The interaction of ER stress and autophagy in trophoblasts: navigating pregnancy outcome.

The endoplasmic reticulum (ER) is a complex and dynamic organelle that initiates unfolded protein response (UPR) and endoplasmic reticulum stress (ER Stress) in response to the accumulation of unfolded or misfolded proteins within its lumen. Autophagy is a paramount intracellular degradation system that facilitates the transportation of proteins, cytoplasmic components, and organelles to lysosomes for degradation and recycling. Preeclampsia (PE) and intrauterine growth retardation (IUGR) are two common complications of pregnancy associated with abnormal trophoblast differentiation and placental dysfunctions and have a major impact on fetal development and maternal health. The intricate interplay between ER Stress, and autophagy and their impact on pregnancy outcomes, through mediating trophoblast differentiation and placental development, has been highlighted in various reports. Autophagy controls trophoblast regulation through a variety of gene expressions and signalling pathways while excessive ER Stress triggers downstream apoptotic signalling, culminating in trophoblast apoptosis. This comprehensive review delves into the intricacies of placental development and explores the underlying mechanisms of PE and IUGR. In addition, this review will elucidate the molecular mechanisms of ER Stress and autophagy, both individually and in their interplay, in mediating placental development and trophoblast differentiation, particularly highlighting their roles in PE and IUGR development. This research seeks to the interplay between ER Stress and impaired autophagy in the placental trophoderm, offering novel insights into their contribution to pregnancy complications.

Effects of the maternal gut microbiome and gut-placental axis on melatonin efficacy in alleviating cadmium-induced fetal growth restriction.

Cadmium (Cd) is a major environmental stressor that induces fetal growth restriction (FGR). Also, changes in gut microbiome diversity-which can be modulated positively by melatonin (Mel) have implications on fetal development and placental functions. Therefore, this study aimed to explore whether the role of Mel in counteracting the Cd-induced FGR by regulating placental barrier injury, endoplasmic reticulum stress (ERS) and mitophagy in pregnant mice is mediated-in part- via the gut microbiota modulations. Pregnant mice were intraperitoneally injected with CdCl2 (5 mg/kg) and Mel (5 mg/kg) once daily, respectively, at the same time from gestational day (GD) 8 to GD18, and then the maternal colon and placental tissues were collected for detection. To investigate the inner relationship between intestinal flora and the protection of Mel on FGR caused by Cd, gut microbiota transplantation (GMT) was carried out from GD0 to GD18 after the removal of intestinal microbiota by antibiotics. Results indicated that Mel relieved barrier injury, ERS and mitophagy in the placenta, and reversed the maternal gut microbiota dysbiosis. The GMT approach suggested a role of intestinal microbiota in placental barrier injury, ERS and mitophagy induced by Cd. Overall, the results highlighted that the intestinal microbiota and gut-placental axis play a central role in the protective effect of Mel against Cd-induced FGR.

Gut microbiota contributes to bisphenol A-induced maternal intestinal and placental apoptosis, oxidative stress, and fetal growth restriction in pregnant ewe model by regulating gut-placental axis.

BACKGROUND: Bisphenol A (BPA) is an environmental contaminant with endocrine-disrupting properties that induce fetal growth restriction (FGR). Previous studies on pregnant ewes revealed that BPA exposure causes placental apoptosis and oxidative stress (OS) and decreases placental efficiency, consequently leading to FGR. Nonetheless, the response of gut microbiota to BPA exposure and its role in aggravating BPA-mediated apoptosis, autophagy, mitochondrial dysfunction, endoplasmic reticulum stress (ERS), and OS of the maternal placenta and intestine are unclear in an ovine model of gestation. RESULTS: Two pregnant ewe groups (n = 8/group) were given either a subcutaneous (sc) injection of corn oil (CON group) or BPA (5 mg/kg/day) dissolved in corn oil (BPA group) once daily, from day 40 to day 110 of gestation. The maternal colonic digesta and the ileum and placental tissue samples were collected to measure the biomarkers of autophagy, apoptosis, mitochondrial dysfunction, ERS, and OS. To investigate the link between gut microbiota and the BPA-induced FGR in pregnant ewes, gut microbiota transplantation (GMT) was conducted in two pregnant mice groups (n = 10/group) from day 0 to day 18 of gestation after removing their intestinal microbiota by antibiotics. The results indicated that BPA aggravates apoptosis, ERS and autophagy, mitochondrial function injury of the placenta and ileum, and gut microbiota dysbiosis in pregnant ewes. GMT indicated that BPA-induced ERS, autophagy, and apoptosis in the ileum and placenta are attributed to gut microbiota dysbiosis resulting from BPA exposure. CONCLUSIONS: Our findings indicate the underlying role of gut microbiota dysbiosis and gut-placental axis behind the BPA-mediated maternal intestinal and placental apoptosis, OS, and FGR. The findings further provide novel insights into modulating the balance of gut microbiota through medication or probiotics, functioning via the gut-placental axis, to alleviate gut-derived placental impairment or FGR. Video Abstract.

Mechanisms underlying the role of endoplasmic reticulum stress in the placental injury and fetal growth restriction in an ovine gestation model.

BACKGROUND: Exposure to bisphenol A (BPA), an environmental pollutant known for its endocrine-disrupting properties, during gestation has been reported to increase the risk of fetal growth restriction (FGR) in an ovine model of pregnancy. We hypothesized that the FGR results from the BPA-induced insufficiency and barrier dysfunction of the placenta, oxidative stress, inflammatory responses, autophagy and endoplasmic reticulum stress (ERS). However, precise mechanisms underlying the BPA-induced placental dysfunction, and subsequently, FGR, as well as the potential involvement of placental ERS in these complications, remain to be investigated. METHODS: In vivo experiment, 16 twin-pregnant (from d 40 to 130 of gestation) Hu ewes were randomly distributed into two groups (8 ewes each). One group served as a control and received corn oil once a day, whereas the other group received BPA (5 mg/kg/d as a subcutaneous injection). In vitro study, ovine trophoblast cells (OTCs) were exposed to 4 treatments, 6 replicates each. The OTCs were treated with 400 µmol/L BPA, 400 µmol/L BPA + 0.5 µg/mL tunicamycin (Tm; ERS activator), 400 µmol/L BPA + 1 µmol/L 4-phenyl butyric acid (4-PBA; ERS antagonist) and DMEM/F12 complete medium (control), for 24 h. RESULTS: In vivo experiments, pregnant Hu ewes receiving the BPA from 40 to 130 days of pregnancy experienced a decrease in placental efficiency, progesterone (P4) level and fetal weight, and an increase in placental estrogen (E2) level, together with barrier dysfunctions, OS, inflammatory responses, autophagy and ERS in type A cotyledons. In vitro experiment, the OTCs exposed to BPA for 24 h showed an increase in the E2 level and related protein and gene expressions of autophagy, ERS, pro-apoptosis and inflammatory response, and a decrease in the P4 level and the related protein and gene expressions of antioxidant, anti-apoptosis and barrier function. Moreover, treating the OTCs with Tm aggravated BPA-induced dysfunction of barrier and endocrine (the increased E2 level and decreased P4 level), OS, inflammatory responses, autophagy, and ERS. However, treating the OTCs with 4-PBA reversed the counteracted effects of Tm mentioned above. CONCLUSIONS: In general, the results reveal that BPA exposure can cause ERS in the ovine placenta and OTCs, and ERS induction might aggravate BPA-induced dysfunction of the placental barrier and endocrine, OS, inflammatory responses, and autophagy. These data offer novel mechanistic insights into whether ERS is involved in BPA-mediated placental dysfunction and fetal development.

Autophagy attenuates placental apoptosis, oxidative stress and fetal growth restriction in pregnant ewes.

Bisphenol A (BPA)-induced oxidative stress (OS) and its potentially associated autophagy and apoptosis have not been studied previously in pregnant ewes. Accordingly, this study investigated the underlying mechanisms of BPA-induced autophagy and apoptosis in the placenta and primary trophoblasts of pregnant ewes exposed to BPA both in vivo and in vitro. In vivo experiment, pregnant Hu ewes (n = 8) were exposed to 5 mg/kg/d of BPA compared to control ewes (n = 8) receiving only corn oil from day 40 through day 110 of gestation. Exposure to BPA during gestation resulted in placental insufficiency, fetal growth restriction (FGR), autophagy, endoplasmic reticulum stress (ERS), mitochondrial dysfunction, OS, and apoptosis in type A placentomes. Regarding in vitro model, primary ovine trophoblasts were exposed to BPA, BPA plus chloroquine (CQ; an autophagy inhibitor) or BPA plus rapamycin (RAP; an autophagy activator) for 12 h. Data illustrated that exposure to BPA enhanced autophagy (ULK1, Beclin-1, LC3, Parkin, and PINK1), ERS (GRP78, CHOP10, ATF4, and ATF6) and apoptosis (Caspase 3, Bcl-2, Bax, P53) but decreased the antioxidant (CAT, Nrf2, HO-1, and NQO1)-related mRNA and protein expressions as well as impaired the mitochondrial function. Moreover, treatment with CQ exacerbated the BPA-mediated OS, mitochondrial dysfunction, apoptosis, and ERS. On the contrary, RAP treatment counteracted the BPA-induced trophoblast dysfunctions mentioned above. Overall, the findings illustrated that BPA exposure could contribute to autophagy in the ovine placenta and trophoblasts and that autophagy, in turn, could alleviate BPA-induced apoptosis, mitochondrial dysfunction, ERS, and OS. These results offer new mechanistic insights into the role of autophagy in mitigating BPA-induced placental dysfunctions and FGR.

Dietary rumen-protected L-arginine or N-carbamylglutamate enhances placental amino acid transport and suppresses angiogenesis and steroid anabolism in underfed pregnant ewes.

This study aimed to investigate the effects of dietary supplementation of underfed Hu ewes from d 35 to 110 of gestation with either rumen-protected L-arginine (RP-Arg) or N-carbamylglutamate (NCG) on placental amino acid (AA) transport, angiogenic gene expression, and steroid anabolism. On d 35 of gestation, 32 Hu ewes carrying twin fetuses were randomly divided into four treatment groups, each consisting of eight ewes, and were fed the following diets: A diet providing 100% of NRC's nutrient requirements for pregnant ewes (CON); A diet providing 50% of NRC's nutrient requirements for pregnant ewes (RES); RES diet plus 5 g/d NCG (RES + NCG); or RES diet plus 20 g/d RP-Arg (RES + ARG). On the d 110 of pregnancy, blood samples were taken from the mother, and samples were collected from type A cotyledons (COT; the fetal portions of the placenta). The levels of 17ß-estradiol and progesterone in the maternal serum and both the capillary area density (CAD) and capillary surface density (CSD) in type A COT were decreased in response to Arg or NCG supplementation when compared to the RES group. The concentrations of arginine, leucine, putrescine and spermidine in type A COT were higher (P < 0.05) in the RES + ARG or RES + NCG group than in the RES group. The mRNA expression levels of inducible nitric oxide synthase (iNOS) and solute carrier family 15, member 1 (SLC15A1) were increased (P < 0.05) while those of progesterone receptor (PGR) and fibroblast growth factor 2 (FGF2) were decreased in type A COT by supplementation with either NCG or RP-Arg compared to the RES group. The results suggest that providing underfed pregnant ewes from d 35 to 110 of gestation with a diet supplemented with NCG or RP-Arg improves placental AA transport, and reduces the expression of angiogenic growth factor genes and steroid anabolism, leading to better fetal development.

Dietary L-Arginine or N-Carbamylglutamate Alleviates Colonic Barrier Injury, Oxidative Stress, and Inflammation by Modulation of Intestinal Microbiota in Intrauterine Growth-Retarded Suckling Lambs.

Our previous studies have revealed that dietary N-carbamylglutamate (NCG) and L-arginine (Arg) supplementation improves redox status and suppresses apoptosis in the colon of suckling Hu lambs with intrauterine growth retardation (IUGR). However, no studies have reported the function of Arg or NCG in the colonic microbial communities, barrier function, and inflammation in IUGR-suckling lambs. This work aimed to further investigate how dietary Arg or NCG influences the microbiota, barrier function, and inflammation in the colon of IUGR lambs. Forty-eight newborn Hu lambs of 7 d old were assigned to four treatment groups (n = 12 per group; six male, six female) as follows: CON (normal birth weight, 4.25 ± 0.14 kg), IUGR (3.01 ± 0.12 kg), IUGR + Arg (2.99 ± 0.13 kg), and IUGR + NCG (3.03 ± 0.11 kg). A total of 1% Arg or 0.1% NCG was supplemented in a basal diet of milk replacer, respectively. Lambs were fed the milk replacer for 21 d until 28 d after birth. Compared to the non-supplemented IUGR lambs, the transepithelial electrical resistance (TER) was higher, while fluorescein isothiocyanate dextran 4 kDa (FD4) was lower in the colon of the NCG- or Arg-supplemented IUGR lambs (p < 0.05). The IUGR lambs exhibited higher (p < 0.05) colonic interleukin (IL)-6, IL-1ß, tumor necrosis factor (TNF)-α, reactive oxygen species (ROS), and malondialdehyde (MDA) levels than the CON lambs; the detrimental effects of IUGR on colonic proinflammatory cytokine concentrations and redox status were counteracted by dietary Arg or NCG supplementation. Both IUGR + Arg and IUGR + NCG lambs exhibited an elevated protein and mRNA expression of Occludin, Claudin-1, and zonula occludens-1 (ZO-1) compared to the IUGR lambs (p < 0.05). Additionally, the lipopolysaccharide (LPS) concentration was decreased while the levels of acetate, butyrate, and propionate were increased in IUGR + Arg and IUGR + NCG lambs compared to the IUGR lambs (p < 0.05). The relative abundance of Clostridium, Lactobacillus, and Streptococcus was lower in the colonic mucosa of the IUGR lambs than in the CON lambs (p < 0.05) but was restored upon the dietary supplementation of Arg or NCG to the IUGR lambs (p < 0.05). Both Arg and NCG can alleviate colonic barrier injury, oxidative stress (OS), and inflammation by the modulation of colonic microbiota in IUGR-suckling lambs. This work contributes to improving knowledge about the crosstalk among gut microbiota, immunity, OS, and barrier function and emphasizes the potential of Arg or NCG in health enhancement as feed additives in the early life nutrition of ruminants.

Dietary N-carbamylglutamate and L-arginine supplementation improves redox status and suppresses apoptosis in the colon of intrauterine growth-retarded suckling lambs.

Previous studies have revealed that dietary N-carbamylglutamate (NCG) or L-arginine (Arg) improves small intestinal integrity and immune function in suckling Hu lambs that have experienced intrauterine growth retardation (IUGR). Whether these nutrients alter redox status and apoptosis in the colon of IUGR lambs is still unknown. This study, therefore, aimed at investigating whether dietary supplementation of Arg or NCG alters colonic redox status, apoptosis and endoplasmic reticulum (ER) stress and the underlying mechanism of these alterations in IUGR suckling Hu lambs. Forty-eight 7-d old Hu lambs, including 12 with normal birth weight (4.25 ± 0.14 kg) and 36 with IUGR (3.01 ± 0.12 kg), were assigned to 4 treatment groups (n = 12 each; 6 males and 6 females) for 3 weeks. The treatment groups were control (CON), IUGR, IUGR + Arg and IUGR + NCG. Relative to IUGR lambs, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) content, as well as proliferation index, were higher (P < 0.05) whereas reactive oxygen species (ROS), malondialdehyde (MDA) levels and apoptotic cell numbers were lower (P < 0.05) in colonic tissue for both IUGR + Arg and NCG lambs. Both mRNA and protein levels of C/EBP homologous protein 10 (CHOP10), B-cell lymphoma/leukaemia 2 (Bcl-2) -associated X protein (Bax), apoptosis antigen 1 (Fas), activating transcription factor 6 (ATF6), caspase 3, and glucose-regulated protein 78 (GRP78) were lower (P < 0.05) while glutathione peroxidase 1 (GPx1), Bcl-2 and catalase (CAT) levels were higher (P < 0.05) in colonic tissue for IUGR + Arg and IUGR + NCG lambs compared with IUGR lambs. Based on our results, dietary NCG or Arg supplementation can improve colonic redox status and suppress apoptosis via death receptor-dependent, mitochondrial and ER stress pathways in IUGR suckling lambs.

Guggulsterone inhibits human cholangiocarcinoma Sk-ChA-1 and Mz-ChA-1 cell growth by inducing caspase-dependent apoptosis and downregulation of survivin and Bcl-2 expression.

Guggulsterone has recently been reported to demonstrate anti-tumor effects in a variety of cancers. The present study aims to investigate the biological roles and underlying mechanism of the action of guggulsterone in cholangiocarcinoma. The immortalized human cholangiocarcinoma Sk-ChA-1 and Mz-ChA-1 cell lines were treated with various concentrations of the trans isomer of guggulsterone, Z-guggulsterone. Cellular proliferation was determined using the XTT assay. The apoptotic status of cholangiocarcinoma cells was assessed by Hoechst 33258 staining, DNA fragmentation assay and flow cytometry. Specific caspase inhibitor was used to explore the role of caspase in guggulsterone-induced apoptosis. A colorimetric assay was performed to measure the alterations of the activities of caspase-3, -8 and -9. Western blot analysis was used to detect the protein expression of survivin, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein and cleaved poly (adenosine diphosphate-ribose) polymerase (PARP). As revealed by the present data, guggulsterone significantly inhibited the growth of the two human cholangiocarcinoma cell lines by inducing cellular apoptosis. In addition, guggulsterone-induced apoptosis of cholangiocarcinoma cells was demonstrated to be partially inhibited by the caspase inhibitors z-VAD-fmk, z-LEHD-fmk and z-IETD-fmk, accompanied by the activation of caspases-3, -8 and -9, accumulation of cleaved PARP and decreased expression of survivin and Bcl-2. In conclusion, the present study demonstrated that guggulsterone was able to suppress the proliferation of cholangiocarcinoma by inducing caspase-dependent apoptosis and downregulating survivin and Bcl-2.