Punicalagin promotes autophagy to protect primary human syncytiotrophoblasts from apoptosis

in Reproduction

Punicalagin is a prominent polyphenol in pomegranate juice that protects cultured syncytiotrophoblasts from stress-induced apoptosis. Here, we test the hypothesis that punicalagin has this effect by inhibiting the mTOR kinase pathway to enhance autophagic turnover and limit apoptosis in cultured primary human syncytiotrophoblasts. In syncytiotrophoblasts, starvation, rapamycin, or punicalagin all decreased the expression of phosphorylated ribosomal protein S6, a downstream target of the mTOR kinase, and of the autophagy markers, LC3-II and p62. In contrast, in the presence of bafilomycin, an inhibitor of late stages of autophagy and degradation in the autophagolysosome, syncytiotrophoblasts exposed to starvation, rapamycin, or punicalagin all showed increased levels of LC3-II and p62. The number of LC3-II punctae also increased in punicalagin-treated syncytiotrophoblasts exposed to chloroquine, another inhibitor of autophagic degradation, and punicalagin increased the number of lysosomes. The apoptosis-reducing effect of punicalagin was attenuated by inhibition of autophagy using bafilomycin or knockdown of the autophagy related gene, ATG16L1. Collectively, these data support the hypothesis that punicalagin modulates the crosstalk between autophagy and apoptosis to promote survival in cultured syncytiotrophoblasts.

Abstract

Punicalagin is a prominent polyphenol in pomegranate juice that protects cultured syncytiotrophoblasts from stress-induced apoptosis. Here, we test the hypothesis that punicalagin has this effect by inhibiting the mTOR kinase pathway to enhance autophagic turnover and limit apoptosis in cultured primary human syncytiotrophoblasts. In syncytiotrophoblasts, starvation, rapamycin, or punicalagin all decreased the expression of phosphorylated ribosomal protein S6, a downstream target of the mTOR kinase, and of the autophagy markers, LC3-II and p62. In contrast, in the presence of bafilomycin, an inhibitor of late stages of autophagy and degradation in the autophagolysosome, syncytiotrophoblasts exposed to starvation, rapamycin, or punicalagin all showed increased levels of LC3-II and p62. The number of LC3-II punctae also increased in punicalagin-treated syncytiotrophoblasts exposed to chloroquine, another inhibitor of autophagic degradation, and punicalagin increased the number of lysosomes. The apoptosis-reducing effect of punicalagin was attenuated by inhibition of autophagy using bafilomycin or knockdown of the autophagy related gene, ATG16L1. Collectively, these data support the hypothesis that punicalagin modulates the crosstalk between autophagy and apoptosis to promote survival in cultured syncytiotrophoblasts.

Introduction

The syncytiotrophoblast layer of human placental villi interfaces the maternal and fetal circulations and regulates maternal–fetal exchange of nutrients, wastes, and gases. Syncytiotrophoblasts are exposed to oxidative and nitrative stress and must have mechanisms that minimize nuclear and cytoplasmic injury.

We recently assessed the ability of pomegranate juice to protect cytotrophoblasts and syncytiotrophoblasts from stress-induced damage, with the idea that this dietary supplement has a low risk for adverse effects and may serve as a therapeutic agent (Chen et al. 2012, 2013). We chose this juice because pomegranate fruit has been consumed for hundreds of years as a nutrient supplement and is purported to have anti-atherosclerotic, anti-cancer, and anti-oxidant effects (Faria & Calhau 2011). We found that pomegranate juice reduces oxidative stress in human villous trophoblasts both in vivo and in vitro (Chen et al. 2012, 2013). The bioprotective effects of pomegranate juice are commonly attributed to its high concentration of polyphenols. Punicalagin (2,3-hexahydroxy-diphenoyl-4, 6-gallagyl-glucose) accounts for more than one-half of the polyphenols in pomegranate juice (Gil et al. 2000), and punicalagin by itself has anti-inflammatory (Jean-Gilles et al. 2013), anti-oxidative (Aqil et al. 2012), and anti-tumor activities (Wang et al. 2013). We tested punicalagin and found that this polyphenol was able to protect cultured human syncytiotrophoblasts from stress-induced apoptosis (Chen et al. 2012).

Here, we sought to uncover the mechanism by which punicalagin exerts this protective effect. We targeted autophagy as a pivotal cellular recycling pathway, for several reasons (Klionsky et al. 2012). First, autophagy is the pathway by which lysosomes degrade cytoplasmic components, including defective organelles, misfolded proteins, and invading microorganisms. Secondly, the mammalian target of rapamycin (mTOR) kinase pathway triggers autophagy in times of nutrient deficiency to generate amino acids and free fatty acids that pass through the tricarboxylic acid cycle to replenish cellular ATP levels. Thirdly, autophagy is often induced in response to stress as a means of promoting cell survival and reducing apoptosis, and multiple mediators of autophagy also play roles in apoptosis, indicating crosstalk between these pathways. Fourthly, autophagy is known to occur in trophoblasts of human placental villi (Oh et al. 2008). We test the hypothesis that punicalagin modulates autophagy to limit apoptosis in syncytiotrophoblasts.

Materials and methods

Isolation and culture of primary human trophoblasts

This study was approved by the Institutional Review Board of Washington University School of Medicine in St Louis, MO, and all subjects signed written consent forms. Primary human trophoblasts were isolated from placentas of uncomplicated singleton pregnancies delivered by cesarean section at 39 weeks' gestation, as previously described (Kliman et al. 1986, Chen et al. 2010). Cells were cultured in phenol-red-free DMEM (Sigma) containing 10% charcoal-stripped fetal bovine serum (Life Technologies, Inc., Grand Island, NY, USA), 20 mM HEPES, pH 7.4, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 5% CO2-air atmosphere. Cytotrophoblasts were plated at 300 000/cm2, cultured for 4 h to allow cell attachment, rinsed with medium multiple times to eliminate syncytial fragments, and cultured for a total of 76 h, with prominent syncytiotrophoblasts present for 52 h. For starvation experiments, trophoblasts were cultured in Earle's balanced salt solution (Thermo Scientific, Hudson, NH, USA) for the last 2, 4, or 6 h of the 76-h culture period. For the data on punicalagin's effects on autophagy, lysosomes and apoptosis, the syncytiotrophoblasts were treated with punicalagin (33.8 μM; Sigma) at 37 °C in 5% CO2-air atmosphere for the last 24 h before collection. In some experiments, cells were treated before collection with vehicle control (DMSO), 30 μM chloroquine (Sigma), 10 nM bafilomycin (Alexis Biochemicals, San Diego, CA, USA), or 10 nM bafilomycin and 200 nM rapamycin (Sigma), for the times indicated in the Figure Legends.

Human chorionic gonadotropin analysis

Supernatants from four primary human trophoblast cultures were assayed for Human chorionic gonadotropin (HCG) by enzyme-linked immunosorbent assay (DRG International, Mountainside, NJ, USA) according to the manufacturer's instructions. HCG levels per 24-h interval were expressed as the mean±s.e.m.

Western blotting

Primary human trophoblasts were lysed in radioimmunoprecipitation buffer (1% Nonie P-40, 0.5% deoxycholate, and 0.1% SDS in PBS) in the presence of protease and phosphatase inhibitors (Sigma). Proteins were separated by SDS–PAGE (25 μg of protein per lane) and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Membranes were blocked in 5% (w/v) nonfat dry milk in PBST (0.05% Tween 20 in PBS) and incubated overnight at 4 °C with primary antibody in PBST plus 5% (w/v) nonfat dry milk. Primary antibodies used were: rabbit monoclonal anti-phospho-S6 ribosomal protein (Cell Signaling Technologies (CST) #2215, 1:1000, 0.035 μg/ml), rabbit monoclonal anti-cleaved poly (ADP-ribose) polymerase (CST, #9541, 1:1000, 0.009 μg/ml), mouse monoclonal anti-cleaved cytokeratin 18 (Roche), rabbit monoclonal anti-Atg16L1 (Sigma, SAB2103567, 2 μg/ml), mouse monoclonal anti-p62 (Santa Cruz Biotechnology), goat polyclonal anti-actin (Santa Cruz Biotechnology, SC1616; 0.2 μg/ml), and rabbit polyclonal anti-LC3-II (Novus Biologicals, NB600-1384, 0.5 μg/ml). After incubation with primary antibodies, membranes were washed in PBST and then probed with HRP conjugated secondary antibody (Santa Cruz Biotechnology, 1:5000) for 4 h and then washed in PBST. Signals were detected by enhanced chemiluminescence (Thermo Scientific, Rockford, IL, USA), and relative protein levels were determined by using Image J software to measure densitometry of bands on film, with normalization to actin levels.

Immunofluorescence and lysosome staining

After culture and treatment for times as indicated in the Figure Legends, cells were fixed with methanol for 20 min at −20 °C, blocked with 5% BSA in PBS for 1 h at room temperature, and incubated at 4 °C overnight with antibodies to p62 (Santa Cruz Biotechnology, 1:200, 1 μg/ml), E-cadherin (Abcam, Cambridge, MA, USA, 40772, 1:100, 2 μg/ml) or LC3-II (Novus Biologicals, 1:200, 2.5 μg/ml), singly or in the combinations noted in the Figure Legends. After washing with PBS three times, cells were incubated with appropriate anti-mouse or anti-rabbit Alexa-labeled secondary antibodies (Life Technologies, 1:200) and nuclei were counter-stained with Draq 5 (Biostatus Limited, Shepsted, UK, 1:1000) for 1 h at room temperature. The cells were then washed three times and mounted. For staining of lysosomes, trophoblasts were treated with 50 nM LysoTracker Red (Life Technologies, Inc.) for 30 min at 37 °C, and nuclei were counter-stained with Draq 5. To quantify lysosomes, ten random confocal images were obtained from each of four primary cultures, using a Nikon E800 epifluorescence microscope and a total magnification of 600×, capturing a 512 pixel×512 pixel image covering 212 μm×212 μm. For each image, lysosomes were counted, defining each lysosome as a LysoTracker-Red positive region encompassing >30 square pixels in area. The lysosomes for each image were then divided by the number of nuclei in that image, providing a lysosome/nucleus measure. Then the ten lysosome/nuclei values were averaged as the final number for each placenta. Finally, the resultant four average numbers from four different placentas were used in the Student's t test comparison.

TUNEL

TUNEL staining was performed used the In Situ Cell Death Detection Kit (Roche). Briefly, cultured primary trophoblasts were rinsed once in PBS, fixed in 4% paraformaldehyde, and permeabilized in 0.1% Triton X-100 for 2 min on ice. The cells were incubated in TUNEL reaction mixture containing enzyme solution and label solution for one hour at 37 °C, rinsed with PBS, and nuclei were counter-stained with Draq 5. Images were acquired as described above.

siRNA-mediated gene silencing in primary trophoblasts

ATG16L1 Silencer Select siRNA (no. s30071: UAUCAUUGCAACUGGAUCCtg) and scrambled negative control #1 siRNA were purchased from Ambion (Norwalk, CT, USA). Trophoblasts were plated for 4 h, and transfected for 48 h using DharmaFECT 1 (Dharmacon, Lafayette, CO, USA) according to the manufacturer's instructions, with punicalagin or vehicle control present in the last 24 h.

Statistical analysis

Each experiment was repeated with the number of primary cell cultures listed in the Figure Legends, each from different placentas. Student's t test was performed when comparing between two conditions, and ANOVA with Bonferroni correction was applied when comparing three or more conditions. P<0.05 was deemed significant. Data are mean±s.e.m. KaleidaGraph (Synergy Software, Reading, PA, USA) software was used for statistical analyses.

Results

Primary cytotrophoblast cultures differentiate into syncytiotrophoblasts

As diagramed in Fig. 1A, cultured primary cytotrophoblasts spontaneously undergo fusion, forming multinucleated syncytiotrophoblasts. This differentiation process is also characterized by altered hormone secretion, and we found β-HCG level in the culture medium increased by an average of tenfold as cultured cells transitioned from cytotrophoblasts to syncytia (Fig. 1B). This hormonal differentiation was accompanied by morphological differentiation, with E-cadherin membrane staining of cultures beyond 52 h outlined multiple nuclei in the same cytoplasmic mass, indicating formation of multinucleated syncytiotrophoblasts (Fig. 1C and D). Taken together, these data confirmed our previous findings that the majority of cells in culture for less than 28 h maintained the cytotrophoblast phenotype, while >85% of the nuclei of cells cultured over 52 h underwent fusion, forming multinucleated syncytiotrophoblasts (Chen et al. 2010).

Figure 1
Figure 1

Differentiation of primary human trophoblasts in culture. Primary human cytotrophoblasts were plated at time 0. Unattached cells and fragments were removed by rinsing at 4 h, and cultures were maintained up to 76 h, during which time cells fused to form syncytiotrophoblasts, as diagramed in (A). (B) The accumulated content for β-HCG in medium during the immediate 24 h prior to the times of harvest noted on the graph. *P<0.05, ANOVA with Bonferroni correction; n=4 primary cultures. (C and D) Representative images of mononucleated cytotrophoblasts in culture at 28 h and syncytiotrophoblasts in cultures at 52 h. Green staining reflects E-Cadherin on the plasma membrane. The arrows show cytotrophoblasts (Cyt; red) and syncytiotrophoblast (Syn; yellow).

Citation: REPRODUCTION 151, 2; 10.1530/REP-15-0287

Starvation, rapamycin and punicalagin induce autophagy in cultured syncytiotrophoblasts

The mTOR kinase is expressed in human syncytiotrophoblasts and serves as a nutrient sensor (Roos et al. 2007). Inactivation of mTOR kinase activity by rapamycin (Klionsky et al. 2012) or by starvation (Desideri et al. 2012, Li et al. 2013) reduces the level of a downstream target of mTOR, phosphorylated ribosomal protein S6 (P-S6), indicative of mTOR inhibition, which can induce autophagy in multiple cell types (Klionsky et al. 2012). We sought to determine if autophagy occurs in syncytiotrophoblasts, and we used both starvation (Fig. 2A, B, C and D) and rapamycin (Fig. 2E, F, G and H) to study this phenomenon. As expected, syncytiotrophoblasts showed a time-dependent decrease in the levels of P-S6 under starvation conditions (Fig. 2A and B) or in the presence of rapamycin in unstarved cells (Fig. 2E and F). LC3-II is an autophagosomal-membrane-associated protein that is commonly reduced with enhanced autophagy, as it is degraded in the autophagolysosome. Expression of LC3-II was lower in starvation conditions, but not noticeably with rapamycin alone, compared to control (Fig. 2C and G). Moreover, the expression of a third marker of autophagy, the autophagosome adaptor protein p62, is typically reduced upon induction of autophagy as its degradation in autophagolysosomes, was significantly reduced in syncytiotrophoblasts exposed to starvation (Fig. 2D), but not exposed to rapamycin (Fig. 2H). Autophagic flux can be assessed by treating cells with bafilomycin, which blocks fusion of autophagosomes and lysosomes and thereby, avoids degradation of proteins in the autophagolysosome. In the presence of bafilomycin, increased autophagic flux is expected to result in increased levels of both LC3-II and p62. Indeed, in the presence of bafilomycin both starvation and rapamycin dramatically increased expression of these two proteins (Fig. 2C, D, E, G, H). As expected because bafilomycin does not prevent inactivation of mTOR, bafilomycin did not prevent the rapamycin-mediated reduction of P-S6 levels (Fig. 2B and F). Collectively, these results are consistent with autophagy occurring in syncytiotrophoblasts.

Figure 2
Figure 2

Starvation and rapamycin induce autophagy in primary human syncytiotrophoblasts. Cells were subjected to starvation (A, B, C and D) or cultured in nutrient replete conditions (E, F, G and H) and exposed to DMSO, rapamycin (Rap), or Rap and bafilomycin (Baf), for the times indicated. All cells were cultured for a total of 76 h and exposed to the condition tested for the times indicated. (A) Western blot of P-S6, p62, LC3-II, and actin from trophoblasts exposed to starvation conditions. Cells in the last lane were treated for 4 h with bafilomycin before collection. (B, C and D) Densitometric analyses of western blots of the indicated proteins, normalized to actin. (E) Western blot of P-S6, LC3-II, and actin exposed to vehicle control, Rap or Rap and Baf. (F and G) Densitometric analyses of western blots of the indicated proteins, normalized to actin. For all densitometric analyses, *P<0.05; **P<0.01; ANOVA with Bonferroni correction. n=4 cultures from different placentas. (H) Immunofluorescence for p62 and E-cadherin in trophoblasts exposed for the final 4 h of culture to DMSO, Rap, or Rap and Baf. Red: E-cadherin; green: p62; blue: DNA; arrows: p62 puncta.

Citation: REPRODUCTION 151, 2; 10.1530/REP-15-0287

Having demonstrated autophagy occurs in primary human syncytiotrophoblasts, we next asked whether punicalagin could induce autophagy in these cells. Similar to our observations in starved and rapamycin-treated cells, we found that punicalagin treatment resulted in reduced P-S6, LC3-II, and p62 levels (Fig. 3A, B, C and D). Treatment with punicalagin in the presence of bafilomycin yielded increased levels of LC3-II and p62 suggesting increased autophagic flux. As expected, bafilomycin had no effect on the reduced P-S6 levels observed in response to punicalagin (Fig. 3E, F, G and H). We next treated cultures with punicalagin or vehicle control in the presence of chloroquine, another agent that inhibits fusion of autophagosomes with lysosomes, and thus, degradation in the autophagolysosome. Syncytiotrophoblasts treated with chloroquine and punicalagin had substantially more LC3-II puncta (Fig. 3J) than those treated with chloroquine and vehicle (Fig. 3I). Finally, we determined whether or not punicalagin increased the number of lysosomes in trophoblasts, as would be expected if punicalagin resulted in increased autophagic flux. We stained syncytiotrophoblasts cultured in the presence or absence of punicalagin with LysoTracker Red and found that the number of lysosomes per nucleus was indeed higher in syncytiotrophoblasts exposed to punicalagin, compared to vehicle control (Fig. 4). Together, these findings strongly support the conclusion that punicalagin induces autophagy in syncytiotrophoblasts.

Figure 3
Figure 3

Punicalagin induces autophagy in syncytiotrophoblasts. Representative western blots (A and E) and densitometry (B, C and D; F, G and H) of P-S6, LC3-II, p62, and actin from primary human trophoblasts cultured for 76 h, in the presence or absence of punicalagin, DMSO or bafilomycin during the 24 h prior to harvest. *P<0.05; one-tail t test. n=4 primary cultures from different placentas. (I and J) Representative immunofluorescence image of LC3-II in syncytiotrophoblasts treated with chloroquine in the presence or absence of punicalagin (Pun).

Citation: REPRODUCTION 151, 2; 10.1530/REP-15-0287

Figure 4
Figure 4

Punicalagin increases lysosomes in syncytiotrophoblasts. Representative images of syncytiotrophoblasts treated with vehicle control (A) or punicalagin (B) for the last 24 h with a 30-min (prior to harvest) exposure to 50 nM LysoTracker Red. Nuclei are stained blue with Draq 5. (C) Enlarged image of selected area in A. (D) Quantification of lysosomes. *P<0.05; t test. n=4 primary cultures from different placentas.

Citation: REPRODUCTION 151, 2; 10.1530/REP-15-0287

Punicalagin-induced autophagy protects syncytiotrophoblasts from apoptosis

Depending on the cell type and culture conditions, autophagy can either protect or promote cell death (Loos et al. 2013). Our previous work showed that punicalagin decreases hypoxia-induced apoptosis in cultured syncytiotrophoblasts (Chen et al. 2013). We hypothesized that punicalagin regulates the interplay between autophagy and apoptosis in cultured syncytiotrophoblasts, favoring survival. Using TUNEL staining, we found that punicalagin-treated syncytiotrophoblasts exhibited lower levels of apoptosis than vehicle-control treated cells (Fig. 5A), confirming our previous findings (Chen et al. 2013). We then examined the expression of two proteins that serve as markers of caspase-mediated apoptosis, the caspase-cleaved forms of nuclear poly-ADP-ribose polymerase (cl-Parp) and of cytoplasmic cytokeratin 18 (cl-Cyt18). We found that the levels of both cl-Parp and cl-Cyt18 decreased in punicalagin-treated cells (Fig. 5B, C and D), indicating reduced apoptosis. To assess the role of autophagy in this process, we treated syncytiotrophoblasts with either punicalagin or vehicle control in the presence of bafilomycin. The apoptosis-reducing effect of punicalagin, as measured by expression of cl-Parp and cl-Cyt18, were reversed by inhibition of autophagy using bafilomycin (Fig. 5B, C and D). These data suggest that autophagy is required for punicalagin-mediated protection from apoptosis in syncytiotrophoblasts.

Figure 5
Figure 5

Punicalagin decreases apoptosis in syncytiotrophoblasts. (A) Representative TUNEL images of syncytiotrophoblasts treated with or without punicalagin (Pun). (B) Western blots of cl-Parp and cl-Cyt18 in syncytiotrophoblasts in the presence or absence of punicalagin or bafilomycin (Baf). (C and D) Densitometric analyses of cl-Parp and cl-Cyt18. *P<0.05; ANOVA with Bonferroni correction; n=4 primary cultures from different placentas.

Citation: REPRODUCTION 151, 2; 10.1530/REP-15-0287

To further test the hypothesis that punicalagin can regulate the interplay between autophagy and apoptosis in cultured syncytiotrophoblasts, we inhibited autophagy using siRNA to knockdown ATG16L1, which participates in the elongation step of autophagy and does not directly contribute to regulation of apoptosis (Klionsky et al. 2008). Compared to vehicle control, punicalagin modestly enhanced the expression of Atg16L1 (by 22%) and reduced the expression of cl-Parp (by 65%) in syncytiotrophoblasts transfected with scrambled siRNA control (Fig. 6A, B and C). Punicalagin-treated syncytiotrophoblasts transfected with specific siATG16L siRNA showed about 50% reduction in Atg16L levels and about 60% more cl-Parp, compared to syncytiotrophoblasts transfected with scrambled siRNA control (Fig. 6A, B and C). These data indicate that punicalagin-mediated protection of syncytiotrophoblasts involves increased autophagy.

Figure 6
Figure 6

Knockdown of ATG16L1 reduces the effect of punicalagin to decrease apoptosis in syncytiotrophoblasts. (A) Representative western blots of Atg16L1, actin, and cl-Parp in primary syncytiotrophoblasts transfected with ATG16L1-specific siRNA or scrambled control siRNA in the presence or absence of punicalagin. (B and C) Densitometric analyses of Atg16L1 (B) and cl-Parp (C), normalized to actin. *P<0.05; ANOVA with Bonferroni correction; n=4 primary cultures from different placentas.

Citation: REPRODUCTION 151, 2; 10.1530/REP-15-0287

Discussion

The data show that syncytiotrophoblasts undergo autophagy in response to starvation, rapamycin, and punicalagin, the latter being a prominent polyphenol in pomegranate juice. We present multiple lines of evidence that indicate punicalagin-induced autophagy protects syncytiotrophoblasts from apoptosis. First, in the presence of bafilomycin, punicalagin decreases the level of the mTOR downstream target, P-S6, and increases the levels of the autophagy markers, p62 and LC3-II. Secondly, treatment with punicalagin and chloroquine result in accumulation of LC3-II. Thirdly, punicalagin increases the number of lysosomes in syncytiotrophoblasts. Fourthly, punicalagin decreases apoptosis, as indicated by decreased TUNEL staining and decreased expression of cl-Parp and cl-Cyt18. Finally, inhibition of autophagy with bafilomycin or by knockdown of ATG16L1, diminishes the ability of punicalagin to reduce the level of apoptosis in syncytiotrophoblasts. Together, these results support the hypothesis that punicalagin modulates crosstalk between apoptosis and autophagy thereby promoting cell survival in cultured primary syncytiotrophoblasts.

No clinical therapeutic options currently exist to limit injury to human placental trophoblast exposed to stressors in vivo. The protective effects of punicalagin on human cultures of syncytiotrophoblasts suggests that this polyphenol may serve as such a therapeutic agent, as no harmful effects of pomegranate juice or its polyphenols have been identified (Cerda et al. 2003a, Ly et al. 2015). Antioxidant properties of phytochemicals in fruits and vegetables generally, and pomegranate juice in particular, are commonly attributed to provide protective effects from injury by reducing the levels of reactive oxygen species and of nitrative species generated within in cells exposed to exogenous stimuli. Such an assumption has recently been challenged (Poljsak & Milisav 2012, Forman et al. 2014, Rahal et al. 2014), and the beneficial effects of punicalagin actually are likely to result from a plethora of pathways that may or may not involve antioxidant properties (Adams et al. 2006). For example, we reported punicalagin modulates the mRNA levels of p53, p21, and MDM2, while inhibiting p53 activity and decreasing apoptosis in trophoblasts (Chen et al. 2013). We also have found that pomegranate juice selectively modulates gene expression in human trophoblasts (Chen et al. 2015). Resveratrol is a polyphenol that was recently shown to improve fetal growth in a mouse model of preeclampsia, and this beneficial effect derives, in part, from nitric oxide effects that improve uterine artery blood flow to the fetal-placental unit (Gurusamy et al. 2010, Poudel et al. 2013). Similar to our observation that punicalagin can induce autophagy and protect syncytiotrophoblasts from apoptosis, resveratrol has been shown to induce autophagy and improve cell survival in cardiac myocytes (Gurusamy et al. 2010).

The kinase mTOR is a nutrient sensor that monitors glucose concentrations and growth factors and regulates the activity of placental amino acid transporters in trophoblasts (Roos et al. 2007). Moreover, mTOR regulates autophagy to eliminate damaged proteins and organelles and to generate substrates for critical cellular functions when nutrients are limited (Loos et al. 2013). Multiple autophagy-related proteins, including Apg9L2, LC3-II, and Beclin 1 (Oh et al. 2008, Bildirici et al. 2012), are expressed in human placental tissues. Autophagy could likely be valuable to syncytiotrophoblasts, especially during complicated pregnancies, as turnover of cytoplasmic components within the syncytium could occur without breach of syncytial barrier function. Moreover, the substrates generated within the syncytiotrophoblast could potentially supply the fetus with nutrients under conditions of limited supplies. Studies of trophoblast in animal models and villi from pregnancy disorders will help elucidate what role, if any, autophagy contributes to syncytiotrophoblast integrity, function, and survival in vivo.

One concern of in vitro experiments is whether or not the studies use a physiologically relevant concentration of the agent investigated. In most situations, this is difficult to precisely determine, as in vivo metabolic studies are not available for most compounds. Although only a few such studies exist for pomegranate juice (Cerda et al. 2003a,b, Mertens-Talcott et al. 2006, Seeram et al. 2006, 2008, Nielsen et al. 2008), what is clear is that ingestion of 235 ml/day (about 8 ounces) of pomegranate juice results in detectable metabolites in the circulation (Mertens-Talcott et al. 2006, Seeram et al. 2006, 2008, Nunez-Sanchez et al. 2014). We showed previously that antenatal ingestion of 235 ml/day of pomegranate juice is sufficient to reduce in vivo oxidative stress in the placentas of women who underwent labor (Chen et al. 2012). In non-pregnant patients on hemodialysis, 100 ml/day of pomegranate juice reduced oxidative stress levels (Shema-Didi et al. 2012). Punicalagin is the predominant polyphenol in pomegranate juice (Basu & Penugonda 2009) and, in juice from POM Wonderful, LLC, Los Angeles, CA, punicalagin is present at ∼310 μM, greater than the 34 μM concentration we used in this study. We previously found that 34 μm punicalagin is in the middle of a dose-response curve, which we found to protect human cytotrophoblasts from exogenous stressors in culture (Chen et al. 2013). Finally, 34 μM is similar to the punicalagin concentration used by others in in vitro and in vivo studies (Yaidikar et al. 2014, Peng et al. 2015, Xu et al. 2015).

Clearly, the metabolism of punicalagin and its interactions with other phytochemicals in fruits and vegetables, like pomegranate juice, contribute to the collective effects of the diet consumed. Future studies will be required to further dissect such interactions and to determine if any agent might serve as a therapeutic option for placental protection.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by the Department of Obstetrics and Gynecology at Washington University School of Medicine, St Louis, MO, and in part by an unrestricted gift to Washington University School of Medicine from POM Wonderful, LLC.

Acknowledgements

We thank Dr Deborah J Frank for critical reading of the manuscript.

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  • FormanHJDaviesKJUrsiniF2014How do nutritional antioxidants really work: nucleophilic tone and para-hormesis versus free radical scavenging in vivo. Free Radical Biology & Medicine662435. (doi:10.1016/j.freeradbiomed.2013.05.045)

    • Search Google Scholar
    • Export Citation
  • GilMITomas-BarberanFAHess-PierceBHolcroftDMKaderAA2000Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing. Journal of Agricultural and Food Chemistry4845814589. (doi:10.1021/jf000404a)

    • Search Google Scholar
    • Export Citation
  • GurusamyNLekliIMukherjeeSRayDAhsanMKGherghiceanuMPopescuLMDasDK2010Cardioprotection by resveratrol: a novel mechanism via autophagy involving the mTORC2 pathway. Cardiovascular Research86103112. (doi:10.1093/cvr/cvp384)

    • Search Google Scholar
    • Export Citation
  • Jean-GillesDLiLVaidyanathanVGKingRChoBWorthenDRChichesterCOIIISeeramNP2013Inhibitory effects of polyphenol punicalagin on type-II collagen degradation in vitro and inflammation in vivo. Chemico-Biological Interactions2059099. (doi:10.1016/j.cbi.2013.06.018)

    • Search Google Scholar
    • Export Citation
  • KlimanHJNestlerJESermasiESangerJMStraussJFIII1986Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology11815671582. (doi:10.1210/endo-118-4-1567)

    • Search Google Scholar
    • Export Citation
  • KlionskyDJAbeliovichHAgostinisPAgrawalDKAlievGAskewDSBabaMBaehreckeEHBahrBABallabioA2008Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy4151175. (doi:10.4161/auto.5338)

    • Search Google Scholar
    • Export Citation
  • KlionskyDJAbdallaFCAbeliovichHAbrahamRTAcevedo-ArozenaAAdeliKAgholmeLAgnelloMAgostinisPAguirre-GhisoJA2012Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy8445544. (doi:10.4161/auto.19496)

    • Search Google Scholar
    • Export Citation
  • LiLChenYGibsonSB2013Starvation-induced autophagy is regulated by mitochondrial reactive oxygen species leading to AMPK activation. Cellular Signalling255065. (doi:10.1016/j.cellsig.2012.09.020)

    • Search Google Scholar
    • Export Citation
  • LoosBEngelbrechtAMLockshinRAKlionskyDJZakeriZ2013The variability of autophagy and cell death susceptibility: Unanswered questions. Autophagy912701285. (doi:10.4161/auto.25560)

    • Search Google Scholar
    • Export Citation
  • LyCYockell-LelievreJFerraroZMArnasonJTFerrierJGruslinA2015The effects of dietary polyphenols on reproductive health and early development. Human Reproduction Update21228248. (doi:10.1093/humupd/dmu058)

    • Search Google Scholar
    • Export Citation
  • Mertens-TalcottSUJilma-StohlawetzPRiosJHingoraniLDerendorfH2006Absorption, metabolism, and antioxidant effects of pomegranate (Punica granatum L.) polyphenols after ingestion of a standardized extract in healthy human volunteers. Journal of Agricultural and Food Chemistry5489568961. (doi:10.1021/jf061674h)

    • Search Google Scholar
    • Export Citation
  • NielsenRPedersenTAHagenbeekDMoulosPSiersbaekRMegensEDenissovSBorgesenMFrancoijsKJMandrupS2008Genome-wide profiling of PPARγ:RXR and RNA polymerase II occupancy reveals temporal activation of distinct metabolic pathways and changes in RXR dimer composition during adipogenesis. Genes and Development2229532967. (doi:10.1101/gad.501108)

    • Search Google Scholar
    • Export Citation
  • Nunez-SanchezMAGarcia-VillalbaRMonedero-SaizTGarcia-TalaveraNVGomez-SanchezMBSanchez-AlvarezCGarcia-AlbertAMRodriguez-GilFJRuiz-MarinMPastor-QuiranteFA2014Targeted metabolic profiling of pomegranate polyphenols and urolithins in plasma, urine and colon tissues from colorectal cancer patients. Molecular Nutrition & Food Research5811991211. (doi:10.1002/mnfr.201300931)

    • Search Google Scholar
    • Export Citation
  • OhSYChoiSJKimKHChoEYKimJHRohCR2008Autophagy-related proteins, LC3 and Beclin-1, in placentas from pregnancies complicated by preeclampsia. Reproductive Sciences15912920. (doi:10.1177/1933719108319159)

    • Search Google Scholar
    • Export Citation
  • PengJWeiDFuZLiDTanYXuTZhouJZhangT2015Punicalagin ameliorates lipopolysaccharide-induced acute respiratory distress syndrome in mice. Inflammation38493499. (doi:10.1007/s10753-014-9955-5)

    • Search Google Scholar
    • Export Citation
  • PoljsakBMilisavI2012The neglected significance of "antioxidative stress". Oxidative Medicine and Cellular Longevity2012480895. (doi:10.1155/2012/480895)

    • Search Google Scholar
    • Export Citation
  • PoudelRStanleyJLRueda-ClausenCFAnderssonIJSibleyCPDavidgeSTBakerPN2013Effects of resveratrol in pregnancy using murine models with reduced blood supply to the uterus. PLoS ONE8e64401. (doi:10.1371/journal.pone.0064401)

    • Search Google Scholar
    • Export Citation
  • RahalAKumarASinghVYadavBTiwariRChakrabortySDhamaK2014Oxidative stress, prooxidants, and antioxidants: the interplay. BioMed Research International2014761264. (doi:10.1155/2014/761264)

    • Search Google Scholar
    • Export Citation
  • RoosSJanssonNPalmbergISaljoKPowellTLJanssonT2007Mammalian target of rapamycin in the human placenta regulates leucine transport and is down-regulated in restricted fetal growth. Journal of Physiology582449459. (doi:10.1113/jphysiol.2007.129676)

    • Search Google Scholar
    • Export Citation
  • SeeramNPHenningSMZhangYSuchardMLiZHeberD2006Pomegranate juice ellagitannin metabolites are present in human plasma and some persist in urine for up to 48 hours. Journal of Nutrition13624812485.

    • Search Google Scholar
    • Export Citation
  • SeeramNPZhangYMcKeeverRHenningSMLeeRPSuchardMALiZChenSThamesGZerlinA2008Pomegranate juice and extracts provide similar levels of plasma and urinary ellagitannin metabolites in human subjects. Journal of Medicinal Food11390394. (doi:10.1089/jmf.2007.650)

    • Search Google Scholar
    • Export Citation
  • Shema-DidiLSelaSOreLShapiroGGeronRMosheGKristalB2012One year of pomegranate juice intake decreases oxidative stress, inflammation, and incidence of infections in hemodialysis patients: a randomized placebo-controlled trial. Free Radical Biology & Medicine53297304. (doi:10.1016/j.freeradbiomed.2012.05.013)

    • Search Google Scholar
    • Export Citation
  • WangSGHuangMHLiJHLaiFILeeHMHsuYN2013Punicalagin induces apoptotic and autophagic cell death in human U87MG glioma cells. Acta Pharmacologica Sinica3414111419. (doi:10.1038/aps.2013.98)

    • Search Google Scholar
    • Export Citation
  • XuXLiHHouXLiDHeSWanCYinPLiuMLiuFXuJ2015Punicalagin Induces Nrf2/HO-1 Expression via Upregulation of PI3K/AKT Pathway and Inhibits LPS-Induced Oxidative Stress in RAW264.7 Macrophages. Mediators of Inflammation2015380218. (doi:10.1155/2015/380218)

    • Search Google Scholar
    • Export Citation
  • YaidikarLBynaBThakurSR2014Neuroprotective effect of punicalagin against cerebral ischemia reperfusion-induced oxidative brain injury in rats. Journal of Stroke and Cerebrovascular Diseases2328692878. (doi:10.1016/j.jstrokecerebrovasdis.2014.07.020)

    • Search Google Scholar
    • Export Citation

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    Society for Reproduction and Fertility

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    Differentiation of primary human trophoblasts in culture. Primary human cytotrophoblasts were plated at time 0. Unattached cells and fragments were removed by rinsing at 4 h, and cultures were maintained up to 76 h, during which time cells fused to form syncytiotrophoblasts, as diagramed in (A). (B) The accumulated content for β-HCG in medium during the immediate 24 h prior to the times of harvest noted on the graph. *P<0.05, ANOVA with Bonferroni correction; n=4 primary cultures. (C and D) Representative images of mononucleated cytotrophoblasts in culture at 28 h and syncytiotrophoblasts in cultures at 52 h. Green staining reflects E-Cadherin on the plasma membrane. The arrows show cytotrophoblasts (Cyt; red) and syncytiotrophoblast (Syn; yellow).

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    Starvation and rapamycin induce autophagy in primary human syncytiotrophoblasts. Cells were subjected to starvation (A, B, C and D) or cultured in nutrient replete conditions (E, F, G and H) and exposed to DMSO, rapamycin (Rap), or Rap and bafilomycin (Baf), for the times indicated. All cells were cultured for a total of 76 h and exposed to the condition tested for the times indicated. (A) Western blot of P-S6, p62, LC3-II, and actin from trophoblasts exposed to starvation conditions. Cells in the last lane were treated for 4 h with bafilomycin before collection. (B, C and D) Densitometric analyses of western blots of the indicated proteins, normalized to actin. (E) Western blot of P-S6, LC3-II, and actin exposed to vehicle control, Rap or Rap and Baf. (F and G) Densitometric analyses of western blots of the indicated proteins, normalized to actin. For all densitometric analyses, *P<0.05; **P<0.01; ANOVA with Bonferroni correction. n=4 cultures from different placentas. (H) Immunofluorescence for p62 and E-cadherin in trophoblasts exposed for the final 4 h of culture to DMSO, Rap, or Rap and Baf. Red: E-cadherin; green: p62; blue: DNA; arrows: p62 puncta.

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    Punicalagin induces autophagy in syncytiotrophoblasts. Representative western blots (A and E) and densitometry (B, C and D; F, G and H) of P-S6, LC3-II, p62, and actin from primary human trophoblasts cultured for 76 h, in the presence or absence of punicalagin, DMSO or bafilomycin during the 24 h prior to harvest. *P<0.05; one-tail t test. n=4 primary cultures from different placentas. (I and J) Representative immunofluorescence image of LC3-II in syncytiotrophoblasts treated with chloroquine in the presence or absence of punicalagin (Pun).

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    Punicalagin increases lysosomes in syncytiotrophoblasts. Representative images of syncytiotrophoblasts treated with vehicle control (A) or punicalagin (B) for the last 24 h with a 30-min (prior to harvest) exposure to 50 nM LysoTracker Red. Nuclei are stained blue with Draq 5. (C) Enlarged image of selected area in A. (D) Quantification of lysosomes. *P<0.05; t test. n=4 primary cultures from different placentas.

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    Punicalagin decreases apoptosis in syncytiotrophoblasts. (A) Representative TUNEL images of syncytiotrophoblasts treated with or without punicalagin (Pun). (B) Western blots of cl-Parp and cl-Cyt18 in syncytiotrophoblasts in the presence or absence of punicalagin or bafilomycin (Baf). (C and D) Densitometric analyses of cl-Parp and cl-Cyt18. *P<0.05; ANOVA with Bonferroni correction; n=4 primary cultures from different placentas.

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    Knockdown of ATG16L1 reduces the effect of punicalagin to decrease apoptosis in syncytiotrophoblasts. (A) Representative western blots of Atg16L1, actin, and cl-Parp in primary syncytiotrophoblasts transfected with ATG16L1-specific siRNA or scrambled control siRNA in the presence or absence of punicalagin. (B and C) Densitometric analyses of Atg16L1 (B) and cl-Parp (C), normalized to actin. *P<0.05; ANOVA with Bonferroni correction; n=4 primary cultures from different placentas.

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  • AqilFMunagalaRVadhanamMVKausarHJeyabalanJSchultzDJGuptaRC2012Anti-proliferative activity and protection against oxidative DNA damage by punicalagin isolated from pomegranate husk. Food Research International49345353. (doi:10.1016/j.foodres.2012.07.059)

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  • ChenBTuuliMGLongtineMSShinJSLawrenceRInderTMichael NelsonD2012Pomegranate juice and punicalagin attenuate oxidative stress and apoptosis in human placenta and in human placental trophoblasts. American Journal of Physiology. Endocrinology and Metabolism302E1142E1152. (doi:10.1152/ajpendo.00003.2012)

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  • ChenBLongtineMSNelsonDM2013Punicalagin, a polyphenol in pomegranate juice, downregulates p53 and attenuates hypoxia-induced apoptosis in cultured human placental syncytiotrophoblasts. American Journal of Physiology. Endocrinology and Metabolism305E1274E1280. (doi:10.1152/ajpendo.00218.2013)

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  • ChenBZaveriPGLongtineMSNelsonDM2015N-myc downstream-regulated gene 1 (NDRG1) mediates pomegranate juice protection from apoptosis in hypoxic BeWo cells but not in primary human trophoblasts. Placenta36847853. (doi:10.1016/j.placenta.2015.05.009)

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  • DesideriEFilomeniGCirioloMR2012Glutathione participates in the modulation of starvation-induced autophagy in carcinoma cells. Autophagy817691781. (doi:10.4161/auto.22037)

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  • FariaACalhauC2011The bioactivity of pomegranate: impact on health and disease. Critical Reviews in Food Science and Nutrition51626634. (doi:10.1080/10408391003748100)

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    • Export Citation
  • FormanHJDaviesKJUrsiniF2014How do nutritional antioxidants really work: nucleophilic tone and para-hormesis versus free radical scavenging in vivo. Free Radical Biology & Medicine662435. (doi:10.1016/j.freeradbiomed.2013.05.045)

    • Search Google Scholar
    • Export Citation
  • GilMITomas-BarberanFAHess-PierceBHolcroftDMKaderAA2000Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing. Journal of Agricultural and Food Chemistry4845814589. (doi:10.1021/jf000404a)

    • Search Google Scholar
    • Export Citation
  • GurusamyNLekliIMukherjeeSRayDAhsanMKGherghiceanuMPopescuLMDasDK2010Cardioprotection by resveratrol: a novel mechanism via autophagy involving the mTORC2 pathway. Cardiovascular Research86103112. (doi:10.1093/cvr/cvp384)

    • Search Google Scholar
    • Export Citation
  • Jean-GillesDLiLVaidyanathanVGKingRChoBWorthenDRChichesterCOIIISeeramNP2013Inhibitory effects of polyphenol punicalagin on type-II collagen degradation in vitro and inflammation in vivo. Chemico-Biological Interactions2059099. (doi:10.1016/j.cbi.2013.06.018)

    • Search Google Scholar
    • Export Citation
  • KlimanHJNestlerJESermasiESangerJMStraussJFIII1986Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology11815671582. (doi:10.1210/endo-118-4-1567)

    • Search Google Scholar
    • Export Citation
  • KlionskyDJAbeliovichHAgostinisPAgrawalDKAlievGAskewDSBabaMBaehreckeEHBahrBABallabioA2008Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy4151175. (doi:10.4161/auto.5338)

    • Search Google Scholar
    • Export Citation
  • KlionskyDJAbdallaFCAbeliovichHAbrahamRTAcevedo-ArozenaAAdeliKAgholmeLAgnelloMAgostinisPAguirre-GhisoJA2012Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy8445544. (doi:10.4161/auto.19496)

    • Search Google Scholar
    • Export Citation
  • LiLChenYGibsonSB2013Starvation-induced autophagy is regulated by mitochondrial reactive oxygen species leading to AMPK activation. Cellular Signalling255065. (doi:10.1016/j.cellsig.2012.09.020)

    • Search Google Scholar
    • Export Citation
  • LoosBEngelbrechtAMLockshinRAKlionskyDJZakeriZ2013The variability of autophagy and cell death susceptibility: Unanswered questions. Autophagy912701285. (doi:10.4161/auto.25560)

    • Search Google Scholar
    • Export Citation
  • LyCYockell-LelievreJFerraroZMArnasonJTFerrierJGruslinA2015The effects of dietary polyphenols on reproductive health and early development. Human Reproduction Update21228248. (doi:10.1093/humupd/dmu058)

    • Search Google Scholar
    • Export Citation
  • Mertens-TalcottSUJilma-StohlawetzPRiosJHingoraniLDerendorfH2006Absorption, metabolism, and antioxidant effects of pomegranate (Punica granatum L.) polyphenols after ingestion of a standardized extract in healthy human volunteers. Journal of Agricultural and Food Chemistry5489568961. (doi:10.1021/jf061674h)

    • Search Google Scholar
    • Export Citation
  • NielsenRPedersenTAHagenbeekDMoulosPSiersbaekRMegensEDenissovSBorgesenMFrancoijsKJMandrupS2008Genome-wide profiling of PPARγ:RXR and RNA polymerase II occupancy reveals temporal activation of distinct metabolic pathways and changes in RXR dimer composition during adipogenesis. Genes and Development2229532967. (doi:10.1101/gad.501108)

    • Search Google Scholar
    • Export Citation
  • Nunez-SanchezMAGarcia-VillalbaRMonedero-SaizTGarcia-TalaveraNVGomez-SanchezMBSanchez-AlvarezCGarcia-AlbertAMRodriguez-GilFJRuiz-MarinMPastor-QuiranteFA2014Targeted metabolic profiling of pomegranate polyphenols and urolithins in plasma, urine and colon tissues from colorectal cancer patients. Molecular Nutrition & Food Research5811991211. (doi:10.1002/mnfr.201300931)

    • Search Google Scholar
    • Export Citation
  • OhSYChoiSJKimKHChoEYKimJHRohCR2008Autophagy-related proteins, LC3 and Beclin-1, in placentas from pregnancies complicated by preeclampsia. Reproductive Sciences15912920. (doi:10.1177/1933719108319159)

    • Search Google Scholar
    • Export Citation
  • PengJWeiDFuZLiDTanYXuTZhouJZhangT2015Punicalagin ameliorates lipopolysaccharide-induced acute respiratory distress syndrome in mice. Inflammation38493499. (doi:10.1007/s10753-014-9955-5)

    • Search Google Scholar
    • Export Citation
  • PoljsakBMilisavI2012The neglected significance of "antioxidative stress". Oxidative Medicine and Cellular Longevity2012480895. (doi:10.1155/2012/480895)

    • Search Google Scholar
    • Export Citation
  • PoudelRStanleyJLRueda-ClausenCFAnderssonIJSibleyCPDavidgeSTBakerPN2013Effects of resveratrol in pregnancy using murine models with reduced blood supply to the uterus. PLoS ONE8e64401. (doi:10.1371/journal.pone.0064401)

    • Search Google Scholar
    • Export Citation
  • RahalAKumarASinghVYadavBTiwariRChakrabortySDhamaK2014Oxidative stress, prooxidants, and antioxidants: the interplay. BioMed Research International2014761264. (doi:10.1155/2014/761264)

    • Search Google Scholar
    • Export Citation
  • RoosSJanssonNPalmbergISaljoKPowellTLJanssonT2007Mammalian target of rapamycin in the human placenta regulates leucine transport and is down-regulated in restricted fetal growth. Journal of Physiology582449459. (doi:10.1113/jphysiol.2007.129676)

    • Search Google Scholar
    • Export Citation
  • SeeramNPHenningSMZhangYSuchardMLiZHeberD2006Pomegranate juice ellagitannin metabolites are present in human plasma and some persist in urine for up to 48 hours. Journal of Nutrition13624812485.

    • Search Google Scholar
    • Export Citation
  • SeeramNPZhangYMcKeeverRHenningSMLeeRPSuchardMALiZChenSThamesGZerlinA2008Pomegranate juice and extracts provide similar levels of plasma and urinary ellagitannin metabolites in human subjects. Journal of Medicinal Food11390394. (doi:10.1089/jmf.2007.650)

    • Search Google Scholar
    • Export Citation
  • Shema-DidiLSelaSOreLShapiroGGeronRMosheGKristalB2012One year of pomegranate juice intake decreases oxidative stress, inflammation, and incidence of infections in hemodialysis patients: a randomized placebo-controlled trial. Free Radical Biology & Medicine53297304. (doi:10.1016/j.freeradbiomed.2012.05.013)

    • Search Google Scholar
    • Export Citation
  • WangSGHuangMHLiJHLaiFILeeHMHsuYN2013Punicalagin induces apoptotic and autophagic cell death in human U87MG glioma cells. Acta Pharmacologica Sinica3414111419. (doi:10.1038/aps.2013.98)

    • Search Google Scholar
    • Export Citation
  • XuXLiHHouXLiDHeSWanCYinPLiuMLiuFXuJ2015Punicalagin Induces Nrf2/HO-1 Expression via Upregulation of PI3K/AKT Pathway and Inhibits LPS-Induced Oxidative Stress in RAW264.7 Macrophages. Mediators of Inflammation2015380218. (doi:10.1155/2015/380218)

    • Search Google Scholar
    • Export Citation
  • YaidikarLBynaBThakurSR2014Neuroprotective effect of punicalagin against cerebral ischemia reperfusion-induced oxidative brain injury in rats. Journal of Stroke and Cerebrovascular Diseases2328692878. (doi:10.1016/j.jstrokecerebrovasdis.2014.07.020)

    • Search Google Scholar
    • Export Citation