Shucheng Zhang

Cyclooxygenase-2 Regulated by the Nuclear Factor-κB Pathway Plays an Important Role in Endometrial Breakdown in a Female Mouse Menstrual-like Model

The role of prostaglandins (PGs) in menstruation has long been proposed. Although evidence from studies on human and nonhuman primates supports the involvement of PGs in menstruation, whether PGs play an obligatory role in the process remains unclear. Although cyclooxygenase (COX) inhibitors have been used in the treatment of irregular uterine bleeding, the mechanism involved has not been elucidated. In this study, we used a recently established mouse menstrual-like model for investigating the role of COX in endometrial breakdown and its regulation. Administration of the nonspecific COX inhibitor indomethacin and the COX-2 selective inhibitor DuP-697 led to inhibition of the menstrual-like process. Furthermore, immunostaining analysis showed that the nuclear factor (NF)κB proteins P50, P65, and COX-2 colocalized in the outer decidual stroma at 12 to 16 hours after progesterone withdrawal. Chromatin immunoprecipitation analysis showed that NFκB binding to the Cox-2 promoter increased at 12 hours after progesterone withdrawal in vivo, and real-time PCR analysis showed that the NFκB inhibitors pyrrolidine dithiocarbamate and MG-132 inhibited Cox-2 mRNA expression in vivo and in vitro, respectively. Furthermore, COX-2 and NFκB inhibitors similarly reduced endometrial breakdown, suggesting that NFκB/COX-2-derived PGs play a critical role in this process. In addition, the CD45(+) leukocyte numbers were sharply reduced following indomethacin (COX-1 and COX-2 inhibitor), DuP-697 (COX-2 inhibitor), and pyrrolidine dithiocarbamate (NFκB inhibitor) treatment. Collectively, these data indicate that NFκB/COX-2-induced PGs regulate leukocyte influx, leading to endometrial breakdown.

Paeoniflorin improves regional cerebral blood flow and suppresses inflammatory factors in the hippocampus of rats with vascular dementia

ObjectiveTo explore the delayed neuroprotection induced by paeoniflorin (PF), the principal component of Paeoniae radix prescribed in Chinese medicine, and its underlying mechanisms in rats subjected to vascular dementia (VD).MethodsA rat model of VD was induced by bilateral common carotid arteries occlusion (BCCAO). Low-dose or high-dose PF (20 or 40 mg/kg once per day) was administrated for 28 days after VD. The behavioral analysis of rat was measured by water morris. Regional cerebral blood volume (rCBV), regional cerebral blood flflow (rCBF) and mean transit time (MTT) were measured in the bilateral hippocampus by perfusion-weighted imaging (PWI). The levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) were measured by commercially available enzyme-linked immunosorbent assay kits. Protein levels were evaluated by western blot analysis. mRNA levels were evaluated by real time-polymerase chain reaction. Western blotting was used to estimate p65 translocation.ResultsThe behavioral analysis showed that PF could decrease the escape latency time (P<0.05), and increase the residence time of the original platform quadrant and the across platform frequency in water maze in VD rats (P<0.05). Likewise, PF remarkably promoted the rCBV (P<0.05), rCBF and decreased per minute MTT (P<0.05) in hippocampus of VD rats. Furthermore, PF decreased the release of IL-1β, IL-6 and TNF-α as well as inhibited the mRNA expression of IL-1β, IL-6 and TNF-α in the hippocampus of VD rats (P<0.05 or P<0.01). PF also could decrease the protein expressions of inducible nitric oxide synthase and cyclooxygenase-2 in the hippocampus of VD rats (P<0.05 or P<0.01). In addition, PF signifificantly inhibited the nuclear factor κB (NF-κB) pathway in the hippocampus of VD rats.ConclusionsPF signifificantly attenuates cognitive impairment, improves hippocampus perfusion and inhibits inflflammatory response in VD rats. In addition, the anti-inflflammatory effects of PF might be due to inhibiting the NF-κB pathway. PF may be a potential clinical application in improving VD.

ROS are critical for endometrial breakdown via NF-κB-COX-2 signaling in a female mouse menstrual-like model.

Progesterone withdrawal triggers endometrial breakdown and shedding during menstruation. Menstruation results from inflammatory responses; however, the role of reactive oxygen species (ROS) in menstruation remains unclear. In this study, we explored the role of ROS in endometrial breakdown and shedding. We found that ROS levels were significantly increased before endometrial breakdown in a mouse menstrual-like model. Vaginal smear inspection, morphology of uterine horns, and endometrial histology examination showed that a broad range of ROS scavengers significantly inhibited endometrial breakdown in this model. Furthermore, Western blot and immunohistochemical analysis showed that the intracellular translocation of p50 and p65 from the cytoplasm into the nucleus was blocked by ROS scavengers and real-time PCR showed that cyclooxygenase-2 (COX-2) mRNA expression was decreased by ROS scavengers. Similar changes also occurred in human stromal cells in vitro. Furthermore, Western blotting and real-time PCR showed that one ROS, hydrogen peroxide (H2O2), promoted translocation of p50 and p65 from the cytoplasm to the nucleus and increased COX-2 mRNA expression along with progesterone maintenance. The nuclear factor κB inhibitor MG132 reduced the occurrence of these changes in human stromal cells in vitro. Viewed as a whole, our results provide evidence that certain ROS are important for endometrial breakdown and shedding in a mouse menstrual-like model and function at least partially via nuclear factor-κB/COX-2 signaling. Similar changes observed in human stromal cells could also implicate ROS as important mediators of human menstruation.

Expression of PRB, FKBP52 and HB-EGF Relating with Ultrasonic Evaluation of Endometrial Receptivity

Background To explore the molecular basis of the different ultrasonic patterns of the human endometrium, and the molecular marker basis of local injury. Methodology/Principal Findings The mRNA and protein expression of FKBP52, progesterone receptor A (PRA), progesterone receptor B (PRB), and HB-EGF were detected in different patterns of the endometrium by real-time RTPCR and immunohistochemistry. There were differences in the mRNA and protein expression of FKBP52, PRB, and HB-EGF in the triple line (Pattern A) and homogeneous (Pattern C) endometrium in the window of implantation. No difference was detected in PRA expression. After local injury, the mRNA expression of HB-EGF significantly increased. In contrast, there was no difference in the mRNA expression of FKBP52, PRB, or PRA. The protein expression of FKBP52, PRB, and HB-EGF increased after local injury. There was no difference in the PRA expression after local injury. Conclusions PRB, FKBP52, and HB-EGF may be the molecular basis for the classification of the ultrasonic patterns. HB-EGF may be the molecular basis of local injury. Ultrasonic evaluation on the day of ovulation can be effective in predicting the outcome of implantation.

Tanshinol protects hippocampus and attenuates vascular dementia development

Tanshinol (3-(3′,4′-dihydroxyphenyl)-(2R)-lactic acid, TSL) is widely used in traditional Chinese medicine for the treatment of cardiovascular and cerebrovascular diseases. Here, we assessed whether TSL protected hippocampus and attenuated vascular dementia (VD) development in rats. The behavioral analysis showed that TSL could decrease the distance and latency time, and increase the swim speed in water maze in rats subjected to VD. TSL remarkably increased acetylcholine level and decreased acetylcholinesterase activity in rats subjected to VD. Likewise, TSL remarkably decreased malondialdehyde and increased superoxide dismutase levels in rats subjected to VD. Furthermore, treatment with TSL reduced the level of dead neurons in dentate gyrus. In addition, TSL upregulated growth-associated protein 43 (GAP43) and vascular endothelial growth factor (VEGF) expression and downregulated phosphorylated Akt (p-AKt) and phosphorylated glycogen synthase kinase (p-GSK3β) expression in hippocampus in rats subjected to VD. These results suggest that TSL may be a potential compound in VD model.

Effect of daily milk supplementation on serum and umbilical cord blood folic acid concentrations in pregnant Han and Mongolian women and birth characteristics in China

Many studies have demonstrated the efficacy of folic acid (FA) supplementation in prevention of neural tube defects (NTDs), although the extent of NTDs varies among individuals of different races and ethnic origin. China is a multi-ethnic country with no standard practice for FA-fortified food. Milk is consumed by women, but little is known about the effects of milk on folate concentration in maternal blood and neonatal umbilical cord blood in Han and Mongolian women after stopping taking the supplement for a month and five month, respectively. The objective of this study was to determine whether only daily consumption of liquid milk can increase the blood folate concentration in pregnant women and whether there are differences in blood folate concentrations between Han and Mongolian women after cessation of FA supplementation. Of the 4052 women enrolled in the parallel group design study. Three thousand five hundred and twenty-six women had confirmed pregnancies and were randomized to receive liquid milk or not until delivery. Women who consumed the liquid milk had significantly increased serum folate concentrations at 16 and 32 weeks of gestation as well as cord blood at birth compared to control groups in both ethnic groups. Infants born to women drinking milk also had better the term birth weight and height, which may be related to the increased concentration of folate. In conclusion, daily consumption of milk can increase the serum folate concentration in pregnant Han and Mongolian women in China (differences in the efficacy of FA and milk supplementation) and may enhance birth outcomes.

Sperm Head Vacuoles—Light Microscopic and Ultrastructural Observations: A Case Report

Background: Sperm head vacuoles are easily detectable in human spermatozoa under the electron microscope. A sperm head vacuole is considered abnormal when it exceeds 20% of the head’s cross-sectional area. The authors report a rare case of primary spermatozoa deformity with 100% vacuolated head and evaluate the correlation between presence of head vacuoles/nucleus vacuoles and abnormal transformation of nucleoprotamine types, defects of nucleoprotamine, and gene disorders of chromatin/chromosome/spermatogenesis. Methods: A 43-year-old male patient with infertility came to the Reproduction Health Center, Hebei, China. Semen was examined in accordance with the WHO criteria, and the spermatozoa were counted. Two hundred spermatozoa were observed both under light microscope and the electronic microscope. Results: About 50% of the spermatozoa had head deformities. In the intact spermatozoa, the heads were 100% vacuolated. Under ultrastructural observation, abnormalities were observed and two major types of spermatozoa were detected. In the head of those incompletely mature spermatozoa, four kinds of the nucleus vacuoles were observed. Conclusion: Abnormal spermatozoa with head vacuoles account for the patient infertility.

Repeated Abortion Affects Subsequent Pregnancy Outcomes in BALB/c Mice

Aim In this study, we aimed to establish a mouse model of repeated medical termination of pregnancy in order to determine subsequent outcomes. Methods A model of mifepristone (RU 486)-induced medical abortion was established in BALB/c mice to facilitate the investigation of the impact of medical abortion on subsequent pregnancies, including litter sizes and newborn birth weights. Pregnant mice were sacrificed to examine midterm pregnancy status, investigate the frequency of fetal resorption, and measure placental function gene expression by real-time PCR and immunohistochemistry. Offspring liver mRNA was harvested for real-time PCR to determine gene expression and assess the effects of abortion on offspring development. Results Mice subjected to 2 previous medical abortions experienced spontaneous abortions in subsequent pregnancies. Medical abortion caused reduced reproductive capacity and affected placental dysfunction, with reduced expression of tissue factor (TF) and genes encoding proteins involved in metabolic functions relevant to pregnancy, such as 11β-hydroxysteroid dehydrogenase 1/2 (11β-HSD1/2) and glucocorticoid receptor (GR). Reduced expression was also observed for platelet endothelial cell adhesion molecule-1 (CD31) and vascular endothelial growth factor (VEGF). In offspring from subsequent pregnancies, genes involved in lipid metabolism, which may enhance key lipid transcription factors, such as PPARA and PPARG, as well as GR/11β-HSD1, were downregulated in the liver. In addition, the sperm motility of the F1 males reduced. Conclusion Repeated medical abortion impaired the reproductive function of female mice, significantly affecting the outcomes of subsequent pregnancies. The impact of repeated abortions on the offspring of subsequent pregnancies was also noteworthy and deserves further exploration. Thus, this model provides a useful means to study the mechanisms underlying the above phenomena, which will ultimately benefit the health of women and their children.

Vascular endothelial growth factor (VEGF) regulation by hypoxia inducible factor-1 alpha (HIF1A) starts and peaks during endometrial breakdown, not repair, in a mouse menstrual-like model

STUDY QUESTION How is vascular endothelial growth factor (VEGF) expression regulated by hypoxia inducible factor 1 alpha (HIF1A) during menstruation? SUMMARY ANSWER After progesterone (P4) withdrawal, HIF1A was activated and it directly up-regulated VEGF mRNA expression and this regulation was the highest during endometrium breakdown in the mouse menstrual-like model. WHAT IS KNOWN ALREADY VEGF, an important angiogenic factor, is known to be essential for endometrial repair, particularly in angiogenesis and re-epithelialization. However, its upstream regulation has not been fully clarified. HIF1 is the first transcription factor response to hypoxia and is closely associated with angiogenesis; it is also an upstream regulator of VEGF mRNA. STUDY DESIGN, SIZE, DURATION We investigated the changes in the expression of HIF1A and VEGF after P4 withdrawal and after HIF1A inhibition. The total number of mice used was 62. The treatment duration in the mouse menstrual-like model was 8 days. PARTICIPANTS/MATERIALS, SETTING, METHODS The mouse menstrual-like model and mouse and human decidual endometrial stromal cells were established to mimic menstruation. Protein and mRNA expressions of HIF1A and VEGF were investigated by immunohistochemistry, Western blot and quantitative PCR. The direct interaction between HIF1A and the Vegf promoter was also investigated by chromatin immunoprecipitation. HIF1A inhibition in vivo and in vitro was achieved by administration of an HIF1A inhibitor and by siRNA knockdown, respectively. MAIN RESULTS AND THE ROLE OF CHANCE HIF1A was translocated to the nucleus from 8 to 16 h after P4 withdrawal, while VEGF mRNA expression was the highest at 12 h. HIF1A directly bound to Vegf promoter during endometrial breakdown, which peaked at 12 h. HIF1A inhibition suppressed VEGF mRNA and protein expression in the mouse menstrual-like model and decidualized stromal cells. Inhibition of HIF1A also suppressed endometrial breakdown. LIMITATIONS, REASONS FOR CAUTION Although HIF1A regulation of VEGF mRNA was confirmed in the mouse menstrual-like model and decidual endometrium stromal cells, the functional regulation of VEGF protein was not further determined. WIDER IMPLICATIONS OF THE FINDINGS Here, we report that the functional regulation of VEGF was complicate in menstruation. We also report that HIF1A plays a key role in endometrial breakdown. STUDY FUNDING/COMPETING INTERESTS The National Nature Science Foundation of China (No. 30901608), the National Basic Research Program of China (2010CB530403) and the National Science and Technology Support Program (No. 2012BAI32B05). The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER This study is not a clinical trial.

Tanshinol suppresses inflammatory factors in a rat model of vascular dementia and protects LPS-treated neurons via the MST1-FOXO3 signaling pathway.

Neuroinflammation plays an important role in vascular dementia(VD). Our previous work showed that mammalian Ste20-like kinase 1 (MST1) and the gene for a downstream transcription factor, FOXO3, play major roles in lipopolysaccharide (LPS)-induced apoptosis in hippocampal neurons. The neurotoxic effects of LPS are derived from its ability to cause an inflammatory response. We also previously showed that Tanshinol (TSL) provides neuro-protection in a rat model of VD. The present study further explores the effects of TSL on the neuroinflammatory aspects of VD and investigates whether TSL affects the MST1-FOXO3signaling pathway. VD was induced in rats using transient bilateral coronary artery occlusion. Interleukin(IL)-1β, IL-6, and tumor necrosis factor (TNF)-α levels were measured using enzyme-linked immunoabsorbent assay kits. Cell apoptosis was assessed by Hoechst 33342 staining. Protein and mRNA levels were evaluated by western blotting and quantitative polymerase chain reaction, respectively. TSL improved working memory and significantly inhibited plasma and hippocampal protein levels of IL-1β, IL-6, and TNF-α in a rat model of VD. LPS induced apoptosis in hippocampal neurons and increasedMST1 and p-FOXO3 protein expression, whereas MST1 siRNA transfection almost completely reversed LPS-induced neuronal apoptosis, indicating that LPS-induced cytotoxicity in hippocampal neurons is associated with MST1. TSL protected against LPS-induced cell apoptosis and suppressed IL-1β, IL-6, and TNF-α mRNA and protein expression as well as MST1 and p-FOXO3 protein expression in neurons. The present study provided novel mechanisms by which TSL exerts its neuroprotective activity and indicates that TSL may be a potential neuro-protective agent in VD.