through direct interaction with MCL-1, a critical anti-apoptotic protein of the Bcl-2 family. Sesamin suppressed IL-2 expression, CD69 upregulation, and proliferation in activated human and murine T cells. Molecular docking predicted strong binding of sesamin to the BH3-binding groove of MCL-1, which was validated by pull-down and co-immunoprecipitation assays. Sesamin inhibited MCL-1 phosphorylation at Ser64 and disrupted its heterodimerization with Bak, promoting caspase-3/8 cleavage and apoptotic death selectively in activated, but not resting, T cells. In a murine model of atopic dermatitis, oral administration of sesamin ameliorated pathological skin symptoms, reduced Th2/Th17 cytokine expression, serum IgE, mast cell infiltration, and lymph node hypertrophy. These effects correlated with suppressed MCL-1 activity and enhanced apoptosis in inflamed tissue. Our findings suggest that sesamin modulates immune responses via a novel MCL-1-dependent mechanism and represents a promising dietary-derived therapeutic strategy for T cell-driven chronic inflammatory diseases.

Keywords: MCL-1; Sesamin; T cell regulation; activated T cells; apoptosis; atopic dermatitis; immunomodulation.

Figure 1

Sesamin suppresses T cell activation marker and cytokine production. (A) The structure of sesamin. (B) Jurkat T cells (5 X 10⁵/well, 12-well plate) or mouse CD4⁺ T cells (3 X 10⁶/well, 12-well plate) pre-treated with indicated concentration of sesamin for 1 h were stimulated with immobilized anti-CD3 (10 μg/ml) and soluble anti-CD28 (2 μg/ml) antibodies for 6 h. Harvested cells were lysed for isolation of total RNA. The mRNA level of IL-2 gene was determined by qPCR analysis. The expression of IL-2 was normalized with the expression of GAPDH. (C) Jurkat T cells (1 X 10⁴/well, 96-well plate) or mouse CD4⁺ T cells (5 X 10⁴/well, 96-well plate) pre-treated with indicated concentration of sesamin for 1 h were stimulated with immobilized anti-CD3 (10 μg/ml) and soluble anti-CD28 (2 μg/ml) antibodies for 24 h. The amount of released IL-2 was evaluated by ELISA from harvested supernatants. (D) Jurkat T cells (5 X 10⁵/well, 12-well plate) pre-treated with 40 μM sesamin for 1 h were stimulated with immobilized anti-CD3 (10 μg/ml) and soluble anti-CD28 (2 μg/ml) antibodies for 16 h. Collected cells were stained with anti-CD69 antibodies conjugated with FITC and mean fluorescence intensity was measured by flow cytometry. The mean fluorescence intensity of control group was considered as 1X and fold increase was presented compared to control group. (E) 0.5 μM CFSE-stained mouse CD4⁺ T cells (1 X 10⁶/well, 96-well plate) pre-treated with 40 μM sesamin for 1 h were stimulated with immobilized anti-CD3 (10 μg/ml) and soluble anti-CD28 (2 μg/ml) antibodies for 72 h. Harvested cells were acquired by flow cytometry to obtain CFSE fluorescence. Mean fluorescence intensity and population of each division were presented. The concentration used in (D) and (E) was selected based on prior dose-response analysis (see Methods). All results are expressed as mean ± SEM of three independent experiments. Statistical comparisons among groups were performed using one-way ANOVA with Tukey's post hoc test. A p-value less than 0.05 was considered statistically significant (*, P < 0.05).

Figure 2

Sesamin interacts physically with MCL-1, a predicted target molecule in T cells. (A) The flow chart how target proteins of sesamin were discovered from Genecards databank and swisstargetprediction analysis. (B) The structure of MCL-1 active domains (above) and molecular docking between BH3 domain of MCL-1 and sesamin by AMdock and pyMOL software. BH3-only protein Bim is marked as orange color following as sequence. A binding residue of sesamin at Arg 222 is marked as blue color. (C) Resting Jurkat T cells (1 X 10⁶) were lysed with RIPA buffer for pull-down assay. Lysates were incubated with control bead (Con bead) or/and bead conjugated with sesamin (SS-bead) and precipitated by centrifugation. Con beads and SS-beads were mixed in 100:0, 50:50, 0:100 ratios, respectively, to assess the dose-dependent enrichment of MCL-1 and Bak. Mcl-1 and Bak were detected from precipitated lysate by bead centrifugation (pull-down) and MCL-1 and β-actin were detected from whole lysate. The expression of detected MCl-1 from precipitated lysate by bead centrifugation (pull-down) was normalized with the expression of β-actin from whole lysate. Bar graph was presented as fold increase. All results are expressed as mean ± SEM of three independent experiments. Statistical comparisons among groups were performed using one-way ANOVA with Tukey's post hoc test. A p-value less than 0.05 was considered statistically significant (*, P < 0.05).

Figure 3

Sesamin inhibits MCL-1 activity in activated T cells, controlling T cell activation. (A) Jurkat T cells (1 X 10⁶/well, 12-well plate) pre-treated with 40 μM sesamin or 5 μM Umi-77 for 1 h were stimulated with immobilized anti-CD3 (10 μg/ml) and soluble anti-CD28 (2 μg/ml) antibodies for 1 to 6 h. Harvested cells were lysed in RIPA buffer and expressions of phosphorylated MCL-1 at Ser64 and MCL-1 were detected by Western blotting analysis. Expressions were normalized with the expression of β-actin. (B) Mouse CD4⁺ T cells (3 X 10⁶/well, 12-well plate) pretreated with 20 or 40 μM sesamin for 1 h were stimulated with anti-CD3 (10 μg/ml) and soluble anti-CD28 (2 μg/ml) antibodies for 6 h. Harvested cells were lysed in RIPA buffer and expressions of phosphorylated MCL-1 at Ser64 and MCL-1 were detected by Western blotting analysis. Expressions were normalized with the expression of β-actin. (C-E) Jurkat T cells (1 X 10⁶/well, 12-well plate) pre-treated with 10 or 40 μM sesamin for 1 h and then 5 μM Umi-77 for additional 1 h were stimulated with immobilized anti-CD3 (10 μg/ml) and soluble anti-CD28 (2 μg/ml) antibodies for 6 h (C), 24 h (D) and 72 h (E). Harvested cells were lysed in RIPA buffer and the expression of phosphorylated MCL-1 at Ser64 and MCL-1 were detected by Western blotting analysis. Expressions were normalized with the expression of β-actin (C). The amount of released IL-2 was evaluated by ELISA from harvested supernatants (D). WST-8 assay was performed to obtain proliferative ratio compared to control. OD value of control group was considered as 1X and fold increase was presented compared to control group (E). All results are expressed as mean ± SEM of three independent experiments. Statistical comparisons among groups were performed using one-way ANOVA with Tukey's post hoc test. A p-value less than 0.05 was considered statistically significant (*, P < 0.05).

Figure 4

Sesamin disrupts the interaction of MCL-1 with Bak in activated T cells. (A) Jurkat T cells (5 X 10⁵/well, 12-well plate) pre-treated with 40 μM sesamin for 1 h were stimulated with immobilized anti-CD3 (10 μg/ml) and soluble anti-CD28 (2 μg/ml) antibodies for 6 h. Harvested cells were lysed in RIPA buffer and the expression of MCL-1, Bak, Bax and Bcl-2 was detected by Western blotting analysis. Expressions were normalized with the expression of β-actin. (B) A prediction result showing binding partners of MCL-1 by GeneMANIA database. (C) Co-IP assay experiments were performed with lysates demonstrated in (A). IP experiments with anti-Bak antibodies were performed and the expression of precipitated MCL-1 and Bak was detected by Western blotting. Precipitated MCL-1 was normalized with the expression of precipitated Bak and the expression of MCL-1 in whole lysate. All results are expressed as mean ± SEM of three independent experiments. Statistical comparisons among groups were performed using one-way ANOVA with Tukey's post hoc test. A p-value less than 0.05 was considered statistically significant (*, P < 0.05).

Figure 5

Sesamin selectively induces caspase-dependent apoptosis in activated T cells. (A) Jurkat T cells (1 X 10⁴/well, 96-well plate) pre-treated with 40 μM sesamin for 1 h were stimulated with immobilized anti-CD3 (10 μg/ml) and soluble anti-CD28 (2 μg/ml) antibodies for 72 h. WST-8 assay was performed to obtain cell viability. (B) Jurkat T cells (1 X 10⁶/well, 12-well plate) pre-treated with 20 or 40 μM sesamin for 1 h were stimulated with immobilized anti-CD3 (10 μg/ml) and soluble anti-CD28 (2 μg/ml) antibodies for 48 h. Harvested cells were lysed in RIPA buffer and the expression of cleaved caspase3 and caspase8 were detected by Western blotting analysis. Expressions were normalized with the expression of β-actin. (C) Jurkat T cells (5 X 10⁵/well, 12-well plate) pre-treated with 20 or 40 μM sesamin for 1 h were stimulated with immobilized anti-CD3 (10 μg/ml) and soluble anti-CD28 (2 μg/ml) antibodies for 48 h. Harvested cells were stained with AnnexinV and 7AAD for AnnexinV apoptosis assay. From Contour Plot, the population of AnnexinV⁺7AAD^- and AnnexinV⁺7AAD⁺ was analyzed. (D) The DIC images of Jurkat T cells from Trypan blue exclusion method. Black dots indicate Trypan blue-stained cells in the images. White bar represents 100 μm. All results are expressed as mean ± SEM of three independent experiments. Statistical comparisons among groups were performed using one-way ANOVA with Tukey's post hoc test. A p-value less than 0.05 was considered statistically significant (*, P < 0.05).

Figure 6

Orally administered sesamin ameliorates AD-model symptoms in vivo. (A) Representative images of mouse ears at day 28 post-induction. (B) Changes of ear thickness of each mouse group during induction (left) and ratio of ear thickness at day 28 (right). (C) Body weight of each mouse at day 0, 14 and 28 post-induction. (D) Scratching number of each mouse per 1 min. Control: Untreated mice reveiving only vehicle (0.5% CMC) without AD induction or sesamin administration. SS: Mice administrated sesamin (25 mg/kg) without AD induction to assess the baseline immunological effect of sesamin. AD: Mice sensitized and challenged with DNCB/HDM to induce AD, receiving vehicle only. AD+SS (10 mg/kg): Mice treated with DNCB/HDM and administered sesamin at 10 mg/kg to evaluate low-dose therapeutic effects. AD+SS (25 mg/kg): Mice treated with DNCB/HDM and administered sesamin at 25 mg/kg to evaluate high-dose therapeutic effects. AD+Tofa (20 mg/kg): Mice treated with DNCB/HDM and administered tofacitinib at 20 mg/kg as a positive control for anti-inflammatory efficacy. All results are expressed as mean ± SEM of three independent experiments. Statistical comparisons among groups were performed using one-way ANOVA with Tukey's post hoc test. A p-value less than 0.05 was considered statistically significant (*, P < 0.05).

Figure 7

Orally administered sesamin attenuates pathological manifestations in the AD model. (A) Representative H&E histopathological images of mice ears. Black bar represents 200 μm. (B) Thickness of epidermis and dermis of each mouse from (A). (C) Clinical scores according to inflammation and dermal papilla of each mouse were evaluated from (A). Measuring standard is described in Materials and methods. (D) Representative Toluidine blue histopathological images of mice ears. Black bar represents 400 μm. (E) The number of infiltrated mast cells to the lesion was counted. (F) Levels of total IgE in serum were assessed by ELISA. All results are expressed as mean ± SEM of three independent experiments. Statistical comparisons among groups were performed using one-way ANOVA with Tukey's post hoc test. A p-value less than 0.05 was considered statistically significant (*, P < 0.05).

Figure 8

Orally administered sesamin reduces ear-tissue atopic gene expression in the AD model. (A-C) mRNA levels of Th2 type cytokines (A), Th1 type cytokines (B) and Th17 type cytokines (C) from ear tissues of each mouse. The expression of target genes was normalized with the expression of Gapdh. All results are expressed as mean ± SEM of three independent experiments. Statistical comparisons among groups were performed using one-way ANOVA with Tukey's post hoc test. A p-value less than 0.05 was considered statistically significant (*, P < 0.05).

Figure 9

Orally administered sesamin ameliorates the systemic immune response associated with T cell activation in the AD model. (A) Anatomical localization of cervical lymph nodes and inguinal lymph nodes on mouse. (B) Representative images of cervical lymph nodes of each mouse group (left), their weights (middle) and lengths (right). (C) Representative images of inguinal lymph nodes of each mouse group (left), their weights (middle) and lengths (right). (D) mRNA levels of Th2 type cytokines (IL-4, IL-5 and IL-13) and master transcription factor of Th2 effector cells (GATA3) from cervical lymph nodes of each mouse. The expression of target genes was normalized with the expression of Gapdh. All results are expressed as mean ± SEM of three independent experiments. Statistical comparisons among groups were performed using one-way ANOVA with Tukey's post hoc test. A p-value less than 0.05 was considered statistically significant (*, P < 0.05).

Figure 10

MCL-1 activity is suppressed in local and distal organ following sesamin treatment. (A, B) ear tissue (A) and cervical lymph nodes (B) were lysed in RIPA buffer and the expression of phosphorylated MCL-1 at Ser64, cleaved caspase3 and cleaved caspase8 were detected by Western blotting analysis. Expressions were normalized with the expression of β-actin. All results are expressed as mean ± SEM of three independent experiments. Statistical comparisons among groups were performed using one-way ANOVA with Tukey's post hoc test. A p-value less than 0.05 was considered statistically significant (*, P < 0.05).