Activation of SGK1/ENaC Signaling Pathway Improves the Level of Decidualization in Unexplained Recurrent Spontaneous Abortion.

Recurrent spontaneous abortion (RSA) is one of the most common complications during pregnancy and seriously affects women's physical and mental health. About 50% of RSA cases are of unknown etiology. Our previous study found that the decidual tissue of patients with unexplained recurrent spontaneous abortion (URSA) had low expression levels of serum and glucocorticoid-induced protein kinase (SGK) 1. Endometrial decidualization is a key link in the early stage of pregnancy and is crucial to the development and maintenance of pregnancy. Decidualization is the proliferation and differentiation of endometrial stromal cells into deciduals, which involves a complex physiological process such as ovarian steroid hormones (estrogen, progesterone, prolactin, etc.), growth factors, and intercellular signaling. The binding of estrogen and its receptor stimulates the synthesis of endometrial deciduating markers prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP-1), which mediates the occurrence of decidualization. Among them, SGK1/ENaC is a signaling pathway closely related to decidualization. The purpose of this study was to further investigate the expression of SGK1 and decidualization-related molecules in the decidual tissue of URSA patients and to explore the potential mechanism of SGK1's protective effect in URSA patients and in mouse models. Decidual tissue samples from 30 URSA patients and 30 women who actively terminated pregnancy were collected, and a URSA mouse model was established and treated with dydrogesterone. Expression levels of SGK1 and its signaling pathway-related proteins (p-Nedd4-2, 14-3-3 protein and ENaC-a), estrogen and progesterone receptors (ERß, PR), and decidualization markers (PRLR, IGFBP-1) were assessed. Our study found that SGK1, p-Nedd4-2, 14-3-3 proteins, and ENaC-a expression levels were reduced in the decidual tissue, the SGK1/ENaC signaling pathway was inhibited, and the expression levels of the decidualization markers PRLR and IGFBP-1 were downregulated in the URSA group compared with the controls. Additionally, the concentrations of E2, P, and PRL in the serum of mice were decreased in the URSA group compared with the controls. However, SGK1/ENaC pathway-related proteins, estrogen and progesterone and their receptors, and decidualization-related molecules were upregulated by dydrogesterone. These data suggest that estrogen and progesterone can induce decidualization by activating the SGK1/ENaC signaling pathway; disruption of this pathway can lead to the development of URSA. Dydrogesterone can increase the expression level of SGK1 protein in decidual tissue.

Effects of multicycle gonadotropin-releasing hormone antagonist protocols on oxidative stress of follicular fluid and ovarian granulosa cells.

The effect of repeated multicycle gonadotropin-releasing hormone antagonist (GnRH-ant) protocols on oxidative stress (OS) in follicular fluid (FF) and ovarian granulosa cells (GCs) remains unclear. This study investigated the effects of repeated multicycle GnRH-ant protocols on OS markers of FF and ovarian GCs. A total of 145 patients were enrolled and divided into four groups: 1 cycle group (n = 42), 2 cycles group (n = 37), 3 cycles group (n = 45), and 4-5 cycles group (n = 21). The FF and ovarian GCs of the patients were collected on the day of last oocyte retrieval and the levels of 8-hydroxy-2-deoxyguanosine (8-OHdG), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were tested by ELISA. The results showed that the serum estradiol levels on hCG injection day in the 3 and 4-5 cycles were significantly (P < 0.05) lower than in the 1 and 2 cycles. The number of retrieved oocytes (12.1 ± 3.3 in cycle 1, 11.7 ± 3.1 in cycle 2, 10.4 ± 2.4 in cycle 3, and 9.4 ± 2.4 in cycles 4-5), embryos with two pronuclei (7.6 ± 3.0 in cycle 1, 7.0 ± 2.5 in cycle 2, 6.2 ± 2.6 in cycle 3, and 5.5 ± 2.1 in cycles 4-5), and the rates of high-quality embryos (52.2% in cycle 1, 47.9% in cycle 2, 38.6% in cycle 3, and 36.5% in cycles 4-5), implantation (35.4% in cycle 1, 32.4% in cycle 2, 23.8% in cycle 3, and 22.9% in cycles 4-5) and clinical pregnancy (50.0% in cycle 1, 43.2% in cycle 2, 33.3% in cycle 3, and 23.8% in cycles 4-5) in cycles 3 and 4-5 were significantly (P < 0.05) lower than those in cycles 1 and 2. Compared with 1 and 2 cycles, the 8-OHdG and SOD were significantly increased in the 3-5 cycles, while the CAT and GSH-Px levels were significantly decreased. Together, this study reveals repeated COS with the use of GnRH-ant protocols results in OS and changes the follicle microenvironment of FF and GCs, possibly leading to poor IVF outcomes in patients with 3-5 cycles of COS.

Systematic biological and proteomics strategies to explore the regulation mechanism of Shoutai Wan on recurrent spontaneous Abortion's biological network.

ETHNOPHARMACOLOGICAL RELEVANCE: Shoutai Wan (STW) is a classic herbal formula for the treatment of recurrent spontaneous abortion (RSA), and clinical studies have shown the effectiveness of STW on RSA. However, the molecular mechanism of STW treatment of RSA is still unclear. METHODS: (1) Animal experiments: The normal pregnancy model was established with CBA/J*BALB/C, and the RSA model was established by CBA/J*DBA/2. The RSA model CBA/J*DBA/2 pregnant mice were randomly divided into four groups (RSA model group, STW low, medium and high dose groups) according to the order of pregnancy, respectively. The drug administration starts from the first day of pregnancy to the 14th day of pregnancy. The embryo loss rate (ELR) of each group was calculated. (2) Proteomic analysis of decidual tissue: The total protein of decidual tissue of each group was isolated by solid phase pH gradient 2-DE technique. The differentially expressed protein spots were analyzed and identified by PDQuest images; the peptide quality fingerprinting (PMF) was obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Then, the proteins were identified by Mascot software searching, their functions were identified by bioinformatics strategy. (3) The expression of HSP27, α-enolase and Transferrin was detected by Western blotting and the expression of Annexin A2 and Transferrin was detected by immunohistochemistry. (4) The differential proteins and potential targets were analyzed by systematic biological strategy. RESULTS: (1) Compared with the normal group, the ELR in the RSA model group was significantly higher (P < 0.01). Compared with the model group, the ELR in the STW high, medium dose groups was lower (P < 0.01). (2) A 2-DE map of the decidual tissue of the RSA model group, normal pregnancy group, STW low, medium and high dose groups was established. Thirty proteins were identified. (3) The results of western blot showed that the expression of HSP27 and a-enolase in the RSA model group was higher than that in the normal group, and the expression of transferrin was lower (P < 0.01). Compared with the model group, the expression of HSP27 and a-enolase in STW high, medium dose groups was decreased (P < 0.01); Compared with the model group, the expression of Transferrin in the STW high dose group was increased (P < 0.01). (5) A lot of RSA treatment-related targets, biological processes and pathways were found after the systematic biological analysis. CONCLUSION: (1) STW may reduce the ELR of the RSA mice. (2) The results of proteomics suggest that RSA is a complex process involving multiple proteins. STW can regulate the expression of various proteins in the decidual tissue of RSA mice, suggesting that it can act on multiple targets. (3) The results of western blotting of HSP27, a-enolase, transferrin were consistent with the results of proteomic analysis. (4) STW may achieve therapeutic effects by interfering with the targets, biological processes and signaling pathways discovered in this study.

[Effects of Shoutai Wan on proteome in decidua tissues of recurrent abortion mice].

OBJECTIVE: To analysis the differential expression of decidua tissue proteins and effective mechanisms of recurrent abortion mice with Shoutai Wan, and explore the mechanism of Shoutai Wan in preventing miscarriage. METHOD: The abortion-prone CBA/J x DBA/2 matings were established as the model of recurrent abortion and the nonabortion-prone CBA/J x BALB/c matings were used as the model of normal pregnancy. The model of recurrent abortion CBA/J x DBA/2 of mice pregnant were randomly divided into four groups according to the sequence of pregnancy, including model group, Shoutai Wan low-dose group, Shoutai Wan middle-dose group and Shoutai Wan high-dose group. From the 1st day of pregnant, mice of normal group, model group, Shoutai Wan low-dose group (3 g x kg x d(-1)), Shoutai Wan middle-dose group (6 g x kg x d(-1)) and Shoutai Wan high-dose group (12 g x kg x d(-1)) are oral administration in different doses. On the 14th day of pregnancy, all mice are killed and the embryo loss rate (ELR) was counted. The expression of differential proteins of mice decidua tissues were separated by means of 2-DE and identified by MALDI-TOF-MS. The functions of identified proteins were further analysed according to bioinformatics resources. RESULT: Compared with model group, low-dose Shoutai Wan can not significantly improve the model of recurrent abortion in pregnant mice ELR; Shoutai Wan middle-dose and high-dose group of pregnant mice ELR were significantly decreased (P < 0.01). The results showed that the well-resolved, reproducible 2-DE patterns of mice decidua tissues of model group, normal group and Shoutai Wan low middle high-dose group were obtained. Through comparative proteome analysis of decidua tissues of all groups, 30 differential expression protein spots which maybe related to recurrent abortion and Shoutai Wan intervention were identified by MALDI-TOF-MS. These differential expression proteins mainly refer to invasion of the blastocyst, blood vessel remodeling and cell apoptosis. CONCLUSION: Shoutai Wan can decrease recurrent abortion mice ELR significantly, and play a role in preventing miscarriage. Recurrent abortion is a complicated process refer to diverse proteins participate. For several protein spots expression of decidua tissues in recurrent abortion mice was regulated by Shoutai Wan, it provides contribution to the effect characteristic of multitarget.

[Effect of qingxin kaiqiao formula and saponin on learning and memory abilities and expression of apoptosis signal transducers Abeta and betaAPP in AD rat brain].

OBJECTIVE: To study the effect of qingxin kaiqiao formula and saponin on the learning and memory ability and the expression of the apoptosis signal transducers Abeta and betaAPP in AD rat brain. METHOD: The comparative observation method was adopted for the animal test. Forty male SD rats were randomly divided into five groups, namely the normal group, the model group, the aricept group, the qingxin kaiqiao formula group and the saponin group, with eight rats in each group. Abeta(25-35) (10 g x L(-1)) was injected into their bilateral amygdala to establish the AD rat model. Since the next day, they were intragastrically administered with Aricept (1.67 mg x kg(-1)), Qingxin Kaiqiao decoction (12.67 mL x kg(-1)), saponin (6.30 mg x kg(-1)) and double distilled water filling for 2 weeks to observe their spatial memory ability in a Morris water maze and study the expression of Caspase-3, Abeta and betaAPP in brain tissues by immunohistochemistry. RESULT: Each traditional Chinese medicine groups showed significant improvement in the learning and memory ability of AD rats and notable differences (P < 0.05, P < 0.01) compared with the control group. The qingxin kaiqiao formula group and the saponin group showed a decrease in the expressions of Caspase-3, Abeta and betaAPP in cerebral cortex and hippocampus area, displaying notable differences (P < 0.01, P < 0.05) compared with the control group. CONCLUSION: qingxin kaiqiao formula and saponin can obviously improve the learning and memory ability of AD rats with by decreasing the expression of Caspase-3, Abeta and betaAPP in cortex and hippocampus.

[Effect of qingxin kaiqiao recipe saponin on the expressions of Bax, Bcl-2, Abeta, and betaAPP in the cortex and hippocampus of Alzheimer's disease rats].

OBJECTIVE: To study the effects of Qingxin Kaiqiao Recipe (QKR) saponin on the expressions of Bax, Bcl-2, beta-amyloid (Abeta), and beta-amyloid precursor protein (betaAPP) in the cortex and hippocampus of Alzheimer's disease (AD) rats. METHODS: Thirty-two SD rats of SPF grade were selected. Abeta 25 - 35 was injected into the bilateral amygdala to prepare the AD model. After modeling rats were randomly divided into the model group, the donepezil hydrochloride group (Donepezil Hydrochloride Tablet, 1.67 mg/kg), the QLR group (QLR Decoction, 12.67 mL/kg), and the saponin group (saponin, 6.30 mg/kg), 8 rats in each group. Another 8 rats were selected as the normal group. Rats in the normal group and the model group were given with equal volume of double distilled water by gastrogavage. The intervention was performed once daily for 2 successive weeks. The Morris water maze test was carried out by the end of medication. The escape latency and the platform crossing times were recorded during the 1 -5 days. The expressions of Bax, Bcl-2, Abeta, and betaAPP in the cortex and hippocampus were detected using immunohistochemical assay. RESULTS: Compared with the model group, the 3rd - 5th day escape latency were all shortened in each medication group. The expressions of Bax, Abeta, and betaAPP decreased in the cortex and hippocampus. The numbers of platform crossing increased. The expression of Bcl-2 in the cortex increased. The expression of Bcl-2 in the hippocampus increased in the donepezil hydrochloride group and the QLR group with statistical difference (P<0.01, P<0.05). Compared with the donepezil hydrochloride group, the expression of betaAPP increased in the cortex and hippocampus of the saponin group. The expression of Abeta in the cortex and hippocampus decreased, the expression of Bax in the hippocampus decreased, and the expression of Bcl-2 in the cortex increased in the QLR group. The escape latency was obviously postponed at day 3 -5, the platform crossing times decreased, the expression of Bcl-2 in the cortex and hippocampus decreased in the saponin group, showing statistical difference (P<0.05, P<0.01). CONCLUSION: QKR could significantly improve AD rats' learning, memory, and spatial capabilities, which might be achieved through decreasing the expressions of Bax, Abeta, and betaAPP in the cerebral cortex and hippocampus, and elevating the expression of Bcl-2.