Optimization of bovine embryonic fibroblast feeder layer prepared by Mitomycin C.

Feeder cells play important roles in In-vitro culture of stem cells. However, the preparation protocol of feeder cells produced by bovine embryonic fibroblast cells (bEFs) is still lack. In this study, the preparation of bEF-feeder by Mitomycin C was optimized with different concentrations and treatment time. The cell viability of bEFs was detected by CCK8 and 5-Ethynyl-2'-deoxyuridine. The growth of bESCs in each bEFs-feeder group was assessed by alkaline phosphatase staining and CCK8. Quantitative real time PCR was used to detect the mRNA expression of pluripotency-related genes of bESCs. Results showed that the proliferation of bEFs was significantly repressed while bEFs were treated with 14 ug/mL or 16 ug/mL Mitomycin C for 3 h, and the cell viability within 2-4 days after treatment was consistent with the 1st day. The numbers of bESCs clones in bEF-feeder treated with 14 µg/mL Mitomycin C for 3 h or 16 µg/mL Mitomycin C for 3 h were significantly higher than that in bEF-feeder treated with 8 µg/mL Mitomycin C for 8 h or bEFs treated with 6 µg/mL Mitomycin C for 9 h. The mRNA expression of pluripotency-related genes in bESCs cultured by bEF-feeder were higher than the MEF-feeder, the clone morphology of bESCs cultured in bEF-feeder was rounder and sharper than the MEF-feeder. In conclusion, the bEF-feeder prepared with 14 µg/mL Mitomycin C for 3 h or 16 µg/mL Mitomycin C for 3 h could effectively maintains the growth of bESCs, and bEF-feeder is more suitable for bESCs culture than the MEF-feeder.

Transcriptome analysis reveals transforming growth factor-ß1 prevents extracellular matrix degradation and cell adhesion during the follicular-luteal transition in cows.

Ovarian angiogenesis is an extremely rapid process that occurs during the transition from follicle to corpus luteum (CL) and is crucial for reproduction. It is regulated by numerous factors including transforming growth factor-ß1 (TGFB1). However, the regulatory mechanism of TGFB1 in ovarian angiogenesis is not fully understood. To address this, in this study we obtained high-throughput transcriptome analysis (RNA-seq) data from bovine luteinizing follicular cells cultured in a system mimicking angiogenesis and treated with TGFB1, and identified 455 differentially expressed genes (DEGs). Quantitative real-time PCR results confirmed the differential expression patterns of the 12 selected genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified that the MAPK and ErbB pathways, cell adhesion molecules (CAMs), and extracellular matrix (ECM)-receptor interactions may play pivotal roles in TGFB1-mediated inhibition of CL angiogenesis. TGFB1 phosphorylated ERK1/2 (MAPK1/3) and Akt, indicating that these pathways may play an important role in the regulation of angiogenesis. Several genes with specific functions in cell adhesion and ECM degradation were identified among the DEGs. In particular, TGFB1-induced upregulation of syndecan-1 (SDC1) and collagen type I alpha 1 chain (COL1A1) expression may contribute to the deposition of type I collagen in luteinizing follicular cells. These results indicate that TGFB1 inhibits cell adhesion and ECM degradation processes involving ERK1/2, ErbB, and PI3K/Akt signaling pathways, and leads to inhibition of angiogenesis during the follicular-luteal transition. Our results further reveal the molecular mechanisms underlying the actions of TGFB1 in early luteinization.

Corticosterone induces growth hormone expression in pituitary somatotrophs during goose embryonic development.

Treatment of fetal rat and embryonic chicken with exogenous glucocorticoids induces premature differentiation of growth hormone (GH) secreting cells. The effect of corticosterone (CORT) on somatotroph differentiation was mostly studied in pituitary cells in vitro. Currently, there is no evidence for glucocorticoid-mediated induction of somatotroph differentiation during pituitary development in bird species other than chicken. In this study, we sought to find out if in ovo injection of corticosterone into developing goose embryos could induce premature increase of GH in somatotrophs. On embryonic day (e) 15, the albumen of fertile goose eggs was injected with 300 µl of 0.9% saline, 300 µl 5 × 10-8M CORT, or 300 µl 5 × 10-6 M CORT. Embryos were allowed to develop until e20 and e28 and isolated pituitaries were subjected to quantitative real-time PCR and immunocytochemistry to detect GH mRNA and protein, respectively. At e20 and e28, blood from chorioallantoic vessels was subjected to radioimmunoassay for analysis of plasma GH protein. In ovo administration of exogenous corticosterone brought about a 2.5-fold increase in the expression of GH mRNA and increased the in situ expression of GH protein in goose pituitary cells, and enhanced plasma GH levels compared to that of the respective controls at e20. These findings prove that administration of glucocorticoid could stimulate the expression of GH in somatotrophs during goose embryonic development. Our results suggest the probable involvement of membrane glucocorticoid receptor in the corticosterone mediated expression of GH. Together with previously published data, our results suggest that corticosterone mediated induction of GH expression during embryonic development is relatively conserved among different vertebrates.

Time course effect of lipopolysaccharide on Toll-like receptors expression and steroidogenesis in the Chinese goose ovary.

The ovary of Chinese goose is easily infected by microorganisms because of the mating behaviour in water, which causes decreased laying performance. This study investigated the time course effect of lipopolysaccharide (LPS) on the steroidogenesis and mRNA expression of Toll-like receptors (TLRs), a class of key pattern recognition receptor, in the breeding goose ovary. The laying geese were treated intravenously with LPS for 0, 6, 12, 24 and 36 h, and all birds were slaughtered approximately 8 h after oviposition. The expression levels of TLRs in the white and yellowish follicles, and granulosa and theca layers of hierarchical follicles were examined by real-time PCR. All 10 members of avian TLR family were differentially expressed among the different follicular tissues. Moreover, at 24 and 36 h after LPS treatment, the hierarchical follicle morphological structure was altered, but the expression levels of TLRs were still higher than the control. Furthermore, during LPS treatment period, the expression pattern of TLRs 2A and 4 genes was similar to that of TLR15 in the white follicles, TLRs 1B, 5 and 15 in the yellowish follicles, TLRs 7 and 15 in the granulosa layer, and TLRs 1A, 2B, 3, 7 and 15 in the theca layer, which had a negative correlation with the kinetics of plasma P4 and E2 concentrations. In conclusion, the mechanism by which pathogen infection inhibited goose follicular growth and further decreased egg production may involve a gradually enhanced inflammatory response and reduced endocrine function. This may be due to stimulated TLRs in the ovary.

Reproductiveaxis gene regulation during photostimulation and photorefractoriness in Yangzhou goose ganders.

BACKGROUND: The Yangzhou goose is a long-day breeding bird that has been increasingly produced in China. Artificial lighting programs are used for controlling its reproductive activities. This study investigated the regulations of photostimulation and photorefractoriness that govern the onset and cessation of the breeding period. RESULTS: Increasing the daily photoperiod from 8 to 12 h rapidly stimulated testis development and increased plasma testosterone concentrations, with peak levels being reached 2 months after the photoperiod increase. Subsequently, testicular activities, testicular weight, spermatogenesis, and plasma testosterone concentrations declined steadily and reached to the nadir at 5 months after the 12-hour photoperiod. Throughout the experiment, plasma concentrations of triiodothyronine and thyroxine changed in reciprocal fashions to that of testosterone. The stimulation of reproductive activities caused by the increasing photoperiod was associated with increases in gonadotropin-releasing hormone (GnRH), but decreases in gonadotropin-inhibitory hormone (GnIH) and vasoactive intestinal peptide (VIP) gene messenger RNA (mRNA) levels in the hypothalamus. In the pituitary gland, the levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) mRNA abruptly increased during the longer 12-hour photoperiod. The occurrence of photorefractoriness was associated with increased GnIH gene transcription by over 250-fold, together with increased VIP mRNA levels in the hypothalamus, and then prolactin and thyroid-stimulating hormone in the pituitary gland. FSH receptor, LH receptor, and StAR mRNA levels in the testis changed in ways paralleling those of testicular weight and testosterone concentrations. CONCLUSIONS: The seasonal reproductive activities in Yangzhou geese were directly stimulated by a long photoperiod via upregulation of GnRH gene transcription, downregulation of GnIH, VIP gene transcription, and stimulation of gonadotrophin. Development of photorefractoriness was characterized by hyper-regulation of GnIH gene transcription in the hypothalamus, in addition of upregulation of VIP and TRH gene transcription, and that of their receptors, in the pituitary gland.

Investigating right ovary degeneration in chick embryos by transcriptome sequencing.

In asymmetric chick gonads, the left and right female gonads undergo distinct programs during development, generating a functional ovary on the left side only. Despite some progress being made in recent years, the mechanisms of molecular regulation remain incompletely understood, and little genomic information is available regarding the degeneration of the right ovary in the chick embryo testis. In this study, we performed transcriptome sequencing to investigate differentially expressed genes in the left and right ovaries and gene functions at two critical time points; embryonic days 6 (E6) and 10 (E10). Using high-throughput RNA-sequencing technologies, 539 and 1046 genes were identified as being significantly differentially expressed between 6R-VS-6L and 10R-VS-10L. Gene ontology analysis of the differentially expressed genes revealed enrichment in functional pathways. Among these, candidate genes associated with degeneration of the right ovary in the chick embryo were identified. Identification of a pathway involved in ovarian degeneration provides an important resource for the further study of its molecular mechanisms and functions.

[Effect of Coixenolide on Foxp3+ CD4+ CD25+ Regulatory T Cells in Collagen-induced Arthritis Mice].

OBJECTIVE: To study the effect of coixenolide on Foxp3+ CD4+ CD25+ regulatory T cells (Treg) in collagen induced arthritis (CIA) mice, and to explore its possible mechanism for treating rheumatiol arthritis. METHODS: Five mice were recruited as a normal control group from 25 mice, and the rest 20 were used in CIA modeling. After successful modeling they were randomly divided in the model control group and the coixenolide group, 10 in each group. Coixenolide injection at 25 mL/kg was intraperitoneally injected to mice in the coixenolide group, while normal saline at 25 mL/kg was intraperitoneally injected to mice in the normal control group and the model control group. The injection lasted for 21 days. Scoring for CIA was performed after injection and arthritis index was calculated. The peripheral blood Foxp3+ CD4+ CD25+ Treg ratio was determined by flow cytometry (FCM). RESULTS: Compared with the normal control group, the arthritis index obviously increased in the model control group (P < 0.01). The arthritis index obviously decreased more in the coixenolide group than in the model control group (P < 0.01). Foxp3+ CD4+ CD25+ Treg levels obviously decreased more in the model control group than in the normal control group (P < 0.01 ). Foxp3+ CD4+ CD25+ Treg levels obviously increased more in the coixenolide control group than in the model control group (P < 0.01). CONCLUSION: Coixenolide could up-regulate Foxp3+ CD4+ CD25+ Treg ratios in CIA mice, which might play certain immunoregulation roles in the incidence of CIA.

Molecular mechanisms of enhancing porcine granulosa cell proliferation and function by treatment in vitro with anti-inhibin alpha subunit antibody.

BACKGROUND: This study was conducted to clarify the effect of the inhibiting action of inhibin on porcine granulosa cell proliferation and function, and to investigate the underlying intracellular regulatory molecular mechanisms. METHODS: Porcine granulosa cells were cultured in vitro, and were treated with an anti-inhibin alpha subunit antibody, with or without co-treatment of follicle-stimulating hormone (FSH) in the culture medium. RESULTS: Treatment with anti-inhibin alpha subunit antibody led to a significant increase in estradiol (E2) secretion and cell proliferation. Anti-inhibin alpha subunit antibody worked synergistically with FSH at low concentrations (25 microg/mL) to stimulate E2 secretion, but attenuated FSH action at high concentrations (50 microg/mL). Immunoneutralization of inhibin bioactivity increased FOXL2, Smad3, and PKA phosphorylation, and mRNA expression of the transcription factors CEBP and c-FOS. The expression of genes encoding gonadotropin receptors, FSHR and LHR, and of those involved in steroidogenesis, as well as IGFs and IGFBPs, the cell cycle progression factors cyclinD1 and cyclinD2, and the anti-apoptosis and anti-atresia factors Bcl2, TIMP, and ADAMTS were upregulated following anti-inhibin alpha-subunit treatment. Treatment with anti-inhibin alpha subunit down regulated expression of the pro-apoptotic gene encoding caspase3. Although expression of the pro-angiogenesis genes FN1, FGF2, and VEGF was upregulated, expression of the angiogenesis-inhibiting factor THBS1 was downregulated following anti-inhibin alpha subunit treatment. CONCLUSIONS: These results suggest that immunoneutralization of inhibin bioactivity, through augmentation of the activin and gonadotropin receptor signaling pathways and regulation of gene expression, permits the development of healthy and viable granulosa cells. These molecular mechanisms help to explain the enhanced ovarian follicular development observed following inhibin immunization in animal models.

An all-in-one platform to deplete pathogenic bacteria for rapid and safe enrichment of plant-derived extracellular vesicles.

Plant-derived extracellular vesicles (PDEVs) have exhibited several advantages, such as high biocompatibility, improvement of skin conditions, and the prevention of skin aging. However, traditional methods of extraction for plant substances, such as heating under reflux or solvent extraction, are complicated, time-consuming, and low in purity. Accordingly, a simple and efficient platform is necessary for purely isolating natural substances from plants. In this study, we report a newly designed platform for removing impurities to purify PDEVs. The proposed platform comprises three parts: (i) inflow of samples, (ii) depletion of impurities, and (iii) collection of PDEVs. The platform is designed to flow from top to bottom using gravity without the need for electric components. The platform allows the delimitation of impurities, such as the pathogenic bacteria in PDEVs, by capturing magnetic beads coated with Concanavalin A (Con A). We validate the practicality of our platform using extracellular vesicles derived from liquorice (LdEVs). Notably, the LdEVs purified using the Con A-coated magnetic beads provide better cell uptake and wound recovery than the commercialized extract LdEVs. This highlights the therapeutic potential of fresh LdEVs purified using our platform, particularly in preventing skin aging. The findings of this study hold significant practical implications for the cosmeceutical and therapeutic field, providing a promising approach for the extraction and purification of natural substances from plants to harness their benefits effectively.

Bioactive diterpenoids and sesquiterpenoids with different skeletons from Salvia digitaloides Diels.

Salvia has been regarded as a beneficial healing herb in ancient Egypt, Rome and Greece, and is listed as an official medicine in the pharmacopoeias of many countries worldwide. Currently, Salvia is widely used to flavor and preserve food. Here, two undescribed norabietane-type diterpenoids, sadigitaloides A and B, two undescribed germacrane-type sesquiterpenoids, sadigitaloides C and D, five undescribed guaiane-type sesquiterpenoid lactones, sadigitaloides E-I, two undescribed noreudesmane-type sesquiterpenoids, sadigitaloides J and K, one known diterpenoid, three known sesquiterpenoids, and three other types of known compounds were isolated from the 95% ethanol extract of the whole plants of Salvia digitaloides. Their structures and absolute configurations were characterized using 1D and 2D NMR spectroscopic techniques, electronic circular dichroism (ECD) calculations, HRESIMS experiments, and single-crystal X-ray diffraction analysis. Some compounds were evaluated for their anti-inflammatory activities against lipopolysaccharide (LPS)-induced TNF-α production in rat macrophage NR8383 cells. Sadigitaloide A showed noticeable anti-inflammatory activity at a concentration of 100.0 µM. At a concentration of 60 µM, sadigitaloide B exhibited better protection of dopaminergic neurons than the positive control n-butylidenephthalide in the Caenorhabditis elegans model injured by 6-OHDA. The phytotoxic activities of some compounds were attributed to considerable inhibitory effects on the growth of the roots and hypocotyls of Raphanus sativus L seedlings, especially cis, trans-abscisic acid, whose inhibition rates were much higher than those of glyphosate at concentrations ranging from 50 to 400 ppm. These results indicated that abietane-type diterpenoids possessed excellent anti-inflammatory and neuroprotective activities and further suggested that the low-molecular-weight compounds exhibited outstanding phytotoxic activities.

Highly oxygenated germacrane-type sesquiterpenoids from the whole plant of Salvia cavaleriei H.Lév. and their biological activities.

The entire plant Salvia cavaleriei H.Lév. (Lamiaceae) is used as a traditional Chinese herbal medicine. Its leaves are edible, and the flowers can be soaked in water to make a health-care tea. In an effort to find natural bioactive chemical components, twelve undescribed germacrane-type sesquiterpenoids, salcavalins A-L, were isolated from the whole plant of S. cavaleriei and were identified as analogs. This is the first study to isolate highly oxygenated germacrane-type sesquiterpenoids from this plant. The structures of these undescribed compounds were elucidated by various spectroscopic methods, and their absolute configurations were confirmed by single-crystal X-ray diffraction analysis with Cu Kα radiation and electronic circular dichroism calculations. The biological activity of these undescribed compounds on the production of tumor necrosis factor-alpha in lipopolysaccharide induced NR8383 cells was evaluated, and salcavalins I and K showed anti-inflammatory activity to some extent. Salcavalins A-C, F and L were found to be neuroprotective with antiparkinsonic potential in a nematode (Caenorhabditis elegans) model. In addition, salcavalins F and I displayed marked phytotoxic activity against radish seeds at a low concentration of 50 ppm. Our findings provide scientific justification to show that bioactive sesquiterpenoids from the edible herb have anti-inflammatory in vitro, neuroprotective and phytotoxic activities.

Real-time kinetics and affinity analysis of the interaction between protein A and immunoglobulins G derived from different species on silica colloidal crystal films.

Kinetic and affinity analysis of protein interactions reveals information on their related activities in biological processes. Herein, we established a system for evaluating the kinetics and affinity of the interaction between protein A and various IgG species on the surface of silica spheres of silica colloidal crystal (SCC) films by the extraordinary optical interference capabilities of 190 nm silica spheres after self-assembly. The equilibrium association constant (KA) was calculated by the equilibrium Langmuir model and nonlinear least-squares analysis of time-dependent data. The relative protein A/IgG binding affinity is human > rabbit >cow >goat. In addition, the competitive interaction of distinct species of IgG with protein A at the interface of SCC films was studied and performed. These findings may help with the use of protein A and other recognition components in a number of sensor types. Furthermore, this research might offer a novel approach to determining the kinetics and affinity of proteins on the surface of spheres particles, which may contribute to the development of the application of spheres particles in pharmaceutical science, biomedical engineering, and other techniques.

Transforming growth factor-ß1 disrupts angiogenesis during the follicular-luteal transition through the Smad-serpin family E member 1 (SERPINE1)/serpin family B member 5 (SERPINB5) signalling pathway in the cow.

Intense angiogenesis is critical for the development of the corpus luteum and is tightly regulated by numerous factors. However, the exact role transforming growth factor-ß1 (TGFB1) plays during this follicular-luteal transition remains unclear. This study hypothesised that TGFB1, acting through TGFB receptor 1 (TGFBR1) and Smad2/3 signalling, would suppress angiogenesis during the follicular-luteal transition. Using a serum-free luteinising follicular angiogenesis culture system, TGFB1 (1 and 10ngmL-1 ) markedly disrupted the formation of capillary-like structures, reducing the endothelial cell network area and the number of branch points (P <0.001 compared with control). Furthermore, TGFB1 activated canonical Smad signalling and inhibited endothelial nitric oxide synthase (NOS3 ) mRNA expression, but upregulated latent TGFB-binding protein and TGFBR1 , serpin family E member 1 (SERPINE1 ) and serpin family B member 5 (SERPINB5 ) mRNA expression. SB431542, a TGFBR1 inhibitor, reversed the TGFB1-induced upregulation of SERPINE1 and SERPINB5 . In addition, TGFB1 reduced progesterone synthesis by decreasing the expression of steroidogenic acute regulatory protein (STAR ), cytochrome P450 family 11 subfamily A member 1 (CYP11A1 ) and 3ß-hydroxysteroid dehydrogenase (HSD3B1 ) expression. These results show that TGFB1 regulates NOS3 , SERPINE1 and SERPINB5 expression via TGFBR1 and Smad2/3 signalling and this could be the mechanism by which TGFB1 suppresses endothelial networks. Thereby, TGFB1 may provide critical homeostatic control of angiogenesis during the follicular-luteal transition. The findings of this study reveal the molecular mechanisms underlying the actions of TGFB1 in early luteinisation, which may lead to novel therapeutic strategies to reverse luteal inadequacy.

[A new type of iridium (III) phenylpyrazole complexes: synthesis, photophysical characterization].

New heteroleptic iridium(III) complexes (ppz)2Ir(LX), which consist of two cyclometalated ligands ppz(1-phenylpyrazole) together with an ancillary ligand LX (LX= 2-(2'-hydroxylphenyl)benzothiazole (BTZ), 2-(3'-methyl-2'-hydroxylphenyl) benzothiazole (3-MeBTZ), 2-(4'-methyl-2'-hydroxylphenyl) benzothiazole (4-MeBTZ) and 2-(4'-Trifluoromethyl-2'hydroxylphenyl) benzothiazole (4-tfmBTZ)), were synthesized and characterized. The molecular structures and photophysical properties were characterized and analyzed comparatively. The results show that the four complexes have basically similar UV-Vis absorption spectra, fluorescence excitation and emission spectra. Their maximum emission peaks are located at 583-615 nm, and accompanied by a lower intensity emission band around 400 nm. The weak emissions around 400 nm are ascribed to the radi ation transition of single state excition from ancillary ligand BTZ perturbed by metallic ion, and light emission around long-wave-length to the radiation transition of 3MLCT of Ir(BTZ) fragment. While the triplet state 3 MLCT of Ir(ppz)2 fragment might be quenched at room temperature. For all complexes, the excitations with maximum efficiency are located at 250-310 nm, which indicates that main contributor to light emitting is ligand-centered absorption (1pi-pi*) of ppz and BTZ rather than 3MLCT transitions, and thus provides a striking evidence that there is intersystem crossing from 1pi-pi* state to 3MLCT state in these complexes. Compared with Ir(ppz)3, these complexes not only have stronger phosphorescence at room temperature but also their emission color can be tuned by modifying ancillary ligand.

[Epigenetics and cancer].

Epigenetic modifications, including DNA methylation, histone modifications, RNA epigenetics and nucleosome remodeling, are important for gene transcription, but such modifications do not change the coding sequence of the gene. Although these events are heritable, they are potentially reversible, thus opening up opportunities for the therapy of cancer. So epigenetic modifications have been thrusted into the forefront of new drug discoveries. Current knowledge suggests that the agents that intervene epigenetics by "turning back on" silenced genes may represent a significant advancement in treating many forms of cancer. In this review, we summarized the research progress of epigenetic gene regulation and the proteins which could read epigenetic codes. And the relationship between epigenetics and cancer was discussed comprehensively.

Fermented feed regulates growth performance and the cecal microbiota community in geese.

This study was designed to investigate the effects of fermented feed diets on the growth performance and cecal microbial community in geese, and to examine associations between the gut microbiota and growth performance. A total of 720 healthy, 1-day-old male SanHua geese were used for the 55-D experiment. Geese were randomly divided into 4 groups, each with 6 replicates of 30 geese. Groups were fed a basal diet supplemented with 0.0, 2.5, 5.0, or 7.5% fermented feed. The results showed that 7.5% fermented feed had an increasing trend in the body weight and average daily gain of the geese; however, there was no significant response to increasing dietary fermented feed level with regards to ADFI and FCR. In addition, compared with the control group, there was a higher abundance of bacteria in the phylum Bacteroidetes in the cecal samples of geese in the 7.5% fermented feed group (53.18% vs. 41.77%, P < 0.05), whereas the abundance of Firmicutes was lower in the 7.5% fermented feed group (36.30% vs. 44.13%, P > 0.05). At the genus level, the abundance of Bacteroides was increased by adding fermented feed to geese diets, whereas the abundances of Desulfovibrio, Phascolarctobacterium, Lachnospiraceae_uncultured, Ruminiclostridium, and Oscillospira were decreased. These results indicate that fermented feeds have an important effect on the cecal microflora composition of geese, and may affect host growth, nutritional status, and intestinal health.

Expression of steroidogenic enzymes and synthesis of steroid hormones during development of ovarian follicles in prepubertal goats.

Expression of mRNAs encoding cytochrome P450 side-chain cleavage (P450scc), cytochrome P450 17 alpha-hydroxylase (P450c17), and cytochrome P450 aromatase (P450arom) were characterized by the RT-PCR technique and concentrations of progesterone (P4), testosterone (T0) and estradiol (E2) were measured by radioimmunoassay during follicular development of prepubertal goats. Synthesis of mRNAs encoding P450scc and P450c17 began in preantral follicles, but mRNA encoding P450arom was not detectable until early antral formation. While mRNA for P450scc was expressed in both theca and granulosa cells, mRNA for P450c17 was expressed only in theca cells while P450arom mRNA only in granulosa cells. In nonatretic follicles from prepubertal ovaries, the relative quantity of mRNA expression of all the three enzymes increased with follicle size; however, while the concentration of P4 and E2 increased, that of T0 decreased with follicle size. While expression of mRNA encoding P450scc was unaffected, that of P450c17 mRNA decreased to the lowest level and mRNA for P450arom became undetectable following atresia; accordingly, while the concentration of P4 increased in the atretic medium follicles, that of T0 and E2 decreased to the lowest level after atresia. While the adult follicular stage follicles showed a similar cytochrome expression as the nonatretic follicles of prepubertal goats, the former contained higher levels of E2 and P4 than the latter. The presence of corpus luteum in an ovary decreased expression of P450scc, significantly in large follicles while it increased concentration of P4. These findings indicated that (1) similar to other species, changes in follicular steroid production in goats were explained in large measure by changes in steroidogenic enzyme expression; (2) while mRNA expression was similar, activities of some of the steroidogenic enzymes may differ between sexually mature and immature goats.

Regulatory effect of Langchuang serial recipes on T-lymphocyte subsets Th and Tc in patients with systemic lupus erythematosus.

OBJECTIVE: To study the principle of clearing Fei (), cooling blood, and detoxification as well as nourishing yin and moisening Fei (abbr. as CCD-NM) in regulating the levels of peripheral T-lymphocyte subsets Th and Tc cells to explore its mechanism for lowering the incidence of infection in patients with systemic lupus erythematosus (SLE). METHODS: Sixty SLE patients without complicated infection were assigned to the treatment group and the control group, 30 in each group. The control group was treated with Western medicine alone, while the treatment group was treated with the same program of Western medicine, but additionally administered with either Langchuang No.1 (I) or 2 (II), serial concerted Chinese recipes, applied respectively in patients in the active stage or in the resting stage. The total time of treatment for both groups was 1 year. Further, a healthy control group was set up with 20 healthy subjects. The expressions of Th1, Th2, and Tc1 and Tc2 cells in peripheral blood were detected and compared with those in the healthy control group. RESULTS: (1) As compared with the healthy control group, ratios of Th1/Th2 and Tc1/Tc2 in SLE patients, whether complicated with infection or not, were significantly lower (P<0.05 or P<0.01). (2) Comparison between patients with complications and those uncomplicated with infection showed that the two ratios and Th1 expression were lower and Tc2 was higher in the former than those in the latter (all P<0.05). (3) Ratios of Th1/Th2 and Tc1/Tc2 increased after treatment in patients of both the treatment group and the control group (P<0.05 and P<0.01), but the changes in the treatment group were more significant (P<0.05). CONCLUSION: The principle of CCD-NM could regulate the Th and Tc subsets toward equilibrium in SLE patients, which might be one of the mechanisms of action for alleviating complicated infection.

Protective effect of avicularin on rheumatoid arthritis and its associated mechanisms.

The present study aimed to investigate the effect of avicularin on rheumatoid arthritis (RA) in vitro, and additionally explore the molecular mechanism. To perform this investigation, an in vitro model of RA was established by treatment of the human RA synovial MH7A cell line with tumor necrosis factor-α (TNF-α). MH7A cells were then treated with various concentrations (10, 30, 100 and 300 µM) of avicularin. Then, the levels of inflammatory factors [interleukin (IL)-1ß, IL-6, IL-8, matrix metalloproteinase (MMP)-1 and MMP-13] were measured by ELISA. Cell viability and apoptosis were detected using an MTT assay and flow cytometry, respectively. In addition, the expression levels of genes and proteins were determined reverse transcription quantitative polymerase chain reaction and western blot analysis. The results of the present study indicated that avicularin significantly decreased the levels of inflammatory factors (IL-1ß, IL-6, IL-8, MMP-1 and MMP-13), previously increased by TNF-α, in a dose-dependent manner. Concurrently, avicularin inhibited the mRNA and protein expression levels of iNOS and COX-2 increased by TNF-α. It was also identified that TNF-α administration significantly promoted MH7A cell viability and inhibited cell apoptosis, and avicularin treatment dose-dependently inhibited MH7A cell viability and induced cell apoptosis. In addition, these data suggested that avicularin prevented the activation of the mitogen-activated protein kinase kinase (MEK)/nuclear factor kappa light-chain-enhancer of activated B-cells (NF-κB) pathway activated by TNF-α. Taken together, these results demonstrated that avicularin may inhibit the inflammatory response, prevent cell viability and induce apoptosis in human RA synovial cells through preventing the activation of the MEK/NF-κB pathway.

[Chromosome changes of aged oocytes after ovulation].

In mouse, matured oocytes are arrested at metaphase- (M) after ovulation. If the M oocytes are not fertilized on time, they will increasingly become aged in the oviduct. The aging of oocytes can lead to abnormal development of embryos. So it is important to study the mechanism of oocyte aging. Herein, we studied the change of oocyte chromosome after ovulation into the oviduct in vivo. Results indicated that 65% of old oocytes (34 h post hCG) showed aberrant MII, with chromosome misalignment and dispersal, in contrast to the young oocytes (14 h post hCG). In addition, chromosome changes may be associated with the increase of acetylation of histone 3 and histone 4, at lysine 14 and lysine 16 (H3K14 and H4K16), respectively. On the other hand, the decrease of methylation of histone 3 at lysine 9 (H3K9) presumably facilitated aberrant chromosome formation.