Uncovering the pharmacological mechanism of Shou Tai Wan on recurrent spontaneous abortion: A integrated pharmacology strategy-based research.

ETHNOPHARMACOLOGICAL RELEVANCE: Shou Tai Wan (STW), a traditional Chinese medicine formula, has been historically used for the treatment of recurrent spontaneous abortion (RSA). Despite its long-standing usage, the exact mechanism underlying the therapeutic effects of STW remains unclear in the existing literature. AIMS OF THIS STUDY: To explore the Pharmacological Mechanism of STW on RSA. METHODS: A network pharmacological methodology was utilized to predict the active compounds and potential targets of STW, collect the RSA targets and other human proteins of STW, and analyze the STW related networks. The animal experiments were also performed to validate the effect of STW on RSA. RESULTS: The results of network analysis showed that STW may regulate PI3K/AKT, MAPK, FoxO signaling pathways and so on. Animal experiment established the RSA model with CBA/J × DBA/2 mice. It was found that STW can reduce the embryo absorption rate of RSA group (p < 0.05) and balance the expression of Th 1/Th2 type cytokines compared with the model group. After 14 days of administration, the decidual and placental tissues were taken and the CD4+ T cells were isolated, and the phosphorylation level of signaling pathway was detected by Springbio720 antibody microarray. This experiment found that STW can significantly up-regulate the phosphorylation levels of STAT3 and STAT6 proteins in the STAT signaling pathway, and down-regulating the phosphorylation level of STAT1 protein. STW also significantly up-regulated the phosphorylation levels of Raf1, A-Raf, Ask1, Mek1, Mek2, JKK1, ERK1, ERK2, c-fos, c-Jun and CREB proteins in the MAPK signaling pathway, and down-regulate the phosphorylation levels of MEK6 and IKKb proteins. Compared with the RSA group, the STW group increased the expression levels of ERK1/2 mRNA and proteins and p-ERK1/2 proteins, and there was a statistical difference (p < 0.05). This is consistent with the chip results. CONCLUSION: STW may achieve therapeutic effects by interfering with the signaling pathways, biological processes and targets discovered in this study. It provides a new perspective for revealing the immunological mechanism of STW in the treatment of RSA, and also provides a theoretical basis for the clinical use of STW in the treatment of RSA.

Systematic Pharmacology-Based Strategy to Explore the Molecular Network Mechanism of Modified Taohong Siwu Decoction in the Treatment of Premature Ovarian Failure.

OBJECTIVE: To explore the molecular network mechanism of modified Taohong Siwu Decoction (MTHSWD) to interfere with premature ovarian failure based on systematic pharmacological strategy. METHODS: The network pharmacology strategy was used to explore the potential mechanism of MTHSWD intervention in POF, and then it was verified through animal experiments. Mouse zona pellucida 3 was used as an antigen to subcutaneously immunize BALB/c female mice to establish an immune POF model. Mice were divided into MTHSWD low-, medium-, and high-dose groups, positive control group, model group, and normal group. After 30 days of drug intervention, ovarian tissue was taken for pathological hematoxylin-eosin (HE) staining, and immunohistochemical methods were used to detect the expression of TGF-ß1 and TGF-ßRII and Smad2/3 protein expression in follicular wall granular cells and ovarian tissue, respectively. RESULTS: Network pharmacology studies have shown that MTHSWD may interfere with the TGF-ß signaling pathway. Animal experimental research shows that, compared with the model group, the number of ovarian mature follicles in the MTHSWD groups and the positive group was significantly increased, and the number of atresia follicles decreased. Immunohistochemistry showed that, compared with the control group, the expression of TGF-ß1, TGF-ßRII, and Smad2/3 in the follicular wall granulosa cells and ovarian tissues of MTHSWD groups was significantly higher than that of the model group (P < 0.05). CONCLUSION: MTHSWD may improve the ovarian function of POF mice by upregulating the protein expression of granulosa cells TGF-ß1, TGF-ßRII, and Smad2/3.

l-Argine regulates the proliferation, apoptosis and endocrine activity by alleviating oxidative stress in sheep endometrial epithelial cells.

l-arginine (L-Arg) is a semiessential amino acid that plays crucial roles in the reproductive performance of animals. This research aimed to investigate the effect of supplementing L-Arg on endometrial epithelial cells (EECs) of Hu sheep. In vivo, female Hu sheep were randomly divided into three groups: control group (n = 5), nutrient-restricted group (n = 5), and L-Arg supplemented nutrient-restricted group (n = 5). Then, the effect of L-Arg on ovine endometrial growth and antioxidant capacity was assessed. We found that L-Arg supplementation promoted the growth of endometrial ductal gland invaginations (DGI), and alleviated oxidative stress in nutrient-restricted sheep. In order to investigate its mechanism, a H2O2-induced EECs oxidative stress model was established, and roles of L-Arg in EECs oxidation resistance, proliferation, apoptosis and endocrine activity were studied in vitro. Our results showed that L-Arg markedly decreased the release of reactive oxygen species (ROS) and malonaldehyde (MDA), and enhanced the expression and activity of certain antioxidant enzymes in EECs challenged by the H2O2 (p < 0.05). Supplementation of L-Arg significantly reduced the effect of 200 µM H2O2 on the viability of EECs (p < 0.05). In addition, EECs treated with L-Arg significantly alleviated the G0/G1-phase cell cycle arrest, apoptosis, and the inhibition of endometrial growth factors expression caused by H2O2 (p < 0.05). Overall, the results demonstrate that L-Arg performs crucial roles in maintaining the proliferation of ovine EECs, endocrine activity and inhibiting apoptosis through reducing oxidative stress. This study offers a theoretical basis for using L-Arg to improve sheep the uterine function.

Network pharmacology and molecular docking analysis on molecular targets and mechanism prediction of Huanglian Jiedu Decoction in the treatment of COVID-19 / 数字中医药（英文）

@#Objective To investigate and predict the molecular targets and mechanism of Huanglian Jiedu Decoction (黄连解毒汤, HLJDD) in the treatment of Corona Virus Disease 2019 (COVID-19) through network pharmacology and molecular docking analysis. Methods The chemical constituents and action targets of HLJDD were retrieved on Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), SymMap v2, Encyclopedia of Traditional Chinese Medicine (ETCM), a High-throughput Experiment- and Reference-guided Database of Traditional Chinese Medicine (HERB), and Traditional Chinese Medicine Integrated Database (TCMID). UniProt and GeneCards were used to query the target genes that corresponding to the active compounds, and then a compound-target network was constructed using Cytoscape 3.7.2. Gene Ontology (GO) database was used to annotate GO functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to predict the possible mechanisms of active compounds. The Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to analysis the tissue enrichment. The main active compounds in HLJDD are molecularly docked with their corresponding related targets. Results Seventy-six compounds were screened and 458 corresponding targets in the network were obtained. Gene annotation showed that the targets were involved mainly in 1 953 biological processes. 884 signaling pathways was enriched, involving signaling by interleukins, cytokine signaling in immune system, generic transcription pathway, and RNA polymerase II transcription. The targets mainly distributed in the lung, liver, and placenta, involving a variety of immune cells, such as T cells and B cells. The molecular docking results showed that core compounds such as wogonin, berberine, and baicalein had high affinity with tumor necrosis factor (TNF), insulin (INS), and tumor protein 53 (TP53). Conclusion The active compounds in HLJDD may have a therapeutic effect on COVID-19 through regulating multiple signal pathways by targeting genes such as vascular endothelial growth factor A (VEGFA), INS, interleukin-6 (IL-6), TNF, caspase-3 , TP53, and mitogen-activated protein kinase 3 (MAPK3).

Exploring the Effect of Jiawei Buguzhi Pills on TGF-ß-Smad Pathway in Postmenopausal Osteoporosis Based on Integrated Pharmacological Strategy.

OBJECTIVE: To explore the effect of Jiawei Buguzhi Pills (JWBGZP) on the TGF-ß-Smad pathway in postmenopausal osteoporosis (PMO) based on integrated pharmacological strategy. METHOD: The ETCM database was used to collect JWBGZP. GeneCards and OMIM databases were utilized to obtain PMO-related genes. Cytoscape was used for network construction and analysis, and DAVID was used for GO and KEGG enrichment analysis of key targets. Animal experiments and cell experiments were conducted to further explore the mechanism. The bone mass density was detected by dual-energy X-ray bone densitometer. The TGF-ß1 and Smad4 mRNA in bone tissue were detected by RT-qPCR. The TGF-ß1 and Smad4 protein in bone tissue were detected by the western blot. The TGF-ß1 and Smad4 protein in osteoblasts were determined by immunohistochemistry. RESULT: A total of 721 JWBGZP potential targets and 385 PMO-related genes were obtained. The enrichment analysis showed that JWBGZP may regulate the TGF-beta signaling pathway, oxidation-reduction process, aging, response to hypoxia, response to ethanol, negative regulation of cell proliferation, PI3K-Akt, HIF-1, and other signaling pathways. The animal experiments showed that compared with the model group, the femoral bone mineral density and lumbar bone mineral density of the JWBGZP group increased (P < 0.05); the expression levels of TGF-ß1 and Smad mRNA and proteins in the JWBGZP group were significantly higher (P < 0.05). The cell experiment results showed a large number of osteoblast stained blue-purple and orange-red calcified nodules. The expression levels of TGF-ß1 and Smad proteins in the JWBGZP group were significantly higher than those in the blank control group and the sham operation group, and the protein expression levels in the model group were the lowest (P < 0.05). CONCLUSION: JWBGZP may be involved in PI3K-Akt, HIF-1, estrogen, prolactin, and other signaling pathways and regulate MAPK1, AKT1, PIK3CA, JAK2, and other gene targets, regulate bone metabolism, and thereby treat PMO.

Arginine infusion rescues ovarian follicular development in feed-restricted Hu sheep during the luteal phase.

The aim of this study was to investigate the molecular mechanisms of arginine (Arg) on follicular development of acute feed-restricted ewes during the luteal phase. From day 6 of the estrous cycle, 24 multiparous Hu sheep were randomly assigned into three groups: control group (a maintenance diet; n = 6), feed restriction group (0.5 maintenance diet, saline infusion; n = 9) and Arg treatment group (0.5 maintenance diet, infusion with 155 µmol of Arg-HCl/kg body weight; n = 9). The intravenous administrations were performed three times per day from day 6 to day 15 of the estrous cycle. At the end of treatment, the hypothalamus and pituitary were collected, as well as the follicular fluid (FF) and granulose cells (GCs) in the ≥2.5 mm follicles. The transcription level of NPVF was significantly increased, and the expression level of GNRH was significantly decreased in the hypothalamus with feed restriction. In addition, feed restriction significantly decreased the number of ≥2.5 mm follicles in the ovaries. In the ≥2.5 mm follicles, feed restriction significantly increased estradiol (E2) level in FF and the expression levels of steroidogenesis related genes (STAR, 3BHSD and CYP19A1) in GCs, while significantly decreased the expressions of FSHR and cell proliferation related genes (YAP1, CCND1 and PCNA) in GCs. Moreover, the activities of glucose metabolism enzymes (PFKP and G6PDH) were significantly decreased in GCs of the ≥2.5 mm follicles with feed restriction. Interestingly, as a precursor of nitric oxide, Arg supplementation can rescue the effects of feed restriction on follicular development by enhancing glucose metabolism and cell proliferation of GCs, and alleviating the abnormal E2 secretion in the ≥2.5 mm follicles, accompanied with recovering the expressions of NPVF and GNRH in the hypothalamus. These findings will be helpful for understanding the role of nutrition and Arg in sheep follicular development.

Inhibition of lysine-specific histone demethylase 1A results in meiotic aberration during oocyte maturation in vitro in goats.

Histone methylation is associated with oocyte maturation in several species and is also expected in goat oocytes, while the mechanism is still unclear. Therefore, single-cell RNA sequencing (scRNA-seq) was performed on goat germinal vesicle (GV) and metaphase II (MII) oocytes, and the functions of lysine-specific histone demethylase 1A (LSD1), one of the differentially expressed genes (DEGs) were investigated during in vitro maturation (IVM) of goat oocytes. Through scRNA-seq, 4516 DEGs were identified from GV oocytes and MII oocytes in goats, among which there were 16 histone methyltransferase and demethylase DEGs (including LSD1). The functions of LSD1 during IVM of goat oocytes were investigated through its inhibitor, GSK-LSD1. We found that the first polar body extrusion rate of goat oocytes significantly reduced with an increase in GSK-LSD1 concentration supplemented into IVM medium (0 µM: 58.84 ± 0.95%; 2.5 µM: 52.14 ± 0.51%, P < 0.01; 50 µM: 41.22 ± 0.42%, P < 0.001; 100 µM: 29.78 ± 1.78%, P < 0.001). Moreover, compared with the control group, the level of H3K4me2 methylation and p-H2AX in goat oocytes significantly increased (P < 0.001 and P < 0.01, respectively) upon 50-µM GSK-LSD1 treatment for 12 h. Furthermore, abnormalities in spindle assembly (25.94 ± 1.02% vs. 71.15 ± 3.32%; P < 0.01) and chromosome alignment (22.93 ± 1.11% vs. 76.03 ± 3.25%; P < 0.01) were observed, and cytoskeletal organization (15.31 ± 1.60% vs. 67.50 ± 3.09%; P < 0.001) was disrupted upon treatment with 50-µM GSK-LSD1 for 12 h, which compared with that in the control group. Additionally, the ratio of BCL2:BAX significantly higher (P < 0.01) in oocytes with 50-µM GSK-LSD1 treatment than that in control group. Collectively, these results indicate the important role of LSD1 in meiotic maturation of goat oocytes. Our data not only clarify dynamic changes in mRNA during oocyte maturation but also provide a theoretical basis and technical means for further studies of meiotic maturation of goat oocytes.

Effects of algae supplementation in high-energy dietary on fatty acid composition and the expression of genes involved in lipid metabolism in Hu sheep managed under intensive finishing system.

This study aims to investigate the effect of algae supplementation in high-energy diet (HE diet) on lipid metabolism of intensive feeding sheep. The lambs were assigned to two groups and received a standard diet (ST diet, 8.40 MJ/kg) or a HE diet (9.70 MJ/kg) based on corn, wheat bran, soybean meal. Each group was divided into two subgroups: control and algae supplement (3%, DM basis). The body fat, serum cholesterol, and oleic acid in the liver and muscle increased in lambs receiving the HE diet. However, after supplementing the microalgae (Schizochytrium sp.) in the HE diet, these parameters were all reduced. In addition, EPA, DHA and CLA cis-9, trans-11 in muscle and liver increased. Algae supplementation also altered the expression of lipid metabolism-related genes, including up-regulated FADS2, ELOVL2, SCD, CPT1α and SREBF-1, and down-regulated PPARα and PPARγ. In conclusion, algae supplementation in ST and HE diets increased n-3PUFA and improved metabolic disorder caused by the HE diet.

Effects of l-arginine on endometrial estrogen receptor α/ß and progesterone receptor expression in nutrient-restricted sheep.

This study aimed to determine the effects of l-arginine (L-Arg) supplementation on steroid hormone receptors in non-pregnant ovine endometrium. All experimental ewes were randomly assigned to either a control group (n = 6), a nutrient-restricted group (n = 6), or an L-Arg supplemented nutrient-restricted group (n = 6). The effects of L-Arg on estrogen receptor α/ß (ERα/ß) and progesterone receptor (PGR) expression in the ovine endometrium were assessed. Our results showed that levels of ERß and PGR expression were significantly increased by nutrient restriction, but L-Arg counteracted the effect of nutrient restriction on ERß and PGR expression (p < 0.05). Also, expression of endometrial ERα was substantially increased (p < 0.05) by L-Arg supplementation. Furthermore, ERα/ß and PGR were mainly detected in the endometrial luminal epithelium and glandular epithelium. Therefore, we isolated and identified endometrial epithelial cells (EECs) from sheep. Different concentrations of L-Arg were added to investigate the effects on ERα/ß and PGR in EECs. The expression levels of endothelial nitric oxide synthase, ERß, and PGR were significantly increased in response to low-concentration (200 µmol) L-Arg supplementation, which subsequently decreased with a high concentration (800 µmol) (p < 0.05). Otherwise, ERα expression was remarkably increased at both L-Arg concentrations in EECs (p < 0.05). Overall, the results indicated that L-Arg performed crucial roles in the regulation of ovine steroid hormone receptor expression in the endometrium. The results of this study provide a theoretical basis and technical means for the normal function of endometrium in response to low nutrient levels.

Amino acids profile within peripheral blood and follicular fluid based on high-performance liquid chromatography methods may explain differences in folliculogenesis between short-term under/over-fed treatments during luteal phase of Hu sheep.

The nutritional alteration of amino acids (AAs) profile in physiological fluid was poorly characterized in livestock. After oestrus synchronization, 24 ewes were randomly assigned to two groups based on the nutrient requirement recommended for maintenance (M): the feed-supplemented group (S, 1.5 × M, N = 12) and feed-restricted group (R, 0.5 × M, N = 12) on days 6-12 of their oestrous cycle, which occurred shortly before ovulation. The concentration of 30 AAs in peripheral blood (PB) and follicular fluid (FF) was quantified to calculate the PB-to-FF concentration gap for each AA and determine its correlation with metabolites and hormones in PB and FF. Results showed that the feed restriction enlarged the oestrous cycle length, decreased the number of follicles 2.5-3.5 mm, increased the number of follicles >3.5 mm and augmented the volume of follicles >2.5 mm. Nineteen AAs from PB were significantly different between the groups. The phosphoethanolamine (PEtN) and ration of essential AAs to nonessential AAs (EAA/NEAA) in FF significantly (p < 0.05) increased and decreased in the R group, respectively. Most AAs, except aspartate (Asp) and carnosine (Car) in the R group and alanine (aAla) in both groups, were significantly lower within FF than those within PB. The correlation of AAs with FSH and progesterone (P4 ) was more significant than that of AAs with other endocrine milieu characteristics. In conclusion, our results revealed that the influence of short-term nutritional manipulation during luteal phase on folliculogenesis might not be due to the variation of intrafollicular AAs profile but rather attribute to the peripheral blood AAs profile alteration.

Effects of nitric oxide on steroidogenesis and apoptosis in goat luteinized granulosa cells.

The aim of this study was to investigate effects of nitric oxide (NO) on steroidogenesis and apoptosis in goat luteinized granulosa cells (LGCs). We cultured goat LGCs from healthy follicles in culture medium supplemented with the NO donor sodium nitroprusside (SNP) or the NO synthase inhibitor Nω-Nitro-l-arginine methyl ester hydrochloride (l-NAME), then examined steroid synthesis, oxidative stress and apoptosis in vitro. The results showed that SNP treatment significantly increased the cGMP concentration in the LGCs (P < 0.05), whereas the l-NAME treatment significantly decreased cGMP concentration (P < 0.05). Then Inhibition of NO production significantly inhibited the expression of CYP19A1, a key gene that is involved in sex steroid hormones synthesis and is responsible for the decrease of E2. Inhibition of NO production resulted in an increased percentage of apoptosis, which was accompanied by upregulating expression levels of apoptosis-related markers BAX, CASP3 and CASP9. These data indicate that NO is required for goat LGCs steroidogenesis and cell survival. Furthermore, Inhibition of NO production decreased the expression of mitochondrial biogenesis related genes and proteins (PPARGC1A, NRF-1 and TFAM) and the mtDNA copy number. Simultaneously, inhibition of NO production suppressed the transcription and translation of SOD, GPX1, and CAT, and decreased the glutathione level and increased the 8-OHdG level. However, SNP treatment increased the expression of genes involved in mitochondrial function and biogenesis, and elevated the anti-oxidant stress system and steroid synthesis. Together, our results indicate that NO may up-regulate the expression of PPARGC1A and its downstream factors through the cGMP pathway, thereby decreasing granulosa cell apoptosis, and may participate in the regulation of granulocyte steroid production through the mitochondrial-dependent pathway.

Effects of l-arginine on endometrial microvessel density in nutrient-restricted Hu sheep.

Nutrient deficiency in ruminants can lead to estrus cycle disorders, a decreased pregnancy rate, and reduce birth weight. l-arginine (L-Arg), an important amino acid, can improve uterine homeostasis in pregnant sheep and prevent intrauterine growth restriction (IUGR). However, most studies of L-Arg have been conducted on pregnant sheep and few have reported the effects of L-Arg on microvessel density (MVD) in the non-pregnant ovine endometrium. The processes of normal uterine cyclical development and implantation are dependent on a balanced of endometrial MVD. In this study, female Hu sheep were randomly assigned to either a control group (n = 6), a nutrient-restricted group (n = 6), or an L-Arg supplemented nutrient-restricted group (n = 6). The effects of L-Arg on MVD in ovine endometrium were then studied. Our results showed that ovine endometrial MVD was significantly increased by nutrient restriction, but L-Arg counteracted the effect of nutrient restriction on MVD (P < 0.05). Levels of angiogenic growth factors (including VEGFA, VEGFR2, and FGF2) had significant increases (P < 0.05) in endometrium of nutrient restriction on sheep, but that L-Arg supplementation substantially decreased (P < 0.05) their expressions in nutrient restriction sheep. Furthermore, oxidative stress caused by nutrient restriction was also alleviated by L-Arg supplementation in the ovine endometrium. Overall, the results suggested that L-Arg has crucial roles in maintaining the balance of endometrial MVD and angiogenic growth factors, and increasing anti-oxidation capability in the endometrium of nutrient-restricted sheep. This study will provide a theoretical basis and technical means for the normal development of endometrial microvessels in low nutrition level.

In vitro influence of selenium on the proliferation of and steroidogenesis in goat luteinized granulosa cells.

In this study, we investigated the effects of Selenium (Se) on the proliferation of and steroidogenesis in goat luteinized granulosa cells (LGCs) and elucidated the mechanisms underlying these effects. Our results showed that proliferating cell nuclear antigen (PCNA), Akt, and phosphoinositide 3-kinase (PI3K) were expressed mainly in ovarian oocytes and granulosa cells (GCs). We observed that 5 ng/mL Se significantly stimulated LGC proliferation, which could be attributed to increases in PCNA, cyclin-dependent kinase 1 (CDK1), phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK; Thr172), and phosphorylated Akt (p-Akt; Ser473) and decreases in p21 (P < 0.05). Se treatment also significantly increased estradiol (E2) production, which could be, at least partially, due to increased levels of 3ß-hydroxysteroid dehydrogenase(3ß-HSD), steroidogenic acute regulatory protein (StAR), p-Akt (Ser473), and cyclic adenosine monophosphate (cAMP) (P < 0.05); however, follicle-stimulating hormone (FSH) significantly enhanced the production of E2, progesterone (P4) and cAMP (P < 0.05). Moreover, Se treatment stimulated proliferation and the synthesis of E2 and cAMP in the presence of FSH (P < 0.05). Additionally, the expression of antioxidant-related genes [glutathione peroxidase (GSH-Px) and superoxide dismutase 2 (SOD2)] and the activity of GSH-Px and SOD were progressively elevated by Se treatment (P < 0.05). These data suggested that Se plays an important role in the proliferation of and steroidogenesis in LGC by activating the PI3K/Akt and AMPK pathways, thereby increasing the expression of its downstream cell-cycle- and steroid-synthesis-related genes, as well as regulating cellular oxidative stress.

Effects of nutrient restriction and arginine treatment on oxidative stress in the ovarian tissue of ewes during the luteal phase.

The aim of this study was to determine whether nutrient restriction and arginine treatment affect energy metabolism changes and oxidative stress through the mitochondrial pathway in the ovarian tissue of ewes during the luteal phase. On days 6-15 of the estrous cycle, 24 multiparous Hu sheep (BW = 43.56 ±â€¯1.53 kg) were randomly assigned to three groups: control group (CG; n = 6), restriction group (RG; n = 9), and l-arginine group (AG; n = 9) administered Arg treatment (or vehicle) three times per day. The ewes were slaughtered at the end of treatment, and blood samples and ovaries were collected for analysis. In this study, the expression levels of antioxidase enzymes (SOD2, CAT and GPX1) and mitochondrial biogenesis-related genes (ESRRA and TFAM), as well as antioxidase activity and mitochondrial function were examined in ovarian tissue. Nutrient restriction resulted in activation of ESRRA and TFAM and an increase in relative mtDNA copy number, whereas arginine treatment led to a pronounced recovery of ovarian tissue. In addition, we observed increased AMPK phosphorylation at Thr172 and SIRT3 levels in nutrient restricted ewes, and these effects decreased with arginine treatment. In conclusion, the present results indicated that short-term nutritional restriction led to changes in energy metabolism and oxidative stress. These changes disrupted the redox balance, thus leading to apoptosis through the mitochondria-dependent apoptosis pathway. Arginine treatment altered gene expression in ovarian tissue and increased the resistance to oxidative stress and the anti-apoptosis capacity. The results presented here suggest a potential method to increase agricultural productivity and economic benefits in the sheep industry by using dietary supplementation with arginine to decrease temporary undernutrition of ewes.

A network pharmacology approach to investigate the pharmacological effects of Guizhi Fuling Wan on uterine fibroids.

To investigate the pharmacological mechanism of Guizhi Fuling Wan (GFW) in the treatment of uterine fibroids, a network pharmacology approach was used. Information on GFW compounds was collected from traditional Chinese medicine (TCM) databases, and input into PharmMapper to identify the compound targets. Genes associated with uterine fibroids genes were then obtained from the GeneCards and Online Mendelian Inheritance in Man databases. The interaction data of the targets and other human proteins was also collected from the STRING and IntAct databases. The target data were input into the Database for Annotation, Visualization and Integrated Discovery for gene ontology (GO) and pathway enrichment analyses. Networks of the above information were constructed and analyzed using Cytoscape. The following networks were compiled: A compound-compound target network of GFW; a herb-compound target-uterine fibroids target network of GWF; and a compound target-uterine fibroids target-other human proteins protein-protein interaction network, which were subjected to GO and pathway enrichment analyses. According to this approach, a number of novel signaling pathways and biological processes underlying the effects of GFW on uterine fibroids were identified, including the negative regulation of smooth muscle cell proliferation, apoptosis, and the Ras, wingless-type, epidermal growth factor and insulin-like growth factor-1 signaling pathways. This network pharmacology approach may aid the systematical study of herbal formulae and make TCM drug discovery more predictable.

Vitamin D receptor expression and potential role of vitamin D on cell proliferation and steroidogenesis in goat ovarian granulosa cells.

This study aimed to investigate the expression of the vitamin D receptor (VDR) in goat follicles and to determine the effects of Vit D3 supplementation on goat granulosa cells (GCs) function linked to follicular development. The results demonstrated that VDR was prominently localized in GCs, with expression increasing with follicle diameter. Addition of Vit D3 (1α,25-(OH)2VD3; 10 nM) to GCs caused an increase in VDR and in steroidogenic acute regulator (StAR) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) mRNA expression. Additionally, Vit D3 increased the cyclic adenosine monophosphate (cAMP), estradiol (E2), and progesterone (P4) levels, while it decreased anti-müllerian hormone receptor (AMHR) and follicle-stimulating hormone receptor (FSHR) mRNA expression (P < 0.05). Addition of FSH remarkably increased E2, P4, and cAMP levels (P < 0.05), and Vit D3 further enhanced the E2 and cAMP levels in the presence of FSH (P < 0.05). Vit D3 significantly induced the mRNA expression of CDK4 and CyclinD1, and downregulated P21 gene expression (P < 0.05). In addition, Vit D3 significantly decreased reactive oxygen species (ROS) production and increased the mRNA and protein expression of superoxide dismutase 2 (SOD2) and catalase (CAT) (P < 0.05). In conclusion, VDR is expressed in GCs of the goat ovaries and Vit D3 might play an important role in GCs proliferation by regulating cellular oxidative stress and cell cycle-related genes. Meanwhile, Vit D3 enhances the E2 and P4 output of GCs by regulating the expression of 3ß-HSD and StAR and the level of cAMP, which regulate steroidogenesis, supporting a potential role for Vit D3 in follicular development.

Effects of diet and arginine treatment during the luteal phase on ovarian NO/PGC-1α signaling in ewes.

The aim of this study was to determine whether arginine (Arg) supplementation of malnourished ewes affects the expression of key NO/PGC-1α signaling pathway genes in the ovary. On Day 6-15 of the estrous cycle, 24 multiparous Hu sheep (BW = 43.56 ± 1.53 kg) were randomly assigned to three groups: control group (CG; n = 6), restriction group (RG; n = 9) and l-arginine group (AG; n = 9), and administered Arg treatment (or vehicle) three times per day. The ewes were slaughtered at the end of treatment, and blood samples and ovaries were collected for analysis. The results of our analyses showed that both short-term feed-restriction and/or supplementation with L-Arg-HCl affected the number of different size follicles observed in the ovary, and the relative day of estrus behavior initiation of ewes. Specifically, the relative day of estrus behavior initiation was significantly advanced in AG compared with that in RG ewes (P < 0.05). Both the number of ≤2 mm-ovarian follicles (P < 0.05) and the total number of ovarian follicles (P < 0.05) were significantly increased in the RG and AG compared with that in the CG ewes. RG ewes exhibited a higher proportion of ≤2 mm (P < 0.05), but a lower proportion of >5 mm follicles than did CG ewes (P < 0.05). The mean number of corpus lutea ≥5 mm was significantly increased in AG as compared to that in either CG or RG ewes. Furthermore, the expression of eNOS, nNOS, iNOS, PDE5A, PDE9A, PRKG2, and PPARGC1A varied significantly among the treatment groups (P < 0.05). GUCY1A3 mRNA levels were significantly increased in RG and AG as compared to those in CG ewes (P < 0.05), whereas conversely, GUCY1B3 mRNA levels were significantly decreased in CG and RG as compared to those in AG ewes (P < 0.05). P53 mRNA levels were found to vary significantly among the three experimental treatment groups (P < 0.05), and similarly, the relative expression levels of P53 were greater in AG and RG than in CG ewes (P < 0.05). The levels of eNOS protein were significantly higher in RG than in either CG or AG ewes (P < 0.05). The relative expression levels of PGC-1α were significantly higher in RG (P < 0.05) and significantly lower in AG ewes (P < 0.05) than in CG ewes. In conclusion, the results of the present study indicate that feed-restriction negatively affects follicular development, and that Arg-supplementation may modulate the expression of key NO/PGC-1α signaling pathway genes in the ovary and thereby accelerate ovulatory processes and the estrous rate. Elucidation of mechanisms underlying these effects of Arg on gene expression in the ewe ovary requires further investigation.

[Study on health-seeking behavior and influencing factors among Chinese hepatitis B surface antigen positive adults].

OBJECTIVE: To analyze the characteristics of health-seeking behaviors and related influencing factors of the community-based hepatitis B surface antigen (HBsAg) positive adults, in China. METHODS: Based on the cohort formed by the HBsAg positive patients, in the national sero-survey project in 2006, we conducted a follow-up programs in 2010 and 2014. In the latest follow-up project, we carried out a cross-sectional study to collect information on health-seeking behaviors of the patients. Questionnaires would include information on clinic visits, diagnosis, regular physical examination and treatments,etc. We used the SPSS 18.0 software for data analysis. RESULTS: Totally, 2 478 HBsAg positive adults (≥18 years old) were followed through, with 34.4% (853/2 478) of them had visited the doctors and diagnosed after they were informed the status of HBsAg positivity, in the 2006-sero-survey program. Among patients who ever visiting the clinic, 51.2% (372/727) of them underwent at least medical examination once a year, with 31.5% (229/727) of them received treatment. Furthermore, 34.5% (79/229) of the treated patients adopted the traditional Chinese medicine or medicine for ' liver protection'. 56.8% (130/229) of the treated patients received antiviral drugs. Data from the binary logistic regression showed that the major influencing factors on clinic visits would include: age, level of education received and residencial areas (rural/urban). CONCLUSIONS: Consciousness on health was low in those community-based HBsAg positive people. Standerdized management and clinical treatment programs should be set up accordingly.

N-carbamylglutamate and L-arginine improved maternal and placental development in underfed ewes.

The objectives of this study were to determine how dietary supplementation of N-carbamylglutamate (NCG) and rumen-protected L-arginine (RP-Arg) in nutrient-restricted pregnant Hu sheep would affect (1) maternal endocrine status; (2) maternal, fetal, and placental antioxidation capability; and (3) placental development. From day 35 to day 110 of gestation, 32 Hu ewes carrying twin fetuses were allocated randomly into four groups: 100% of NRC-recommended nutrient requirements, 50% of NRC recommendations, 50% of NRC recommendations supplemented with 20g/day RP-Arg, and 50% of NRC recommendations supplemented with 5g/day NCG product. The results showed that in maternal and fetal plasma and placentomes, the activities of total antioxidant capacity and superoxide dismutase were increased (P<0.05); however, the activity of glutathione peroxidase and the concentration of maleic dialdehyde were decreased (P<0.05) in both NCG- and RP-Arg-treated underfed ewes. The mRNA expression of vascular endothelial growth factor and Fms-like tyrosine kinase 1 was increased (P<0.05) in 50% NRC ewes than in 100% NRC ewes, and had no effect (P>0.05) in both NCG- and RP-Arg-treated underfed ewes. A supplement of RP-Arg and NCG reduced (P<0.05) the concentrations of progesterone, cortisol, and estradiol-17ß; had no effect on T4/T3; and improved (P<0.05) the concentrations of leptin, insulin-like growth factor 1, tri-iodothyronine (T3), and thyroxine (T4) in serum from underfed ewes. These results indicate that dietary supplementation of NCG and RP-Arg in underfed ewes could influence maternal endocrine status, improve the maternal-fetal-placental antioxidation capability, and promote fetal and placental development during early-to-late gestation.

A Network Pharmacology Approach to Explore the Pharmacological Mechanism of Xiaoyao Powder on Anovulatory Infertility.

Aim. To explore the pharmacological mechanism of Xiaoyao powder (XYP) on anovulatory infertility by a network pharmacology approach. Method. Collect XYP's active compounds by traditional Chinese medicine (TCM) databases, and input them into PharmMapper to get their targets. Then note these targets by Kyoto Encyclopedia of Genes and Genomes (KEGG) and filter out targets that can be noted by human signal pathway. Get the information of modern pharmacology of active compounds and recipe's traditional effects through databases. Acquire infertility targets by Therapeutic Target Database (TTD). Collect the interactions of all the targets and other human proteins via String and INACT. Put all the targets into the Database for Annotation, Visualization, and Integrated Discovery (DAVID) to do GO enrichment analysis. Finally, draw the network by Cytoscape by the information above. Result. Six network pictures and two GO enrichment analysis pictures are visualized. Conclusion. According to this network pharmacology approach some signal pathways of XYP acting on infertility are found for the first time. Some biological processes can also be identified as XYP's effects on anovulatory infertility. We believe that evaluating the efficacy of TCM recipes and uncovering the pharmacological mechanism on a systematic level will be a significant method for future studies.