Anti-Apoptotic Effect of Apelin in Human Placenta: Studies on BeWo Cells and Villous Explants from Third-Trimester Human Pregnancy

Figures

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  Figure 1. Effect of apelin on protein expression of apoptotic factors in BeWo cells. The cells were incubated with apelin at doses 2 of (AP2) and 20 (AP20) ng/mL for 24, 48 and 72 h, and subsequently, Western blot analysis was performed to examine the expression of BCL2 (B-cell like lymphoma 2), BAX (Bcl-2-like protein 4), caspase 3, 8 and 9 and p53. Results are shown as stripes on gel image (A) and densitometry analysis relative to β-actin (B–F). Experiments were independently performed and repeated three times (n = 3). The data are arranged as means ± SEM. Different letters indicate significant differences (p < 0.05) among groups; Control (C).

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  Figure 2. Effect of apelin on caspase 3 and 7 activity and histone-associated DNA fragment level in BeWo cells. The cells were incubated with 2 and 20 ng/mL of apelin alone or in combination with 0.1 µL/mL staurosporine for 24, 48 and 72 h, after which caspase 3 and 7 activity (A,B) was analysed using the Caspase-Glo 3/7 assay or the level of histone-associated DNA fragments (C,D) by the Cell Death Detection ELISA kit. Experiments were independently performed and repeated three times (n = 3). The data are arranged as means ± SEM. Different letters indicate significant differences (p < 0.05) among groups; Control (C), Staurosporine (S), Relative Luminescence Unit (RLU), Relative Absorbance Unit (RAU).

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  Figure 3. Effect of apelin on oxidative stress in BeWo cells. The cells were treated with apelin at 2 and 20 ng/mL for 24 h, and the oxygen consumption assay was performed. Experiments were independently performed and repeated three times (n = 3). The data are arranged as means ± SEM. Different letters indicate significant differences (p < 0.05) among groups; Control (C).

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  Figure 4. Involvement of APJ receptor, mitogen-activated protein kinase (ERK1/2/MAP3/1) and protein kinase B (AKT) in the antiapoptotic effect of apelin in BeWo cells: The cells were pre-treated for 1 h with APJ receptor, MAP3/1 and AKT kinase inhibitors ML221 (5 µM), PD098059 (1 µM) and LY290042 (1 µM). Subsequently, apelin at a dose of 2 (AP2) ng/mL was added. After 72 h of incubation, activity of caspase 3 and 7 was analysed using the Caspase-Glo 3/7 assay. Experiments were independently performed and repeated three times (n = 3). The results are presented as a percentage compared to the control (100%). The data are arranged as means ± SEM. Different letters indicate significant differences (p < 0.05) among groups; Control (C).

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  Figure 5. Effect of apelin on DNA fragmentation in villous explants from human placenta. The explants were incubated with apelin at 2 and 20 ng/mL for 24, 48 and 72 h, and the level of histone-associated DNA fragment was measured using the Cell Death Detection ELISA kit. Experiments were performed on five independent cultures of human placenta explants (n = 5). The level of DNA fragmentation was calculated to 100 µg protein of villous explants and then as percentage compared to the 24-h control sample (100%). The data are arranged as means ± SEM. Different letters indicate significant differences (p < 0.05) among groups; Control (C).

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  Figure 6. Effect of apelin on protein expression of apoptotic proteins in villous explants from human placenta. The explants were incubated with 2 (AP2) and 20 (AP20) ng/mL of apelin for 24, 48 and 72 h, and Western blot analysis was performed to determine the expression of BCL2, BAX and caspase 3. Results are shown as stripes on gel image (A) and densitometry analysis relative to β-actin (B). Experiments were performed on five independent cultures of human placenta explants (n = 5). The data are arranged as means ± SEM. Different letters indicate significant differences (p < 0.05) among groups; Control (C).

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  Figure 7. Model of apelin antiapoptotic action in BeWo cell line (A) and human placenta explants (B). Apelin inhibits apoptosis by activation of apelin receptor (APJ), mitogen-activated kinase (MAP3/1) and protein kinase B (AKT); BCL2 (B-cell lymphoma 2), BAX (Bcl-2-like protein 4); BAK1 (Bcl-2 homologous antagonist/killer); BOK (Bcl-2-related ovarian killer protein); NOD1 (Nucleotide-binding oligomerisation domain-containing protein 1); CRADD (Caspase and RIP adapter with death domain); DIABLO (Diablo IAP-Binding Mitochondrial Protein); TNFRSF25 (Tumour necrosis factor receptor superfamily member 25); BIRC6 (Baculoviral IAP repeat-containing protein 6); MCL1 (Induced myeloid leukaemia cell differentiation protein MCL1); XIAP (X-linked inhibitor of apoptosis).