Electroacupuncture improves endometrial receptivity through miRNA-223-3p-mediated regulation of leukemia inhibitory factor / signal transducer and activator of transcription 3 signaling pathway.

Ovulation-inducing drugs such as endogenous steroids could reduce endometrial receptivity during the implantation window, resulting in lower clinical pregnancy rates and higher miscarriage rates. The present study employed electroacupuncture therapy along with different frequencies on elevating impaired endometrial receptivity to elucidate the mechanism therein. The rats were split up into seven groups of normal, model, low-frequency electroacupuncture (LF-EA), high-frequency electroacupuncture (HF-EA), LF-EA+anti-miRNA, HF-EA+anti-miRNA, and anti-miRNA. PCR assays were used to detect miR-223-3p expressions. The effects of electroacupuncture and miR-223-3p on rats' endometrial membrane cell-drinking process in a manner of scanning electron microscopy were recorded. After that we observed on the electroacupuncture effects on the conditions of adhesion molecules and LIF/STAT3 signaling pathway with assays of immunofluorescence and Western Blot. This study was end up with dual luciferase assay, where combination of miR-233-3p onto the 3'-UTR sequence of LIF was determined. PCR assay demonstrated that HF-EA procured an inhibition in miR-223-3p expression, whereas scanning electron microscopy turned out that both electroacupuncture and miR-223-3p were capable of raising the amount of intrauterine pinocytosis and the number of blastocyst implantation in rats. Additionally, assays of Western Blot and immunofluorescence showed that therapy of electroacupuncture brought about decreasing expressions in adhesion molecules of E-cadherin, ß-catenin and claudin-1 (CLDN1). We found that both electroacupuncture and miR-223-3p were able to fortify LIF/STAT3 signaling pathway, then the fact of miR-223-3p combination to LIF 3'-UTR sequence was validated via our dual-luciferase assay. Electroacupuncture therapy inhibited the miR-223-3p expression upon LIF/STAT3 signaling pathway to elevate endometrial receptivity.

Diets enriched in PUFAs at an early postimplantation stage prevent embryo resorptions and impaired mTOR signaling in the decidua from diabetic rats.

Maternal diabetes increases the risk of embryo resorptions and impairs embryo development. Decidualization is crucial for embryo development and regulated by mTOR signaling. However, little is known about how maternal diabetes affects the decidua at early postimplantation stages and whether dietary treatments enriched in polyunsaturated fatty acids (PUFAs) can prevent decidual alterations. Here, we determined resorption rates, decidual mTOR pathways and markers of decidual function and remodeling in diabetic rats fed or not with diets enriched in PUFAs exclusively during the early postimplantation period. Pregestational streptozotocin-induced diabetic Albino Wistar rats and controls were fed or not with diets enriched in 6% sunflower oil or 6% chia oil (enriched in n-6 or n-3 PUFAs, respectively) on days 7, 8 and 9 of pregnancy and evaluated on day 9 of pregnancy. Maternal diabetes induced an 11-fold increase in embryo resorptions, which was prevented by both PUFAs-enriched diets despite no changes in maternal glycemia. The activity of mTOR pathway was decreased in the decidua from diabetic rats, an alteration prevented by the PUFAs-enriched diets. PUFAs-enriched diets prevented increased expression of Foxo1 (a negative regulator of mTOR) and reduced expression of miR-21 (a negative regulator of Foxo1). These diets also prevented reduced markers of decidual function (leukemia inhibitory factor and IGFBP1 expression and MMPs activity) in diabetic rat decidua. We identified the early post implantation as a crucial stage for pregnancy success, in which dietary PUFAs can protect diabetic pregnancies from embryo resorptions, decidual mTOR signaling impairments, and altered markers of decidual function and remodeling.

Impact of Kuntai Capsules on LIF, IGF-1 and EGF expression in the implantation window of endometrium in mice / Impacto de cápsulas de Kuntai en la expresión de LIF, IGF-1 y EGF en la ventana de implantación de endometrio en ratones

To investigate the influence of Kuntai capsules on the expression level of leukemia inhibitory factor (LIF), insulin-like growth factor-I (IGF-1)and epidermal growth factor (EGF) during the mouse's implantation window of superovulation period and controlled ovarian hyperstimulation period. 90 female mice were randomly divided into six groups in control, superovulation and controlled ovarian hyperstimulation (COH) conditions. The RNA expression of EGF, LIF and IGF-1 in the endometrium on the 4th day of pregnancy was detected, and the relative expression was compared. mRNA expression of these three factors in endometrium was significantly lower in superovulation and COH groups than control group (p<0.001). mRNA expression of these three factors in endometrium remained obviously lower in superovulation plus kuntai capsule group and COH plus kuntai capsule group than control group (p<0.01). mRNA expression of these three factors in endometrium was lower in control group than in the NS plus kuntai capsule group (p<0.05). Kuntai capsule cannot completely reverse the endometrial damages caused by superovulation and COH. Thus Kuntai capsule could partially improve a mouse's endometrial receptivity during the implantation window.

Para investigar la influencia de las cápsulas de Kuntai en el nivel de expresión del factor inhibidor de la leucemia (LIF), el factor de crecimiento similar a la insulina I (IGF-1) y el factor de crecimiento epidérmico (EGF) durante la ventana de implantación del ratón del período de superovulación y la hiperestimulación ovárica controlada período, se dividieron aleatoriamente 90 ratones hembra en seis grupos en condiciones de control, superovulación e hiperestimulación ovárica controlada (COH). Se detectó la expresión de ARN de EGF, LIF e IGF-1en el endometrio al cuarto día de embarazo, y se comparó la expresión relativa. La expresión de ARNm de estos tres factores en el endometrio fue significativamente menor en los grupos de superovulación y COH que en el grupo control (p<0,001). La expresión de ARNm de estos tres factores en el endometrio permaneció más baja en el grupo de cápsulas de superovulación más Kuntai y en el grupo de cápsulas de COH más Kuntai respecto del grupo control (p<0,01). La expresión de ARNm de estos tres factores en el endometrio fue menor en el grupo control que en el grupo de cápsula NS más Kuntai (p<0,05). La cápsula de Kuntai no pudo revertir completamente los daños endometriales causados por la superovulación y la COH. Por lo tanto, se sugiere que la cápsula de Kuntai podría mejorar parcialmente la receptividad endometrial de un ratón durante la ventana de implantación.

Buyang Huanwu Decoction () Attenuates Glial Scar by Downregulating the Expression of Leukemia Inhibitory Factor in Intracerebral Hemorrhagic Rats.

OBJECTIVE: To evaluate the effect of Buyang Huanwu Decoction (, BYHWD) on glial scar after intracerebral hemorrhage (ICH) and investigate the underlying mechanism. METHODS: Collagenase type VII (0.5 U) was injected stereotaxically into right globus pallidus to induce ICH model. One hundred and twenty Sprague-Dawley rats were randomly divided into 3 groups according to a random number table, including normal group (n=40), ICH model group (n=40) and BYHWD group (n=40), respectively. After ICH, the rats in the BYHWD group were intragastrically administered with BYHWD (4.36 g/kg) once a day for 21 days, while the rats in ICH group were administered with equal volume of distilled water for 21 days, respectively. Double immunolabeling was performed for proliferating cell nuclear antigen (PCNA)+/glial fibrillary acidic protein (GFAP)+ nuclei. The expression of GFAP and leukemia inhibitory factor (LIF) was evaluated by immunohistochemistry and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The astrocytes with hypertrophied morphology around the hematoma was observed on day 3 after ICH. The number of GFAP positive cells and GFAP mRNA levels increased notably on day 3 and reached the peak on day 14 post-ICH (P<0.01). PCNA+/GFAP+ nuclei were observed around the hematoma and reached the peak on day 14 post-ICH (P<0.01). In addition, LIF-positive astrocytes and LIF mRNA level in the hemorrhagic region increased significantly till day 14 post-ICH (P<0.01). However, BYHWD not only reduced the number of PCNA+/GFAP+ nuclei, but also decreased GFAP and LIF levels (P<0.05). CONCLUSIONS: BYHWD could attenuate ICH-induced glial scar by downregulating the expression of LIF in the rats.

Water-extracted tubers of Cyperus rotundus L. enhance endometrial receptivity through leukemia inhibitory factor-mediated expression of integrin αVß3 and αVß5.

ETHNOPHARMACOLOGICAL RELEVANCE: Cyperus rotundus L. (CR) has been traditionally used as an herbal medicine in Asian countries to treat diverse gynecological disorders. However, the potential therapeutic effect of CR on endometrial receptivity for successful embryo implantation to treat female infertility has not been fully studied. AIM OF STUDY: The aim of this study was to evaluate the effect of water-extracted CR on endometrial receptivity by investigating the expression of leukemia inhibitory factor (LIF) and integrins, cell adhesion, and embryo implantation using mifepristone (RU486; RU)-induced implantation failure model. MATERIALS AND METHODS: The water extract of CR was prepared and fingerprinted using high-performance liquid chromatography (HPLC). For the expression and regulation of LIF, reverse transcription polymerase chain reaction (RT-PCR) and western blotting were performed in CR-stimulated Ishikawa cells. To evaluate LIF-mediated integrin expression, knockdown of LIF by shRNA was performed in Ishikawa cells. The effect of CR on endometrial receptivity was determined by an in vitro adhesion assay between JAr cells and CR-induced Ishikawa cells. In vivo, C57BL/6 female mice (n = 7 per group) orally received CR (31.68mg/kg/day), a similar dose as used clinically. Seven days after CR treatment, all female mice were caged with male mice until pregnancy was verified. On day 4 of pregnancy, RU (4mg/kg) was injected subcutaneously to induce embryo implantation failure. RESULT: CR increased the expression of LIF through the phosphatidylinositol-3-kinase/ protein kinase B (PI-3K/AKT) signaling pathway in Ishikawa cells. In addition, CR enhanced adhesion of JAr cells onto Ishikawa cells by inducing the expression of LIF-dependent integrins αVß3 and αVß5. Furthermore, CR improved the number of implantation sites in pregnant mice despite RU injection. CONCLUSION: CR increased the expression of LIF-mediated integrins αVß3 and αVß5 on the surface of endometrial cells, which is associated with adhesion of trophoblastic cells to endometrial cells for blastocyst implantation. Our findings provide evidence that CR has therapeutic potential against poor endometrial receptivity.

Paeonia lactiflora Enhances the Adhesion of Trophoblast to the Endometrium via Induction of Leukemia Inhibitory Factor Expression.

In the present study, we investigated the role of Paeonia lactiflora Pall. extract on embryo implantation in vitro and in vivo. A polysaccharides depleted-water extract of P. lactiflora (PL-PP) increased LIF expression in human endometrial Ishikawa cells at non-cytotoxic doses. PL-PP significantly increased the adhesion of the human trophectoderm-derived JAr spheroids to endometrial Ishikawa cells. PL-PP-induced LIF expression was decreased in the presence of a p38 kinase inhibitor SB203580 and an MEK/ERK inhibitor U0126. Furthermore, endometrial LIF knockdown by shRNA reduced the expression of integrins ß3 and ß5 and adhesion of JAr spheroids to Ishikawa cells. In vivo administration of PL-PP restored the implantation of mouse blastocysts in a mifepristone-induced implantation failure mice model. Our results demonstrate that PL-PP increases LIF expression via the p38 and MEK/ERK pathways and favors trophoblast adhesion to endometrial cells.

New cancer cachexia rat model generated by implantation of a peritoneal dissemination-derived human stomach cancer cell line.

Cancer cachexia (CC), a syndrome characterized by anorexia and body weight loss due to low fat-free mass levels, including reduced musculature, markedly worsens patient quality of life. Although stomach cancer patients have the highest incidence of cachexia, few experimental models for the study of stomach CC have been established. Herein, we developed stomach CC animal models using nude rats subcutaneously implanted with two novel cell lines, i.e., MKN45c185, established from the human stomach cancer cell line MKN-45, and 85As2, derived from peritoneal dissemination of orthotopically implanted MKN45c185 cells in mice. Both CC models showed marked weight loss, anorexia, reduced musculature and muscle strength, increased inflammatory markers, and low plasma albumin levels; however, CC developed earlier and was more severe in rats implanted with 85As2 than in those implanted with MKN45cl85. Moreover, human leukemia inhibitory factor (LIF), a known cachectic factor, and hypothalamic orexigenic peptide mRNA levels increased in the models, whereas hypothalamic anorexigenic peptide mRNA levels decreased. Surgical removal of the tumor not only abolished cachexia symptoms but also reduced plasma LIF levels to below detectable limits. Importantly, oral administration of rikkunshito, a traditional Japanese medicine, substantially ameliorated CC-related anorexia and body composition changes. In summary, our novel peritoneal dissemination-derived 85As2 rat model developed severe cachexia, possibly caused by LIF from cancer cells, that was ameliorated by rikkunshito. This model should provide a useful tool for further study into the mechanisms and treatment of stomach CC.

Progesterone is essential for protecting against LPS-induced pregnancy loss. LIF as a potential mediator of the anti-inflammatory effect of progesterone.

Lipopolysaccharide (LPS) administration to mice on day 7 of gestation led to 100% embryonic resorption after 24 h. In this model, nitric oxide is fundamental for the resorption process. Progesterone may be responsible, at least in part, for a Th2 switch in the feto-maternal interface, inducing active immune tolerance against fetal antigens. Th2 cells promote the development of T cells, producing leukemia inhibitory factor (LIF), which seems to be important due to its immunomodulatory action during early pregnancy. Our aim was to evaluate the involvement of progesterone in the mechanism of LPS-induced embryonic resorption, and whether LIF can mediate hormonal action. Using in vivo and in vitro models, we provide evidence that circulating progesterone is an important component of the process by which infection causes embryonic resorption in mice. Also, LIF seems to be a mediator of the progesterone effect under inflammatory conditions. We found that serum progesterone fell to very low levels after 24 h of LPS exposure. Moreover, progesterone supplementation prevented embryonic resorption and LPS-induced increase of uterine nitric oxide levels in vivo. Results show that LPS diminished the expression of the nuclear progesterone receptor in the uterus after 6 and 12 h of treatment. We investigated the expression of LIF in uterine tissue from pregnant mice and found that progesterone up-regulates LIF mRNA expression in vitro. We observed that LIF was able to modulate the levels of nitric oxide induced by LPS in vitro, suggesting that it could be a potential mediator of the inflammatory action of progesterone. Our observations support the view that progesterone plays a critical role in a successful pregnancy as an anti-inflammatory agent, and that it could have possible therapeutic applications in the prevention of early reproductive failure associated with inflammatory disorders.

Recombinant rabbit leukemia inhibitory factor and rabbit embryonic fibroblasts support the derivation and maintenance of rabbit embryonic stem cells.

The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs.

Reprogramming pig fetal fibroblasts reveals a functional LIF signaling pathway.

Distinct signaling pathways are reported to maintain pluripotency in embryo-derived stem cells. Mouse embryonic stem cells (ESCs) respond to leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP)-mediated activity, whereas human ESCs depend upon Fibroblast growth factor (FGF) and activin signaling. In the majority of mammals investigated, however, the signals that support stem cell pluripotency are not well defined, as is evident by the persistent difficulties in maintaining authentic stable ESC lines. Induction of pluripotency by transcription factor-mediated reprogramming could provide an alternative way to produce ESC-like cells from nonpermissive species, and facilitate identification of core ESC signaling requirements. To evaluate the effectiveness of this approach in pigs, we transduced porcine foetal fibroblasts with retroviruses expressing Oct4, Sox2, Klf4, and c-Myc, and maintained the resulting cultures in medium containing either LIF or FGF2. Alkaline phosphatase positive colonies with compact, mouse ESC-like morphology were preferentially recovered using serum-free medium supplemented with LIF. These cell lines expressed the endogenous stem cell transcription factors, OCT4, NANOG, and SOX2, and the cell surface marker SSEA-4, consistent with acquisition of an undifferentiated state. However, restricted differentiation potential, and persistent expression of retroviral transgenes indicated that reprogramming was incomplete. Interestingly, LIF activated both the transcription factor STAT3 and its target gene SOCS3, and stimulated cell growth, indicating functional coupling of the signaling pathway in these cells. This demonstration of LIF-dependence in reprogrammed pig cells supports the notion that the connection between LIF/STAT3 signaling and the core regulatory network of pluripotent stem cells is a conserved pathway in mammals.

Effects of acupuncture on LIF and IL-12 in rats of implantation failure.

PROBLEM: Acupuncture has a positive effect on implantation obstacle, but the mechanism is still not clear, so the aim of the experiment is to explore the possible role that acupuncture plays in implantation. METHOD OF STUDY: Early pregnant rats were randomized into normal group (N), group treated with mifepristone (M), acupuncture treatment group (A), and progestin treatment group (P). The model of blastocyst implantation obstacle in groups M, A, and P was established with mifepristone. Bilateral 'Housanli' and 'Sanyinjiao' were needled in group A. The expression of interleukin (IL)-12, leukemia inhibitory factor (LIF), LIFR protein, and mRNA in endometrium were detected. RESULTS: Positivity of the protein expression of IL-12, LIF, and LIFR in the endometrium was significantly higher in groups N, A, and P; positivity of the mRNA of IL-12 and LIF in the endometrium was significantly higher in groups N, A, and P. CONCLUSION: Acupuncture could improve the poor receptive state of endometrium by promoting LIF and IL-12 secretion to improve blastocyst implantation.

Extracts from a traditional Chinese herbal remedy (Zhuyun recipe) improve endometrial receptivity in mice with embryonic implantation dysfunction and ovulation stimulation.

OBJECTIVE: Although ovarian stimulation has an important role in assisted reproductive technologies (ART), it may also have detrimental effects on endometrial receptivity. Traditional Chinese herbal remedy, as a kind of traditional treatments, has been widely and increasingly applied in clinic. In this article, the impact of traditional Chinese medicines (TCM) on embryonic implantation, pregnant rate and underlying mechanisms will be investigated. METHODS: One hundred and sixty-three female pregnant kunming mice were randomly divided into 6 groups, including A, control group; B, ovulation stimulation (OS) group; C, OS+TCM group; D, embryo implantation dysfunction (EID) group; E, EID+TCM group; F, TCM only group. Uterus samples were collected at gestation Day 4 and were detected with immunohistochemistry and Real Time-PCR analyses. Uterine horns were excised to determine the number of pregnant mice and implantation sites on the Day 8 postcoitum. RESULTS: OS group and EID group showed a significant decrease in pregnant rate and the expression of both the endometrial leukaemia inhibitory factor (LIF) and integrin ß3 subunit during the implantation window. OS+TCM group and EID+TCM group showed a higher pregnant rate and endometrial LIF and integrin ß3 subunit expression compared to OS group and EID group. The number of implanted embryo in EID group was lower than in control group, but higher in EID+TCM group than in EID group. No significant difference was found in the measured indices between the TCM only group and control group. CONCLUSIONS: OS model and EID model may have a negative influence on endometrial receptivity and embryonic implantation in mice. Conversely, TCM appears to reverse the expression of endometrial LIF and integrin ß3 subunit, improves the uterine receptivity in mice and increases pregnant rate and embryonic implantation. It provides a new insight into the clinic infertility's treatment.

Leukemia inhibitory factor promotes olfactory sensory neuronal survival via phosphoinositide 3-kinase pathway activation and Bcl-2.

Leukemia inhibitory factor (LIF), a neuropoietic cytokine, has been implicated in the control of neuronal development. We previously reported that LIF plays a critical role in regulating the terminal differentiation of olfactory sensory neurons (OSNs). Here, we demonstrate that LIF plays a complementary role in supporting the survival of immature OSNs. Mature OSNs express LIF, which may be elaborated in a paracrine manner to influence adjacent neurons. LIF null mice display more apoptotic immature neurons than do their wild-type littermates. LIF treatment of dissociated OSNs in vitro significantly reduces the apoptosis of immature OSNs. Double immunocytochemical analysis indicates that the survival of immature OSNs is dependent on the presence of LIF. LIF activates the phosphoinositide 3-kinase (PI3K) pathways and induces the expression of the antiapoptotic molecule Bcl-2 in OSNs, whereas inhibition of the PI3K pathway blocks LIF-dependent OSN survival and Bcl-2 induction. Thus, LIF plays a central role in maintaining the size and integrity of the population of immature neurons within the olfactory epithelium; this population is critical to the rapid recovery of olfactory function after injury. LIF may play a similar role elsewhere in the CNS and thus be important for manipulation of stem cell populations for therapeutic interventions.

Effect of Bushenantai recipe on the expression of endometrial LIF in mice with embryonic implantation dysfunction.

In order to observe the effect of Bushenantai recipe on the expression of endometrial leukemia-inhibitory factor (LIF) in mice with embryonic implantation dysfunction (EID), 120 Kunming mice post coition were randomized into three groups: normal control group, model group and traditional Chinese medicine group (TCM group) (n=40 in each group). Uterus was collected on the pregnancy day (Pd) 4, 5, 6 after an intravenous injection of Evan's blue. The endometrium was dyed by Evan's blue and the mean points of response were observed on Pd 5. The expression of LIF mRNA and protein was detected by RT-PCR and immunohistochemistry respectively and analyzed statistically by image system. The results showed that the number of implantation sites in model group was remarkably less than in normal control group and TCM group. There was no significant difference between normal control group and TCM group. The expression of LIF mRNA and protein in model group was delayed. Bushenantai recipe could increase the expression of LIF mRNA and protein in endometria of mice with EID. It was suggested that Bushenantai recipe could improve embryo implantation of mice with EID by promoting the endometrial LIF expression and endometrial decidualization.

Effect of Bushenantai recipe on the expression of endometrial LIF in mice with embryonic implantation dysfunction / 华中科技大学学报（医学）（英德文版）

In order to observe the effect of Bushenantai recipe on the expression of endometrial leukemia-inhibitory factor (LIF) in mice with embryonic implantation dysfunction (EID), 120 Kunming mice post coition were randomized into three groups: normal control group, model group and traditional Chinese medicine group (TCM group) (n=40 in each group). Uterus was collected on the pregnancy day (Pd) 4, 5, 6 after an intravenous injection of Evan's blue. The endometrium was dyed by Evan's blue and the mean points of response were observed on Pd 5. The expression of LIF mRNA and protein was detected by RT-PCR and immunohistochemistry respectively and analyzed statistically by image system. The results showed that the number of implantation sites in model group was remarkably less than in normal control group and TCM group. There was no significant difference between normal control group and TCM group. The expression of LIF mRNA and protein in model group was delayed. Bushenantai recipe could increase the expression of LIF mRNA and protein in endometria of mice with EID. It was suggested that Bushenantai recipe could improve embryo implantation of mice with EID by promoting the endometrial LIF expression and endometrial decidualization.