Bu-Shen-Yi-Qi Formula Improved Biologic Function Of Human First-trimester Trophoblasts By Regulation Of SOCS3

Recurrent spontaneous abortion is one of the most common complications of pregnancy. Between 15% and 25% of clinically recognized pregnancies culminate in spontaneous abortions, and the abnormal regulation of endocrine-immune at human first-trimester materno-fetal interface plays a key role in abortion. Our serial clinical investigations and animal studies have showed that Bu-Shen-Yi-Qi formula promoted growth and differentiation of the cytotropholblasts in vitro, improved the pregnant rat IL-10/IFN-γ ratio of decidua and increased serum progesterone and PRL level. So it protected pregnancy by regulating endocrine-immune disturbance. However, the exact mechanism of this process is poorly understood.Recently, SOCS(suppressors of cytokine signaling) have been shown to participate in a negative feedback loop to regulate the cytokine and hormone signaling in vitro and in vivo, and controlled the cell's endocrine function and immune reaction response to cytokines. In addition, SOCS3 is recognized that it involves in signaling of cells proliferation. SOCS3 is required for mouse placental development. Abundant expression of SOCS3 is observed in E9.5 and E10.5 placenta of mice. In E9.5 placenta, SOCS3 is expressed in all trophoblast lineage. There are marked decrease in the spongiotrophoblast layer and increase in the number of giant trophoblast cells in mice lacking SOCS3, which result in an embryonic lethality between days 11 and 13 of gestation, but SOCS in the human first-trimester materno-fetal interface had no reported. The present study investigate the regulation of Bu-Shen-Yi-Qi formula on the SOCS3 expression and its possible signaling transduction pathways involved in the effects of on the biological functions such as growth, secretion and crosstalking between cells. It will establish base to explore mechanism of regulating endocrine-immune network at human first-trimester materno-fetal interface and the new medicine effect of Bu-Shen-Yi-Qi formula further.Section I Expression of SOCS1, SOCS2, SOCS3 and its regualtion in materno-fetal interface of human first trimester gestationObjective: To detect the expression of SOCS1, SOCS2, SOCS3 in villous and decidualtissue of normal human first trimester gestation. To culture the human cytotrophoblastsand decidual stromal cells in vitro, and detect their basal expression of SOCS, thenexplore the possible regulation factors inducing SOCS expression in materno-fetalinterface.Methods: The expression of SOCS1, SOCS2, SOCS3 gene and protein were valued byRT-PCR, Western blot respectively. The location of SOCS protein was detected by immunohistochemistry. The first-trimester human cytotrophoblast cells and decidual stromal cells were isolated by trypsin/DNase I digestion, and discontinuous Percoll density gradient centrifugation. After identified by immunocytochemistry with monoclonal antibody (mAb) to Cytokeratin 7 and Vimentin, the trophoblast cells and decidual stromal cells were appeared more than 95% pure. After cultured with 1% FBS, the secretion of IL-10, IFN-γ were detected by ELISA.Results: (1) The expression of SOCS, SOCS2, SOCS3 were founded in mater-fetal interface, while positive expression of SOCS3 in villi/decidua of human first trimester gestation were 73.7%/71.1%, and that of SOCS2 were 50.0%/39.5%, then SOCS1 expression were 34.2%/31.6%. The protein expression character of SOCS1, SOCS2, SOCS3 were similar to that transcription lever. SOCS1, SOCS2, SOCS3 were mainly expressed in villous trophoblasts and decidual stroma, its positive stain were found in plasm or/and nucleus. (2)Trophoblasts and decidual stromal cells(DSC) were not detected SOCS1 transcription, but the low level mRNA of SOCS2 and SOCS3 were found after cultured 12h-14h with 1%FBS, and IL-10 secreted by these cells increased significantly ( P<0.05 ) , and their level of IFN-γ had not found changed significantly (P>0.05) .(3)Cytotrophoblasts and DSC were induced up-regulate of SOCS2 and SOCS3 gene within 8h response to IL-10(0.1ng/ml).Conclusion: The SOCS of materno-fetal interface may play an important biological role in regulation of endocrine-immune network at normal human first-trimester materno-fetal interface, the IL-10 autosecreted and parasecreted by cytoblasts and DSC may be the oneof cause inducing SOCS2 and SOCS3. Hence, to study biologic function of these SOCS at materno-fetal interface will give the valuable information to understanding the complex mechanism of maintaining gestationSection II Bu-Shen-Yi-Qi Formula improved growth of human first trimester cytotrophoblasts by up-regulating expression of SOCS3Objective: To investigate effect of Bu-Shen-Yi-Qi Formula on expression of SOCS3 of the human first trimester cytotrophoblasts and JAR cell line. We explored the mechanism of the Formula on improving cytotrophoblasts growth.Methods: The first-trimester human trophoblast cells were isolated by trypsin/DNase I digestion, and discontinuous Percoll density gradient centrifugation. After identify cells pure, they were cultured in vitro. The expression of SOCS3 gene was valued by RT-PCR. The expression of SOCS3 protein and pSTAT3, pERK were valued by Western blot respectively. Cells proliferation/viability was detected by MTT Assay. PCNA and Annexin V expression were valued by way of the flow cytometry, which stimulated by Bu-Shen-Yi-Qi Formula and/or U0126. SOCS3 expression of JAR cell line was down-regulated or up-regulated by transfecting cells with SOCS3 siRNA or sense SOCS3 gene. The transfection efficiency was proved by RT-PCR, real time PCR and western blot, Activation of STAT3 and ERK1/2 after SOCS3 siRNA transfection were detected by western blot. PCNA expression of JAR was valued by way of the flow cytometry after SOCS3 overexpression.Results: (1) Bu-Shen-Yi-Qi Formula could promote the cells viability and PCNA expression in vitro of the first trimester human cytotrophoblasts in a concentration-dependent manner when concentration of medicine ranged from 10% to 20%, and it has the similar role on JAR cell line. After cultured with 1% fetal cattle serum 48h, cytotrophoblasts of Annexin V-FITC~+PI~-were 28.34±4.48%, their apoptosis percent decreased, and cells of Annexin V-FITC~+PI~- were 14.61±0.66%, 5.79±1.74% after adding 10% and 20% Bu-Shen-Yi-Qi Formula, the inhibition of apoptosis in a concentration-dependent manner, and the role of 20% Formula was better. (2) 10% and 20% Bu-Shen-Yi-Qi Formula could briefly up-regulate the SOCS3 expression of cytotrophoblastas in vitro with the similar trend: SOCS3 expression increased at 1h time point, and reached the peak at 2h time point, then decreased subsequently and return tothe nearly basal level. 20% Formula up-regulated SOCS3 expression was higher than that of 10% concentration drug. (3) 20% Bu-Shen-Yi-Qi Formula could induce cytotrophoblsts ERK1/2 activation in concentration-dependent manner, and the role of 20% Formula was stronger than that of 10% Formula, but STAT3 activation were undetected. It induced JAR cells ERK1/2 activation and SOCS3 expression within 10min and lasted 8h. (4) MEK inhibitor U0126 could down-regulate SOCS3 expression of cytotrophoblasts, and inhibited phospho-ERK1/2 level induced by Bu-Shen-Yi-Qi Formula, then inhibit the function of traditional medicine influencing cells proliferation and apoptosis. (5) mTOR inhibitor rapamycin could inhibit cytotrophoblsts SOCS3 expression, cells viability and proliferation induced by Bu-Shen-Yi-Qi Formula, but rapamycin could not affect on activation of ERK. (6) SOCS3 expression of JAR was down-regulated by transfecting SOCS3 siRNA, the activation of STAT3 and ERK influenced by Bu-Shen-Yi-Qi Formula were not changed after transfection. (7) SOCS3 overexpression was found promote the JAR cells PCNA expression without serum in culture.Conclusion: Bu-Shen-Yi-Qi Formula could promote the cells proliferation and inhibit apoptosis in vitro of the first trimester human cytotrophoblasts in a concentration-dependent manner, and it quickly up-regulated the SOCS3 expression then down-regulated its expression within 4h of Bu-Shen-Yi-Qi Formula treatment. MAPK/ERK1/2 and PI3K/m TOR pathway but not JAK/STAT3 may play an important role in expression of SOCS3 and regulating proliferation and apoptosis in human cytotrophoblasts induced by Bu-Shen-Yi-Qi Formula. The change of SOCS3 expression could not affect on activation of ERK, SOCS3 may involve in the role of improve growth proliferation of cytotrophoblast induced by Bu-Shen-Yi-Qi Formula.Section III Bu-Shen-Yi-Qi Formula improved the secretion of human trophoblasts and decidual stromal cellsObjective: To detecte the relationship of proliferation with secretion level of human first-trimester trophoblasts after cultured with Bu-Shen-Yi-Qi Formula. in vitro. To explore influence factors involved in crosstalking between human trophoblastas and decidual stromal cells, as well as induced trophoblasts SOCS3 expression.Methods: Trophoblastas and decidual stromal cells of the human first trimester were culture in vitro. After stimulated cells by Bu-Shen-Yi-Qi Formula 24h, 36h and 48h,IL-10, IFN-γ, progesterone , PRL secreted by those cells analyzed by ELISA and chemistry luminescent method, the expression of IL-10 receptors of cells were analyzed by way of the flow cytometry, and the vitality of trophoblasts were valued by MTT. The expression of SOCS3 protein were valued by Western blot.Results: (1) Bu-Shen-Yi-Qi Formula could promote the cells viability in a time and concentration-dependent manner when concentration of medicine ranged from 10% to 20% treated trophoblasts after 24h 36h and 48h time point respectively, as well as up-regulated IL-10R expression, and promoted IL-10, progesterone secreted by trophoblasts. The IFN-γ induced by Bu-Shen-Yi-Qi Formula similar as the level of those serum control groups. The relationship analysis showed that the cells viability induced by Bu-Shen-Yi-Qi Formula were in positive relation with the progesterone IL-10/IFN-γ secretion of the trophoblasts. (2) Bu-Shen-Yi-Qi Formula could up-regulate IL-10R expression of DSC at 48h timepoint, and promoted the PRL and IL-10 secretion in a time or/and concentration-dependent manner when concentration of medicine ranged from 10% to 20%. (3) IL-10 (0.1ng/ml, lng/ml) up-regulated SOCS3 protein expression of human trophoblasts from 2h to 8h timepoint.Conclusion: Human cytotrophoblasts and DSC could influence each other by promoting hormone and IL-10 secretion and up-regulating IL-10R expression after cultured with traditional medicine. IL-10 secreted by those cells also induced SOCS3 protein expression of trophoblasts. The first trimester human trophoblasts cells viability and secretion induced by Bu-Shen-Yi-Qi Formula could promote one another. Bu-Shen-Yi-Qi Formula may improve biologic function such as trophoblasts growth and secretion by regulation of SOCS3 expression.In summary, materno-fetal interface of human first trimester expressed SOCS1, SOCS2, SOCS3, and IL-10 secretion in it induced trophoblsts and DSC SOCS3 expression. Bu-Shen-Yi-Qi Formula up-regulated SOCS3 by activation of MAPK/ERK1/2 , PI3K/mTOR signaling pathway and improved trophoblasts growth, SOCS3 may improve trophoblsts growth. The first trimester human trophoblasts cells viability could promote cells secretion induced by Bu-Shen-Yi-Qi Formula, at the same time cytotrophoblasts and DSC influenced each other by secreting hormone and IL-10 induced by traditional medicine. IL-10 secreted by those cells influenced trophoblasts growth by inducingSOSC3. To study the biological function of SOCS3 in human trophoblasts further may be benefit to understand the mechanism of placenta development and regulation of endocrine-immune network at human first-trimester materno-fetal interface.