Vitamin C Induces the Reduction of Oxidative Stress and Paradoxically Stimulates the Apoptotic Gene Expression in Extravillous Trophoblasts Derived From First-Trimester Tissue

Abstract

Aim: To investigate the effects of vitamin C on the expression of the genes related to apoptosis in extravillous trophoblasts (EVTs) in the first trimester.

Methods: Extravillous trophoblasts were cultured under 2% O2 followed by 2% O2 or 8% O2 with or without vitamin C. The level of reactive oxygen species (ROS) in the cultured medium was estimated using electron spin resonance spectroscopy. The expression levels of the genes TP53, BCL2, and BAX were quantified using real-time quantitative polymerase chain reaction.

Results: Reactive oxygen species were found to be decreased after adding vitamin C under increasing oxygen concentrations. In addition, the ratio of BAX/BCL2 also increased after adding vitamin C under conditions of 2% O2, while the gene expression level of BCL2 increased after adding vitamin C under increasing oxygen concentrations. In contrast, the gene expression level of TP53 and the ratio of BAX/BCL2 both decreased.

Conclusion: We have revealed that vitamin C reduces ROS and may promote the apoptosis of EVTs under conditions of 2% O2 while paradoxically preventing apoptosis under increasing oxygen concentrations.

Keywords: ESR; RT-PCR; apoptosis; extravillous trophoblasts; vitamin C.

Figure 1.

Experimental design of the EVTs culture. EVTs were cultured under conditions of 2% O₂ for 24 hours to promote attachment to the Matrigel. The cells were then cultured for an additional 24 hours under conditions of 2% O₂ with or without 200 μmol/L of vitamin C or 8% O₂ with or without 200 μmol/L of vitamin C. The expression levels of genes related to mitochondria-dependent apoptosis and ROS were subsequently analyzed. EVT indicates extravillous trophoblast.

Figure 2.

Medium free radical generation under conditions of increasing oxygen concentrations with vitamin C. The medium was mixed with DMPO and subjected to an ESR spin-trapping analysis. The ESR spectrum of the medium without vitamin C is shown, which revealed the presence of RO^· (A). The signal intensity of RO^· under conditions of an increasing oxygen concentration in the presence of 200 μmol/L of vitamin C was decreased (n = 4; B). # indicates RO^· spin adduct. *, P < .05, between the samples cultured with and without vitamin C; DMPO, 5,5-dimetyl-1-pyrroline-N-oxide; ESR, electron spin resonance; RO·, alkoxyl radical.

Figure 3.

Expression of apoptosis-related genes induced by the generation of oxidative stress under conditions of hypoxia with or without vitamin C. Using real-time quantitative PCR, a significantly increased ratio of BAX/BCL2 mRNA was observed (D). ACTB mRNA was used to normalize the gene expression. The results are expressed as the mean ± SEM. * indicates P < .05, between the samples cultured with and without vitamin C; PCR, polymerase chain reaction; mRNA, messenger RNA; SEM, standard error of the mean.

Figure 4.

Expression of apoptosis-related genes induced by the generation of oxidative stress under conditions of increasing oxygen concentrations with or without vitamin C. Using RT-qPCR, a significantly decreased expression of TP53 mRNA was observed (A) in the samples treated with vitamin C, compared to an increased expression of BCL2 mRNA (C). Vitamin C decreased the ratio of BAX/BCL2 mRNA (D). ACTB mRNA was used to normalize the gene expression. The results are expressed as the mean ± SEM. * indicates P < .05, between the samples cultured with and without vitamin C; RT-qPCR, real-time quantitative polymerase chain reaction; mRNA, messenger RNA; SEM, standard error of the mean.