[Effect of yougui formula granule on ovarian granulosa cells gene expression profiles in IVF patients of shen yang deficiency syndrome].

OBJECTIVE: To observe the effect of Yougui Formula Granule (YFG) on ovarian granulosa cells gene expression profiles in in vitro fertilization-embryo transfer (IVF-ET) patients of Shen yang deficiency syndrome (SYDS) from the viewpoint of genomics. METHODS: Totally 72 infertility patients undergoing IVF-ET were randomly assigned to the treatment group and the control group according to random digit table, 36 in each group. Patients in the treatment group took YFG combined gonadotropin (Gn), while those in the control group took placebos combined Gn. All medication lasted for 3 menstrual cycles before IVF. With high-throughput gene sequencing technology, gene expression profiles of ovarian granulosa cells in the two groups were analyzed to explore the difference by gene ontology (GO) enrichment analysis and kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. RESULTS: Ovarian granulosa cell gene expression profiles from the follicular fluid showed, when compared with the control group, 391 differential genes were found in the syndrome-control group, 153 down-regulated and 238 upregulated. Enrichment of differentially expressed cellular location and molecular function of genes involved cell proliferation and apoptosis associated cyclin, protein ubiquitination, construction of microtubules and microfilament, mitochondrial function and energy-related factors, regulatory factors for hormone synthesis. Participated pathways involved energy metabolism pathway and transforming growth factor-ß (TGF-ß) signaling pathway. CONCLUSIONS: There existed significant difference in gene expression profiles of ovarian granulosa cells between the treatment group and the control group. Differentially expressed genes involved in biological processes correlates with Shen yang deficiency induced proliferation of germ cells, confused apoptosis, and hindered process during which mitochondria produced energy.

Huang Lian Jie Du decoction attenuated colitis via suppressing the macrophage Csf1r/Src pathway and modulating gut microbiota.

Introduction: Ulcerative colitis, a subtype of chronic inflammatory bowel disease (IBD), is characterized by relapsing colonic inflammation and ulcers. The traditional Chinese herbal formulation Huang Lian Jie Du (HLJD) decoction is used clinically to treat diarrhea and colitis. However, the mechanisms associated with the effects of treatment remain unclear. This study aims to elucidate the molecular mechanistic effects of HLJD formulation on colitis. Methods: Chronic colitis in mice was induced by adding 1% dextran sulfate sodium (DSS) to their drinking water continuously for 8 weeks, and HLJD decoction at the doses of 2 and 4 g/kg was administered orally to mice daily from the second week until experimental endpoint. Stool consistency scores, blood stool scores, and body weights were recorded weekly. Disease activity index (DAI) was determined before necropsy, where colon tissues were collected for biochemical analyses. In addition, the fecal microbiome of treated mice was characterized using 16S rRNA amplicon sequencing. Results: HLJD decoction at doses of 2 and 4 g/kg relieved DSS-induced chronic colitis in mice by suppressing inflammation through compromised macrophage activity in colonic tissues associated with the colony-stimulating factor 1 receptor (Csf1r)/Src pathway. Furthermore, the HLJD formula could modify the gut microbiota profile by decreasing the abundance of Bacteroides, Odoribacter, Clostridium_sensu_stricto_1, and Parasutterella. In addition, close correlations between DAI, colon length, spleen weight, and gut microbiota were identified. Discussion: Our findings revealed that the HLJD formula attenuated DSS-induced chronic colitis by reducing inflammation via Csf1r/Src-mediated macrophage infiltration, as well as modulating the gut microbiota profile.

Huang-Lian-Jie-Du decoction alleviates depressive-like behaviors in dextran sulfate sodium-induced colitis mice via Trem2/Dap12 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Huang-Lian-Jie-Du decoction (HLJD), a traditional Chinese medicine prescription, has been implicated as effective in treating colitis, depression and inflammation-related diseases. Whether HLJD decoction could ameliorate colitis-induced depression was still unknown and the underlying mechanism was needed to be clarified. AIM OF THE STUDY: Our study aimed to explore the effect and the underlying mechanism of HLJD treatment on colitis-induced depression and the involvement of the inflammatory factors and microglial-activated related genes. MATERIALS AND METHODS: The chronic colitis model was established by treating male mice with 1% dextran sulfate sodium (DSS) for 8 weeks. One week after DSS-treated, HLJD decoction was administered orally with 2 and 4 g/kg daily for 7 weeks. Behavior tests (Open field/Elevated plus maze/Novel object recognition) and TUNEL staining were then assessed. The expression of inflammatory-related genes and microglial dysregulation were measured by RT-PCR and the expression of Trem2, Danp12 and Iba1 were assessed by immunofluorescence methods. RESULTS: Depressive-like behaviors were observed in mice treated with DSS, which suffered colitis. Compared to normal control (NC-V) mice, the density of TUNEL + cells in the habenula (Hb), hippocampus (HIP), and cortex were significantly higher in colitis (DSS-V) mice, especially in Hb. Compared to NC-V and several brain regions, the expression levels of the Il-1ß, Il-10 and Dap12 mRNA were significantly increased in the lateral habenula (LHb) of colitis mice. Moreover, the expression of Trem2, Dap12 and Iba1 were increased in LHb of DSS-V mice. HLJD treatment could alleviate depressive-like behaviors, reduce the density of TUNEL + cells in Hb and the expression of Il-6, Il-10 and Dap12 mRNA in LHb of DSS-V mice. The overexpression of Trem2, Dap12 and Iba1 in LHb of DSS-V mice were reversed after HLJD treatment. CONCLUSION: These results reveal LHb is an important brain region during the process of colitis-induced depression. HLJD treatment could alleviates depressive-like behaviors in colitis mice via inhibiting the Trem2/Dap12 pathway in microglia of LHb, which would contribute to the precise treatment. It provides a potential mechanistic explanation for the effectiveness of HLJD treatment in colitis patients with depression.

Study on the Intervention Effect of Qi Gong Wan Prescription on Patients with Phlegm-Dampness Syndrome of Polycystic Ovary Syndrome Based on Intestinal Flora.

OBJECTIVE: This was a population-based cohort study, to compare the type and structure of intestinal flora in patients with polycystic ovary syndrome (PCOS) with phlegm-dampness syndrome, nonphlegm-dampness syndrome, and normal population. Besides, changes in the intestinal flora and the clinical curative effects of Jiawei Qi Gong Wan on phlegm-dampness syndrome in patients with polycystic ovary syndrome (PCOS) were evaluated. Patients and Methods. A total of 22 patients with PCOS with phlegm-dampness syndrome, 21 PCOS patients with nonphlegm-dampness syndrome, and 20 control volunteers were selected for this study. The general index, sex hormone index, fasting blood glucose (FPG), and serum fasting insulin (FINS) were determined in each of the groups. The intestinal flora of each group was determined by the 16s rDNA high-throughput sequencing technique. Besides, the PCOS with phlegm-dampness syndrome group was treated with Jiawei Qi Gong Wan, and the changes in TCM syndrome, sex hormone index, BMI, WHR, FPG, FINS, HOMA-IR, and intestinal flora were determined before and after treatment. RESULTS: PCOS patients with phlegm-dampness syndrome showed characteristics of obesity and insulin resistance. There were several differences in both structure and function of the intestinal flora between PCOS patients with phlegm-dampness syndrome, PCOS patients with nonphlegm-dampness syndrome, and the control group. An imbalance in the intestinal flora may be a key factor in the pathogenesis of PCOS with phlegm-dampness syndrome and also has a specific influence on glucose and lipid metabolism, obesity, and the menstrual cycle in PCOS patients with phlegm-dampness syndrome. Besides, the imbalance is associated with a decrease in the number of butyrate-producing bacteria, an increase in the number of lipopolysaccharide-producing bacteria, and an increase in proinflammatory bacteria. The intestinal flora in PCOS patients with phlegm-dampness syndrome was found to be linked to obesity, impaired glucose tolerance, and hyperandrogenemia. Treatment with Jiawei Qi Gong Wan was found to increase the diversity of intestinal flora, increase the number of intestinal probiotics, and improve the structure and functional genes of intestinal flora which improved the state of insulin resistance, regulated endocrine metabolism, and improved the overall symptoms. CONCLUSIONS: Intestinal flora imbalance is a key factor in the pathogenesis of PCOS with phlegm-dampness syndrome. Besides, Jiawei Qi Gong Wan improves insulin resistance which is linked to the status of the intestinal flora in PCOS patients with phlegm-dampness syndrome.

Effects of Electroacupuncture on Expression of PI3K/Akt/Foxo3a in Granulosa Cells from Women with Shen (Kidney) Deficiency Syndrome Undergoing in vitro Fertilization-Embryo Transfer.

OBJECTIVE: To observe the effects of electroacupuncture (EA) on reproductive outcomes in women with Shen (Kidndy) deficiency syndrome after in vitro fertilization-embryo transfer (IVF-ET), and explore the underlying molecular mechanism. METHODS: Sixty-six infertile patients with Shen deficiency syndrome undergoing IVF-ET were divided into EA or control groups according to a random table, 33 cases in each group. Before undergoing IVF, patients in the EA and control groups received EA therapy and placebo needle puncture, respectively, for 3 menstrual cycles. Shen deficiency syndrome scores were assessed. Other outcome measures included the number of retrieved oocytes and fertilization, high-quality embryo and clinical pregnancy rates. Follicular fluid was collected on the day of oocyte retrieval, and granulosa cell expression of phosphatidylinositide 3-kinases (PI3K), serine-threonine kinase (Akt) and forkhead box O3 (Foxo3a) mRNA were measured by reverse transcribed and quantitative real-time polymerase chain reaction. RESULTS: Syndrome scores for pre- versus post-treatments decreased significantly (16.53±1.75 to 8.67±1.61) in the EA group (P<0.05), but showed no significant change in the control group (17.18±1.58 to 14.74±1.58). A significant difference in score change was found between the EA and control groups (P<0.05). High-quality embryo and clinical pregnancy rates were both increased in the EA group compared with the control group [69.15% (195/282) vs. 60.27% (176/292) and 66.67% (22/33) vs. 42.42% (14/33), respectively, P<0.05]. The fertilization rate was equivalent in EA and control groups. No difference was found in the number of retrieved oocytes between the two groups. Granulosa cell expression levels of PI3K and Akt mRNA were significantly increased in the EA group compared with the control group, while the expression of Foxo3a was reduced (all P<0.05). CONCLUSIONS: For infertile patients with Shen deficiency syndrome undergoing IVF, EA for tonifying Shen as an adjunct treatment may alleviate clinical symptoms and improve the high-quality embryo rate. The EA-induced mechanism may involve regulation of PI3K/Akt/Foxo3a expression in granulosa cells to improve the developmental microenvironment of oocytes and inhibit granulosa cell apoptosis, possibly contributing to the improved clinical pregnancy rate (Registration No. ChiCTR 1800016217).

Bone morphogenetic protein 6 affects cell-cell communication by altering the expression of Connexin43 in human granulosa-lutein cells.

Connexin 43 (Cx43)-coupled gap junctions in granulosa cells play an important role in follicular development, oocyte maturation, and corpus luteum maintenance. Bone morphogenetic protein 6 (BMP6) is highly expressed in human oocytes and granulosa cells and is involved in the regulation of female reproduction. Currently, whether oocyte- and granulosa cell-derived BMP6 affects the expression of Cx43 and its related gap junction intercellular communication (GJIC) activity in human granulosa cells remains unknown. In this study, we demonstrate that BMP6 treatment significantly suppressed the expression of Cx43 in both primary and immortalized (SVOG) human granulosa-lutein cells. Using both pharmacological inhibitors and small interfering RNA-mediated knockdown approaches, we demonstrate that ALK2 and ALK3 BMP type I receptors are involved in BMP6-induced suppressive effects on Cx43 expression and GJIC activity in SVOG cells. Furthermore, these cellular activities are most likely mediated by the SMAD1/SMAD5-SMAD4-dependent signaling pathway. Notably, the ChIP analyses demonstrated that phosphorylated SMADs could bind to human Cx43 promoter. Our findings provide new insight into the molecular mechanisms by which an intrafollicular growth factor regulates cell-cell communication in human granulosa cells.

Neurotrophins and glial cell line-derived neurotrophic factor in the ovary: physiological and pathophysiological implications.

BACKGROUND: Neurotrophins [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4)] and glial cell line-derived neurotrophic factor (GDNF) are soluble polypeptide growth factors that are widely recognized for their roles in promoting cell growth, survival and differentiation in several classes of neurons. Outside the nervous system, neurotrophin (NT) and GDNF signaling events have substantial roles in various non-neural tissues, including the ovary. OBJECTIVE AND RATIONALE: The molecular mechanisms that promote and regulate follicular development and oocyte maturation have been extensively investigated. However, most information has been obtained from animal models. Even though the fundamental process is highly similar across species, the paracrine regulation of ovarian function in humans remains poorly characterized. Therefore, this review aims to summarize the expression and functional roles of NTs and GDNF in human ovarian biology and disorders, and to describe and propose the development of novel strategies for diagnosing, treating and preventing related abnormalities. SEARCH METHODS: Relevant literature in the English language from 1990 to 2018 describing the role of NTs and GDNF in mammalian ovarian biology and phenotypes was comprehensively selected using PubMed, MEDLINE and Google Scholar. OUTCOMES: Studies have shown that the neurotrophins NGF, BDNF, NT-3 and NT-4 as well as GDNF and their functional receptors are expressed in the human ovary. Recently, gathered experimental data suggest putative roles for NT and GDNF signaling in the direct control of ovarian function, including follicle assembly, activation of the primordial follicles, follicular growth and development, oocyte maturation, steroidogenesis, ovulation and corpus luteum formation. Additionally, crosstalk occurs between these ovarian regulators and the endocrine signaling system. Dysregulation of the NT system may negatively affect ovarian function, leading to reproductive pathology (decreased ovarian reserve, polycystic ovary syndrome and endometriosis), female infertility and even epithelial ovarian cancers. WIDER IMPLICATIONS: A comprehensive understanding of the expression, actions and underlying molecular mechanisms of the NT/GDNF system in the human ovary is essential for novel approaches to therapeutic and diagnostic interventions in ovarian diseases and to develop more safe, effective methods of inducing ovulation in ART in the treatment of female infertility.

Effects of Liuwei Dihuang Granule ([symbols; see text]) on the outcomes of in vitro fertilization pre-embryo transfer in infertility women with Kidney-yin deficiency syndrome and the proteome expressions in the follicular fluid.

OBJECTIVE: To observe the effects of Liuwei Dihuang Granule ([symbols; see text], LDG) for tonifying Kidney (Shen) on the outcomes of in vitro fertilization pre-embryo transfer (IVF-ET) of infertility women with Kidney-yin deficiency syndrome and to explore its mechanism by detecting the proteome expression in the follicular fluid. METHODS: Sixty-six infertility patients of Kidney-yin deficiency syndrome who would undergo IVF-ET, were randomly assigned to a treatment group and a control group according to a random number table, 33 cases in each group. Another 33 cases of non-Kidney-yin deficiency syndrome was taken as a syndrome-control group. Besides Western routine therapy, LDG was given 3 menstrual cycles before IVF to the treatment group, and a placebo granule to the control and syndrome-control groups. The scores of Kidney-yin deficiency symptoms (sore waist and knees, dry vagina, dysphoria with feverish sensation in the chest, palms and soles, etc.) were assessed, the number of retrieved oocytes, rates of high quality oocytes and embryos, fertility rate and clinical pregnancy rate were recorded, and the follicular fluid was collected on the day when the ovum was picked up, the differential protein expression was detected using two-dimensional gel electrophoresis, and then, matrix assisted laser desorption ionization time-of flight mass spectrometry (MALDI-TOF-MS) was applied to identify the proteins. RESULTS: The syndrome score in the treatment group decreased significantly from 16.09±2.58 to 8.67±2.13, while it changed insignificantly in the control group, with a significant difference in the lowering score between the two groups (P<0.05); the high quality rates of oocytes and embryos and clinical pregnancy rate were all superior in the treatment group to the control group (82.29% vs 78.08%, 76.76% vs 68.79%, 63.64% vs 36.36%, all P<0.05). The protein expression map from the follicular fluid showed that compared with the control group, 33 differential protein expressions were found in the syndrome-control group, among which 18 were down-regulated, and 15 up-regulated; in the treatment group 28 differential protein expressions were found, among which 15 were down-regulated, and 13 up-regulated. Through MALDI-TOF-MS, 14 proteins were identified (P<0.05). CONCLUSIONS: For the infertility patients undergoing IVF, LDG could alleviate clinical symptoms, improve rates of high quality oocytes and embryos, so as to raise clinical pregnancy rate. The mechanism may be through regulating proteome expression in the follicular fluid to improve the developmental microenvironment for oocytes which would lead to a successful embryo implantation.