Curcumin Protects Human Trophoblast HTR8/SVneo Cells from H2O2-Induced Oxidative Stress by Activating Nrf2 Signaling Pathway

Pregnancy complications are associated with oxidative stress induced by accumulation of trophoblastic ROS in the placenta. We employed the human trophoblast HTR8/SVneo cell line to determine the effect of curcumin pre-treatment on H2O2-induced oxidative damage in HTR8/Sveo cells. Cells were pretreated with 2.5 or 5 μM curcumin for 24 h, and then incubated with 400 μM H2O2 for another 24 h. The results showed that H2O2 decreased the cell viability and induced excessive accumulation of reactive oxygen species (ROS) in HTR8/Sveo cells. Curcumin pre-treatment effectively protected HTR8/SVneo cells against oxidative stress-induced apoptosis via increasing Bcl-2/Bax ratio and decreasing the protein expression level of cleaved-caspase 3. Moreover, curcumin pre-treatment alleviated the excessive oxidative stress by enhancing the activity of antioxidative enzymes. The antioxidant effect of curcumin was achieved by activating Nrf2 and its downstream antioxidant proteins. In addition, knockdown of Nrf2 by Nrf2-siRNA transfection abolished the protective effects of curcumin on HTR8/SVneo cells against oxidative damage. Taken together, our results show that curcumin could protect HTR8/SVneo cells from H2O2-induced oxidative stress by activating Nrf2 signaling pathway.


Introduction
Intrauterine growth retardation (IUGR) and preeclampsia (PE) are detrimental pregnancy complications that could cause significant increased perinatal morbidity and mortality [1]. Normal placental development during early pregnancy depends entirely on the differentiation, proliferation, and invasion of trophoblast cells [2]. IUGR refers to impaired growth and development of the fetus or fetal organs, and these consequences are associated with the dysfunction of placental trophoblast cells [3].
Oxidative stress arose from the production of reactive oxygen species (ROS) reduces the antioxidant capacity of cells, which, in turn, results in cell damage and eventually cell death [4,5]. During normal pregnancy, the metabolisms of the mother and fetus are enhanced because of higher energy and oxygen requirements [6]. This could consequently accelerate the accumulation of ROS, and eventually induce excessive oxidative stress in the trophoblast cells. Nevertheless, cells have developed an antioxidant defense system to protect against oxidative stress. The system consists of antioxidant enzymes such as glutathione (GSH), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT), which could scavenge ROS to prevent possible cellular damage [7]. Previous studies have demonstrated that antioxidants could attenuate the oxidative damage via enhancing the activities of these antioxidant enzymes [8,9]. Thus, antioxidants might protect placental trophoblast cells from R: GCGCCCAATACGACCAAATC Nrf2, nuclear factor-erythroid 2-related factor 2; HO-1, home oxygenase-1; GCLC, glutamate-cysteine ligase catalytic; GCLM, glutamate-cysteine ligase modifier; NQO1, NAD(P)H quinone dehydrogenase 1; Bcl-2, Bcl-2 apoptosis regulator; Bax, Bcl-2 associated X, apoptosis regulator; SLC2A1, solute carrier family 2 member 1; SLC2A3, solute carrier family 2 member 3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Small Interfering RNA (siRNA) Transfection
The human-specific siRNAs targeting Nrf2 were designed and synthesized by GenePharma (Shanghai, China). For transfection, the HTR8/SVneo cells were seeded in 6-well culture plates and siRNA-Nrf2 were transfected into the cells using the Lipofectamine™ 2000 Transfection reagent (Thermo Scientific, Wilmington, DE, USA) prior to treatment with curcumin and H 2 O 2 according to the manufacturer's instructions. The target sequences used in this study were shown in Table 2.

Statistical Analysis
All the data are presented as mean ± SEM for at least three independent experiments. The Shapiro-Wilk test was used to estimate the normality distribution of the data. Statistical differences were Antioxidants 2020, 9, 121 5 of 14 determined by one-way ANOVA followed by Tukey's test (Graph Pad Software Inc. San Diego, CA, USA). Differences were considered to be significant at p < 0.05.

Curcumin Protected against H 2 O 2 -Induced Cytotoxicity in HTR8/SVneo Cells
Firstly, to achieve the optimized oxidative stress conditions, we examined HTR8/SVneo cells treated with different concentrations of H 2 O 2 (100, 200, 400, 600, 800 and 1000 µM) for 24 h. Cell viabilities were analyzed using a CCK-8 assay. As shown in Figure 1A, a dose-dependent increase in cytotoxicity of HTR8/SVneo cells was observed in response to H 2 O 2 . The IC50 value of H 2 O 2 concentration was 400 µM which resulted in 50% inhibition of HTR8/SVneo cells. Thus, 400 µM H 2 O 2 treatment for 24 h was chosen to perform subsequent experiments. Secondly, to evaluate cell viability in different concentrations of curcumin and to determine its non-cytotoxic concentration, HTR8/SVneo cells were pretreated with different concentrations of curcumin (0, 2.5, 5, 10, 20 and 40 µM) for 24 h. As shown in Figure 1B, 10 µM curcumin has cytotoxicity to HTR8/SVneo cells, thus we chose 2.5 and 5 µM curcumin for the following experiments. Finally, to assess the cytoprotective effect of curcumin, HTR8/SVneo cells were pretreated with 2.5 and 5 µM curcumin for 24 h, followed by 400 µM H 2 O 2 for another 24 h. As shown in Figure 1C, the 5 µM curcumin pretreatment group significantly increased the cell viability was significantly increased in the 5 µM curcumin pre-treatment group compared with the H 2 O 2 treatment group.

Statistical Analysis
All the data are presented as mean ± SEM for at least three independent experiments. The Shapiro-Wilk test was used to estimate the normality distribution of the data. Statistical differences were determined by one-way ANOVA followed by Tukey's test (Graph Pad Software Inc. San Diego, CA, USA). Differences were considered to be significant at p < 0.05.

Curcumin Protected against H2O2-Induced Cytotoxicity in HTR8/SVneo Cells
Firstly, to achieve the optimized oxidative stress conditions, we examined HTR8/SVneo cells treated with different concentrations of H2O2 (100, 200, 400, 600, 800 and 1000 μM) for 24 h. Cell viabilities were analyzed using a CCK-8 assay. As shown in Figure 1A, a dose-dependent increase in cytotoxicity of HTR8/SVneo cells was observed in response to H2O2. The IC50 value of H2O2 concentration was 400 μM which resulted in 50% inhibition of HTR8/SVneo cells. Thus, 400 μM H2O2 treatment for 24 h was chosen to perform subsequent experiments. Secondly, to evaluate cell viability in different concentrations of curcumin and to determine its non-cytotoxic concentration, HTR8/SVneo cells were pretreated with different concentrations of curcumin (0, 2.5, 5, 10, 20 and 40 μM) for 24 h. As shown in Figure 1B, 10 μM curcumin has cytotoxicity to HTR8/SVneo cells, thus we chose 2.5 and 5 μM curcumin for the following experiments. Finally, to assess the cytoprotective effect of curcumin, HTR8/SVneo cells were pretreated with 2.5 and 5 μM curcumin for 24 h, followed by 400 μM H2O2 for another 24 h. As shown in Figure 1C, the 5 μM curcumin pretreatment group significantly increased the cell viability was significantly increased in the 5 μM curcumin pretreatment group compared with the H2O2 treatment group.

Curcumin Increased H2O2-Induced CAT, GSH-Px Activity and Reduced the Level of Intracellular ROS in HTR8/SVneo Cells under Oxidative Stress
To evaluate the effects of curcumin on the activity of antioxidant enzymes in HTR8/SVneo cells, the activities of CAT and GSH-Px were assessed. As shown in Figure 2 A,B, the activities of CAT and GSH-Px showed no significant difference (p > 0.05) between the control and H2O2 treatment group. Whereas, pre-treatment with 5 μM curcumin followed by 400 μM H2O2 up-regulated (p < 0.05) the activities of CAT and GSH-Px in HTR8/SVneo cells. To further prove the protective of curcumin against oxidative stress, we measured the intracellular level of ROS by using fluorescent probe

Curcumin Increased H 2 O 2 -Induced CAT, GSH-Px Activity and Reduced the Level of Intracellular ROS in HTR8/SVneo Cells under Oxidative Stress
To evaluate the effects of curcumin on the activity of antioxidant enzymes in HTR8/SVneo cells, the activities of CAT and GSH-Px were assessed. As shown in Figure 2A,B, the activities of CAT and GSH-Px showed no significant difference (p > 0.05) between the control and H 2 O 2 treatment group. Whereas, pre-treatment with 5 µM curcumin followed by 400 µM H 2 O 2 up-regulated (p < 0.05) the activities of CAT and GSH-Px in HTR8/SVneo cells. To further prove the protective of curcumin against oxidative stress, we measured the intracellular level of ROS by using fluorescent probe DCFH-DA. As shown in Figure 2C, the accumulation of ROS in HTR8/SVneo cells was significantly increased (p < 0.05) in the H 2 O 2 group compared with the control group, while pre-treatment with 2.5 and 5 µM curcumin remarkably reduced (p < 0.05) the H 2 O 2 -induced ROS accumulation in HTR8/SVneo cells.

Curcumin Inhibited H2O2-Induced Apoptosis of HTR8/SVneo Cells
To further evaluate the inhibitory effect of curcumin on H2O2-induced apoptosis in HTR8/SVneo cells, apoptotic rates were measured by Annexin V-FITC/PI double staining using flow cytometry. As shown in Figure 3, the percentage of apoptotic cells was 4.56% ± 1.04% in the control group, whereas that of the H2O2 treatment group was 14.13% ± 1.56% (p < 0.05). However, pre-treatment with 5 μM curcumin markedly decreased apoptotic rates to 6.00% ± 1.00% (p < 0.05) in HTR8/SVneo cells treated with 400 μM H2O2.

Curcumin Inhibited H 2 O 2 -Induced Apoptosis of HTR8/SVneo Cells
To further evaluate the inhibitory effect of curcumin on H 2 O 2 -induced apoptosis in HTR8/SVneo cells, apoptotic rates were measured by Annexin V-FITC/PI double staining using flow cytometry. As shown in Figure 3, the percentage of apoptotic cells was 4.56% ± 1.04% in the control group, whereas that of the H 2 O 2 treatment group was 14.13% ± 1.56% (p < 0.05). However, pre-treatment with 5 µM curcumin markedly decreased apoptotic rates to 6.00% ± 1.00% (p < 0.05) in HTR8/SVneo cells treated with 400 µM H 2 O 2 .

Curcumin Inhibited H2O2-Induced Apoptosis of HTR8/SVneo Cells
To further evaluate the inhibitory effect of curcumin on H2O2-induced apoptosis in HTR8/SVneo cells, apoptotic rates were measured by Annexin V-FITC/PI double staining using flow cytometry. As shown in Figure 3, the percentage of apoptotic cells was 4.56% ± 1.04% in the control group, whereas that of the H2O2 treatment group was 14.13% ± 1.56% (p < 0.05). However, pre-treatment with 5 μM curcumin markedly decreased apoptotic rates to 6.00% ± 1.00% (p < 0.05) in HTR8/SVneo cells treated with 400 μM H2O2.

Curcumin Regulated mRNA Expression of Multiple Antioxidant Genes and Nutrient Transporter Genes in HTR8/SVneo Cells under Oxidative Stress
Since curcumin has been shown to exert antioxidant effect by inducing several antioxidant defense enzymes in several cell lines [14,23]. Thus, we performed RT-qPCR to detect whether the protective function of curcumin against the oxidative stress induced by H 2 O 2 was associated with antioxidant-associated factors in HTR8/SVneo cells. Our results showed that H 2 O 2 stimulation alone significantly increased the mRNA levels of Nrf2, HO-1, GCLC, GCLM and NQO1 (p < 0.05), whereas pre-treatment of 5 µM curcumin further up-regulated (p < 0.05) the expression of Nrf2, GCLM and NQO1. H 2 O 2 treatment alone increased (p < 0.05) the transcription level of Bax, and pre-treatment with 5 µM curcumin increased (p < 0.05) the expression of Bcl-2. The expression of Bcl-2/Bax was also up-regulated (p < 0.05) in both curcumin pre-treatment groups ( Figure 4F-H). In addition, H 2 O 2 treatment alone had no effect (p > 0.05) on the mRNA expression of solute carrier family 2 member 1 (SLC2A1) and solute carrier family 2 member 3 (SLC2A3), while pre-treatment with 5 µM curcumin increased (p < 0.05) the gene expression of SLC2A3 (Figure 4I,J).

Curcumin Regulated mRNA Expression of Multiple Antioxidant Genes and Nutrient Transporter Genes in HTR8/SVneo Cells under Oxidative Stress
Since curcumin has been shown to exert antioxidant effect by inducing several antioxidant defense enzymes in several cell lines [14,23]. Thus, we performed RT-qPCR to detect whether the protective function of curcumin against the oxidative stress induced by H2O2 was associated with antioxidant-associated factors in HTR8/SVneo cells. Our results showed that H2O2 stimulation alone significantly increased the mRNA levels of Nrf2, HO-1, GCLC, GCLM and NQO1 (p < 0.05), whereas pre-treatment of 5 μM curcumin further up-regulated (p < 0.05) the expression of Nrf2, GCLM and NQO1. H2O2 treatment alone increased (p < 0.05) the transcription level of Bax, and pre-treatment with 5 μM curcumin increased (p < 0.05) the expression of Bcl-2. The expression of Bcl-2/Bax was also up-regulated (p < 0.05) in both curcumin pre-treatment groups ( Figure 4F,G,H). In addition, H2O2 treatment alone had no effect (p > 0.05) on the mRNA expression of solute carrier family 2 member 1 (SLC2A1) and solute carrier family 2 member 3 (SLC2A3), while pre-treatment with 5 μM curcumin increased (p < 0.05) the gene expression of SLC2A3 (Figure 4I,J).

Curcumin Increased Nrf2, HO-1 and NQO1 Protein Expression and Nrf2 Translocation in HTR8/SVneo Cells under Oxidative Stress
As shown in Figure 5, H2O2 stimulation alone up-regulated (p < 0.05) the protein expression of Nrf2 and NQO1, while it did not affect (p > 0.05) the protein expression of HO-1. Pre-treatment with 2.5 or 5 μM curcumin up-regulated (p < 0.05) the protein expression of nuclear Nrf2, total-Nrf2, HO-1 and NQO1. In addition, immunofluorescence staining showed that both H2O2 and curcumin stimulated Nrf2 nuclear translocation in HTR8/SVneo cells ( Figure 6). However, we observed that pre-treatment with curcumin induced more Nrf2 nuclear translocation compared with the H2O2 treatment group. Furthermore, pre-treatment with curcumin significantly increased the Bcl-2/Bax ratio and decreased the expression of active caspase-3 in H2O2-treated HTR8/SVneo cells (Figure 5 A,E,F).

Curcumin Increased Nrf2, HO-1 and NQO1 Protein Expression and Nrf2 Translocation in HTR8/SVneo Cells under Oxidative Stress
As shown in Figure 5, H 2 O 2 stimulation alone up-regulated (p < 0.05) the protein expression of Nrf2 and NQO1, while it did not affect (p > 0.05) the protein expression of HO-1. Pre-treatment with 2.5 or 5 µM curcumin up-regulated (p < 0.05) the protein expression of nuclear Nrf2, total-Nrf2, HO-1 and NQO1. In addition, immunofluorescence staining showed that both H 2 O 2 and curcumin stimulated Nrf2 nuclear translocation in HTR8/SVneo cells ( Figure 6). However, we observed that pre-treatment with curcumin induced more Nrf2 nuclear translocation compared with the H 2 O 2 treatment group. Furthermore, pre-treatment with curcumin significantly increased the Bcl-2/Bax ratio and decreased the expression of active caspase-3 in H 2 O 2 -treated HTR8/SVneo cells ( Figure 5A,E,F).

Nrf2 Knockdown Attenuated the Protective Effect of Curcumin on HTR8/SVneo Cells under Oxidative Stress
To further elucidate the role of Nrf2 in the cytoprotective effects of curcumin against oxidative stress, we transfected HTR8/SVneo cells with a Nrf2 siRNA for 24 h, then pre-treated with 5 µM curcumin followed by the treatment of 400 µM H 2 O 2. As shown in Figure 7, after the transfection with si-Nrf2 in curcumin and H 2 O 2 treatment group, we observed markedly decreased (p < 0.05) mRNA and protein expression level of Nrf2, HO-1 and NQO1 compared with the curcumin + H 2 O 2 transfected with si-NC group. In addition, Nrf2 silencing abolished (p < 0.05) the upregulation of cell viability caused by curcumin pretreatment ( Figure 8A). The activation of CAT and GSH-Px were significantly decreased (p < 0.05) with knockdown of Nrf2 compared with the curcumin + H 2 O 2 transfected with si-NC group ( Figure 8C).

Nrf2 Knockdown Attenuated the Protective Effect of Curcumin on HTR8/SVneo Cells under Oxidative Stress
To further elucidate the role of Nrf2 in the cytoprotective effects of curcumin against oxidative stress, we transfected HTR8/SVneo cells with a Nrf2 siRNA for 24 h, then pre-treated with 5 μM curcumin followed by the treatment of 400 μM H2O2. As shown in Figure 7, after the transfection with si-Nrf2 in curcumin and H2O2 treatment group, we observed markedly decreased (p < 0.05) mRNA

Discussion
Placental trophoblast cells play important roles in the pregnancy and the development of the fetus. Excessive accumulation of trophoblastic ROS induced by greater maternal and fetal metabolism has been considered as an important factor that leads to placental dysfunction [27]. In the present study, we found for the first time that curcumin, a natural antioxidant known for its cytoprotective and anti-apoptotic actions [28,29], ameliorates H2O2-induced oxidative stress and cell apoptosis in human trophoblast HTR8/SVneo cells by activating the Nrf2 signaling pathway.
It is well established that H2O2 could be used to stimulate oxidative stress in vitro [30][31][32]. In the present study, we found that treatment with 400 μM H2O2 resulted in decreased cell viability in HTR8/SVneo cells, which is consistent with a previous study [1]. Curcumin has been reported to be toxic at a high dose, while it exerts a strong antioxidant effect at a low dose [23]. In the present study,

Discussion
Placental trophoblast cells play important roles in the pregnancy and the development of the fetus. Excessive accumulation of trophoblastic ROS induced by greater maternal and fetal metabolism has been considered as an important factor that leads to placental dysfunction [27]. In the present study, we found for the first time that curcumin, a natural antioxidant known for its cytoprotective and anti-apoptotic actions [28,29], ameliorates H2O2-induced oxidative stress and cell apoptosis in human trophoblast HTR8/SVneo cells by activating the Nrf2 signaling pathway.
It is well established that H2O2 could be used to stimulate oxidative stress in vitro [30][31][32]. In the present study, we found that treatment with 400 μM H2O2 resulted in decreased cell viability in HTR8/SVneo cells, which is consistent with a previous study [1]. Curcumin has been reported to be toxic at a high dose, while it exerts a strong antioxidant effect at a low dose [23]. In the present study,

Discussion
Placental trophoblast cells play important roles in the pregnancy and the development of the fetus. Excessive accumulation of trophoblastic ROS induced by greater maternal and fetal metabolism has been considered as an important factor that leads to placental dysfunction [27]. In the present study, we found for the first time that curcumin, a natural antioxidant known for its cytoprotective and anti-apoptotic actions [28,29], ameliorates H 2 O 2 -induced oxidative stress and cell apoptosis in human trophoblast HTR8/SVneo cells by activating the Nrf2 signaling pathway.
It is well established that H 2 O 2 could be used to stimulate oxidative stress in vitro [30][31][32]. In the present study, we found that treatment with 400 µM H 2 O 2 resulted in decreased cell viability in HTR8/SVneo cells, which is consistent with a previous study [1]. Curcumin has been reported to be toxic at a high dose, while it exerts a strong antioxidant effect at a low dose [23]. In the present study, pre-treatment of more than 10 µM curcumin for 24 h can induce obvious cell death in HTR8/SVneo cells, so we chose 2.5 and 5 µM curcumin as the optimal concentrations of curcumin for the subsequent experiments. Similar with the previous findings, pre-treatment with curcumin had the ability to enhance the viability of HTR8/SVneo cells after treatment of H 2 O 2 [23,33]. Moreover, the result of 5 µM curcumin pre-treatment showed a better effect than 2.5 µM, suggesting curcumin might exert the protective effect in a dose-dependent manner in HTR8/SVneo cells. These results indicate that low dose curcumin could protect HTR8/SVneo cells from death induced by H 2 O 2 .
ROS are oxygen free radicals, and the accumulation of ROS could cause oxidative stress and cell apoptosis [33]. Excessive oxidative stress induced by trophoblastic ROS is a pivotal factor for IUGR [27]. Our data showed that the exposure of HTR8/Sveo cells to H 2 O 2 resulted in increased intracellular ROS accumulation, but it was significantly alleviated by pre-treatment with either 2.5 µM or 5 µM curcumin. Consistent with our result, a previous study has also found that curcumin is able to reduce ROS accumulation [34]. This effect might be related with augmented antioxidant defense system promoted by curcumin pre-treatment. In addition, CAT and GSH-Px are the main members of antioxidant defense system. CAT converts hydrogen peroxide into water and oxygen [35]. GSH-Px is a vital antioxidant enzyme that catalyzes the reduction of hydroperoxides at the expense of reduced GSH [36]. In agreement with a previous finding that curcumin could improve the antioxidant capacity, curcumin pre-treatment enhanced the activities of CAT and GSH-Px in the HTR8/Sveo cells treated with H 2 O 2 [37]. The antioxidant property of curcumin has been reported to be mainly associated with its free radical scavenging activity [15]. Thus, our results indicate that pre-treatment with curcumin could reduce oxidative stress in HTR8/Sveo cells treated with H 2 O 2 .
Oxidative stress can induce excessive cell apoptosis through either mitochondria-dependent or independent pathway [38]. Equally, the apoptosis rate can also reflect the degree of oxidative stress [39]. We observed that apoptosis was sharply promoted in the H 2 O 2 treatment group. Supportively, H 2 O 2 treatment reduced the protein expression level of Bcl-2/Bax ratio. Bcl-2 is the core molecule which plays a considerable role of resistance in apoptosis, whereas Bax is a promoting apoptosis protein. Bcl-2/Bax ratio has been reported to be an important determinant factor of apoptosis [40]. Ample pieces of evidence have also proven that H 2 O 2 treatment could increase cell apoptosis [32,41]. However, pre-treatment with curcumin inhibited cell apoptosis, which was demonstrated by declined apoptosis rate, reduced protein expression of cleaved-caspase 3 and increased expression level of Bcl-2/Bax ratio. These results were consistent with the previous studies, which have also affirmed the anti-apoptotic effects of curcumin against oxidative stress-induced apoptosis [42]. Moreover, apoptosis is an essential regulatory cell process that occurs via caspase-independent pathway or caspase-dependent pathway [43]. Therefore, the decline of cleaved-caspase 3 protein expression level suggests that pre-treatment with curcumin can suppress apoptosis in HTR8/Sveo cells through inhibiting caspase-dependent pathway. Further functional investigation should be performed to estimate whether curcumin could reduce apoptosis in trophoblast cells via inhibiting caspase-independent pathway.
Nrf2 is a vital transcription factor that regulates cell survival and maintains redox homeostasis [44,45]. Nrf2 signaling pathway is also a crucial antioxidant pathway, which is responsible for regulating the expression of antioxidant enzymes against oxidative stress in cells [46][47][48]. The transcription of HO-1 and NQO1, the downstream genes of Nrf2, are primarily under the control of Nrf2 in maintaining cell redox balance [49,50]. Under oxidative stress, Nrf2 could translocate into the nucleus to activate the expression of its downstream antioxidant enzymes [32]. Similar with the previous studies, the Nrf2 signaling was activated by H 2 O 2 treatment in order to compensate for the oxidative damage [32,51]. Nrf2 nuclear translocation was also observed in the H 2 O 2 treatment group. In addition, previous researchers have made great effort to prove that curcumin activates Nrf2 and HO-1 signaling leading to protection against oxidative stress in different cell types [17,19,52,53]. In accordance with these findings, pre-treatment with curcumin induced a more obvious Nrf2 nuclear translocation than the H 2 O 2 treatment alone. Combined with the results of more effectively enhanced mRNA and protein expression of Nrf2, HO-1 and NQO1 induced by pre-treatment with curcumin, our data suggested that curcumin exerted its antioxidant effect against oxidative stress in HTR8/SVneo cells by promoting the activation of Nrf2 signaling pathway. The enhanced activation of Nrf2 signaling could also explain for the increased activity of CAT and decreased accumulation of ROS. In addition, up-regulation of nutrient transporters (SLC2A3) can improve the transfer of nutrients (including amino acids, fatty acids, and glucose) from mother to fetus, which plays an important role in placental development and fetal growth [54,55]. In our previous in vivo experiment, we have proven that curcumin has beneficial effects on nutrient transport and placental development, which could be applied for alleviating IUGR of mice. Furthermore, we found that knockdown of Nrf2 by Nrf2-siRNA transfection markedly diminished the curcumin-induced up-regulation of Nrf2, HO-1 and NQO1. Similarly, a previous study has found that knockdown of Nrf2 could decrease the expression level of HO-1 and NQO1 [56]. Here, we have shown that knockdown of Nrf2 decreased the protective effect of curcumin pre-treatment on the cell viability against oxidative stress, suggesting that Nrf2 is a key factor in maintaining the survival of HTR8/SVneo cells. Since Nrf2 controls the transcription of antioxidant enzymes, the increased activities of CAT and GSH-Px by pre-treatment with curcumin were also reduced after knockdown of Nrf2. Collectively, our data strongly proves that the antioxidant effect of curcumin on HTR8/SVneo cells against oxidative stress is achieved by activation of Nrf2 signaling pathway.

Conclusions
In conclusions, this is the first study demonstrating that pre-treatment with curcumin could alleviate H 2 O 2 -induced oxidative stress in human trophoblast HTR8/SVneo cells by improving the antioxidant capacity and activating Nrf2 signaling pathway.