Background: The chronic unpredictable mild stress (CUMS) model has long been considered the best model for exploring the pathophysiological mechanisms underlying depression. However, there are no widely recognised standards for strategies for modeling and for behavioral testing. The present study aimed to optimize the protocols for food deprivation and the sucrose preference test (SPT) for the CUMS model.Methods: We first evaluated the effects of different long periods of food deprivation on the body weight of Sprague Dawley (SD) rats by testing food deprivation for 24 hours (8:00‐8:00⁺), food deprivation for 12 hours during the daytime (8:00‐20:00) and food deprivation for 12 hours at night (20:00‐8:00⁺). Next, we established a SD rat CUMS model with 15 different stimulations, and used body weight measurement, SPT, forced swim test (FST), open field test (OFT) and Morris water maze (MWM) test to verify the success of the modeling. In the SPT, consumption of sucrose and pure water within 1 and 12 hours was measured.Results: Twelve hours of food deprivation during the daytime (8:00‐20:00) had no effect on body weight, while 12 hours of food deprivation at night (20:00‐8:00⁺) and 24 hours of food deprivation (8:00‐8:00⁺) significantly reduced the mean body weight of the SD rats. When SPT was used to verify the successful establishment of the CUMS rat model, sucrose consumption measured within 12 hours was less variable than that measured within 1 hour.Conclusions: Twelve hours of food deprivation in the daytime (8:00‐20:00) may be considered a mild stimulus for the establishment of a CUMS rat model. Measuring sucrose consumption over 12 hours is recommended for SPT.

Human trophoblast progenitor cells differentiate via two distinct pathways, to become the highly invasive extravillous cytotrophoblast (CTB) cells (EVT) or fuse to form syncytiotrophoblast. Inadequate trophoblast differentiation results in poor placenta perfusion, or even complications such as pre-eclampsia (PE). Cullin1 (CUL1), a scaffold protein in cullin-based ubiquitin ligases, plays an important role in early embryonic development. However, the role of CUL1 in trophoblast differentiation during placenta development has not been examined. Here we show that CUL1 was expressed in CTB cells and EVT in the first trimester human placentas by immunohistochemistry. CUL1 siRNA significantly inhibited outgrowth of extravillous explants in vitro, as well as invasion and migration of HTR8/SVneo cells of EVT origin. This inhibition was accompanied by decreased gelatinolytic activities of matrix metalloproteinase (MMP)-9 and increased expression of tissue inhibitors of MMPs (TIMP-1 and -2). Consistently, exogenous CUL1 promoted invasion and migration of HTR8/SVneo cells. Notably, CUL1 was gradually decreased during trophoblast syncytialization and CUL1 siRNA significantly enhanced forskolin-induced fusion of choriocarcinoma BeWo cells. CUL1 protein levels in human pre-eclamptic placental villi were significantly lower as compared to their matched control placentas. Taken together, our results suggest that CUL1 promotes human trophoblast cell invasion and dysregulation of CUL1 expression may be associated with PE.

Gamma-aminobutyric acid (GABA) functions primarily as an inhibitory neurotransmitter through its receptors in the mature central nervous system. The GABA type A receptor π subunit (GABRP) has been identified in the tissues of the reproductive system, particularly in the uterus. In addition, we have previously detected GABRP expression in both human and mouse placentas. To examine the role of GABRP in trophoblastic cell invasion, we constructed a pIRES2-GABRP-EGFP plasmid which was used for the transfection of a human placental cell line derived from first trimester extravillous trophoblasts (HTR-8/SVneo). The number of invaded cells was decreased by GABRP overexpression. Notably, the decrease in the invasive cell number may be due to the increased apoptosis of the HTR-8/SVneo cells following GABRP transfection, which was further confirmed by flow cytometry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Based on the increased apoptosis of trophoblastic cells in pregnancies complicated by preeclampsia (PE) and the fact that GABRP promotes the apoptosis of trophoblastic cells, we hypothesized that GABRP expression is increased in the placental tissues from patients with PE compared with that in the normal groups and this hypothesis was confirmed by RT-qPCR and immunohistochemical analysis. Taken together, these findings imply that GABRP plays an important role in placentation and this pathway may be a promising molecular target for the development of novel therapeutic strategies for PE.

Normal implantation depends on appropriate trophoblast growth and invasion. Inadequate trophoblast invasion results in pregnancy-related disorders, such as early miscarriage and pre-eclampsia, which are dangerous to both the mother and fetus. Msh Homeobox 2 (MSX2), a member of the MSX family of homeobox proteins, plays a significant role in the proliferation and differentiation of various cells and tissues, including ectodermal organs, teeth, and chondrocytes. Recently, MSX2 was found to play important roles in the invasion of cancer cells into adjacent tissues via the epithelial-mesenchymal transition (EMT). However, the role of MSX2 in trophoblastic invasion during placental development has yet to be explored. In the present study, we detected MSX2 expression in cytotrophoblast, syncytiotrophoblast, and extravillous cytotrophoblast cells of first or third trimester human placentas via immunohistochemistry analysis. Furthermore, we found that the in vitro invasive ability of HTR8/SVneo cells was enhanced by exogenous overexpression of MSX2, and that this effect was accompanied by increased protein expression of matrix metalloproteinase-2 (MMP-2), vimentin, and β-catenin. Conversely, treatment of HTR8/SVneo cells with MSX2-specific siRNAs resulted in decreased protein expression of MMP-2, vimentin, and β-catenin, and reduced invasion levels in a Matrigel invasion test. Notably, however, treatment with the MSX2 overexpression plasmid and the MSX2 siRNAs had no effect on the mRNA expression levels of β-catenin. Meanwhile, overexpression of MSX2 and treatment with the MSX2-specific siRNA resulted in decreased and increased E-cadherin expression, respectively, in JEG-3 cells. Lastly, the protein expression levels of MSX2 were significantly lower in human pre-eclamptic placental villi than in the matched control placentas. Collectively, our results suggest that MSX2 may induce human trophoblast cell invasion, and dysregulation of MSX2 expression may be associated with pre-eclampsia.

Well-controlled trophoblast invasion at maternal-fetal interface is a critical event for the normal development of placenta. CD82 is a member of transmembrane 4 superfamily, which showed important role in inhibiting tumor cell invasion and migration. We surmised that CD82 are participates in trophoblast differentiation during placenta development. CD82 was found to be strongly expressed in human first trimester placental villous and extravillous trophoblast cells as well as in trophoblast cell lines. To investigate whether CD82 plays a role in trophoblast invasion and migration, we further utilized human villous explants culture model on matrigel and invasion/migration assay of trophoblast cell line HTR8/SVneo. CD82 siRNA significantly promoted outgrowth of villous explants in vitro (P<0.01), as well as invasion and migration of HTR8/SVneo cells (P<0.05), whereas the trophoblast proliferation was not affected. The enhanced effect of CD82 siRNA on invasion and migration of trophoblast cells was found associated with increased gelatinolytic activities of matrix metalloproteinase MMP9 while over-expression of CD82 markedly decreased trphoblast cell invasion and migration as well as MMP9 activities. These findings suggest that CD82 is an important negative regulator at maternal-fetal interface during early pregnancy, inhibiting human trophoblast invasion and migration.

Photodynamic therapy has been confirmed to an efficient therapeutic modality of malignant tumors. The aim of the present study was to explore the photodynamic action of LED-activated nanoscale photosensitizer-loading hypocrellin in nasopharyngeal carcinoma cells. Nasopharyngeal carcinoma cell line CNE2 cells were subjected to photodynamic therapy with hypocrellinloaded nanophotosensitizer. The uptake of the nanophotosensitizer in the CNE-2 cells was measured using a spectrophotometer and photodynamic toxicity was investigated 18 h after LED radiation treatment. Apoptosis was determined using flow cytometry with propidum iodine staining, and nuclear staining with Hoechst 33258. Active caspase-3 in the CNE2 cells was evaluated using flow cytometry with phycoerythrin (PE)-conjugated anti-active caspase-3 antibodies. The cellular uptake of the nanophotosensitizer in the CNE-2 cells reached optimal at 6 h. LED-activated nanophotosensitizer resulted in doseand light-dependent phototoxicity. Apoptotic rate 18 h after PDT increased to 34.32 ± 1.94% under the light energy of 1 J/cm². Hoechest 33258 staining reinforced the findings above. Condensation of chromatin and nuclear fragmentations was found in many PDT-treated cells. The activated caspase-3 in the CNE2 cells significantly increased up to 43.90% when the CNE2 cells were exposed to the nanophotosensitizer for 6 h and then 1 J/cm² irradiation. LED-activated nanophotosensitizer significantly killed the CNE2 cells and enhanced apoptosis and activated caspase-3 in the CNE2 cells. The hypocrellin-loaded nanophotosensitizer might be efficient photosensitizer and LED-activated nanophotosensitizer can be developed for treating nasopharyngeal carcinoma.