[Key to Indigo Naturalis' oral effectiveness: in vivo and in vitro verification of bile action].

Indigo Naturalis( IN) is mainly composed of 10% organic matter and 90% inorganic matter,with a poor wettability and strong hydrophobicity. Indigo,indirubin and effective ingredients are almost insoluble in water. And how it exerts its effect after oral administration still needs to be revealed. For this reason,this study put forward the hypothesis that " Indigo Naturalis forms a slightly soluble calcium carbonate carrier in a strong acid environment of gastric fluid,and organic substances are solubilized in the bile environment of intestinal fluid",and then verified the hypothesis. First,the dissolution apparatus was used to simulate the change process of IN in different digestive fluid,and the effects of low-dose and normal bile on the dissolution of inorganic substances and the release of organic substances were compared. After the surface morphology and element changes of IN in different digestive fluid were observed,it was found that bile is the key to promoting the dissolution of organic and inorganic substances in IN. Furthermore,the rat fever model induced by 2,4-dinitrophenol was used to study the antipyretic effect of IN in normal rats and bile duct ligation rats. It was found that the antipyretic effect of IN on normal rats was better than that of bile duct ligation rats. The above results indicated that after oral administration of IN,the calcium carbonate carrier was transformed into a slightly soluble state in acidic gastric fluid,and a small amount of organic matter was released. When IN entered the intestinal fluid mixed with bile,the carrier dissolved in a large amount,and indigo and indirubin were dissolved in a large amount,so as to absorb the blood and exert the effect. This study has a certain significance for guiding clinical application of IN. For patients with insufficient bile secretion( such as bile duct resection),oral administration with IN may not be effective and shall be paid attention.

Electrical stimulation boosts seed germination, seedling growth, and thermotolerance improvement in maize (Zea mays L.).

Electrical signaling, similar to chemical signalings such as calcium (Ca2+), reactive oxygen species (ROS, mainly hydrogen peroxide: H2O2), nitric oxide (NO), and hydrogen sulfide (H2S), regulates many physiological processes. However, the effect of electrical stimulation on seed germination, seedling growth, and thermotolerance improvement in maize was little known. In this study, using maize as materials, the effect of electrical stimulation on seed germination, seedling growth, and thermotolerance improvement in maize was explored. The results suggested that electrical stimulation with optimal intensity boosted germination rate and seedling growth (as indicated in the increase in the length of shoots and roots, as well as fresh weight) under normal germination conditions. In addition, electrical stimulation augmented the survival rate of maize seedlings, mitigated the decrease in the tissue vitality, and reduced the peroxidation of membrane lipids under heat stress. These data suggested that electrical stimulation could boost seed germination, seedling growth, and thermotolerance improvement in maize.

Leptin exerts proliferative and anti-apoptotic effects on goose granulosa cells through the PI3K/Akt/mTOR signaling pathway.

Leptin was known as a pivotal regulator for the control of food intake and energy expenditure. However, leptin has also been found to be involved in the regulation of female reproductive system through interactions with pathways in the hypothalamic-hypophyseal axis and direct action at the ovarian level. In the present study, granulosa cells from goose ovarian preovulatory (F1-F3) follicles were cultured with leptin (0, 1, 10 or 100ng/ml). The proliferative and anti-apoptotic actions of leptin in granulosa cells were revealed by CCK-8, BrdU and TUNEL assays. Quantitative real-time PCR and Western blot analyses further indicated that leptin treatment led to increased expression of cyclin D1, cyclin D2, cyclin D3 and bcl-2, and decreased expression of p21 and caspase-3. The effects were involved in the activation of the PI3K/Akt/mTOR signaling pathway, as leptin treatment enhanced the expression of PI3K, Akt1, Akt2, Raptor, mTOR, S6K and p-S6K. Moreover, blockade of the PI3K/Akt/mTOR pathway attenuated the influences of leptin on proliferation and apoptosis of granulosa cells, considering that activated factors by leptin were inhibited in the presence of either 20µM LY294002 (a PI3K inhibitor) or 10µM rapamycin (an mTOR inhibitor). In addition, leptin had a modulatory effect on the expression of its receptor at the transcriptional and translational levels, and blockade of PI3K/Akt/mTOR inhibited both basal and leptin-induced Lepr gene and protein expression. These findings suggest that leptin exerts its proliferative and anti-apoptotic effects on goose granulosa cells through the PI3K/Akt/mTOR signaling pathway via interaction with its receptor.

Isolation, culture and differentiation of duck (Anas platyrhynchos) preadipocytes.

In the present study, we isolated preadipocytes from the adipose tissue of Peking duck and subsequently cultured them in vitro. Cell counting kit-8 assay was employed to establish the growth curve of duck primary preadipocytes. Meanwhile, after the cells reaching full confluency, they were induced to differentiate into mature adipocytes by the addition of a cocktail containing dexamethasone, insulin, 3-isobutyl-1-methylxanthine, and oleic acid for 8 days. Successful differentiation was demonstrated by the development of lipid droplets and the expression of key marker genes including peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer binding protein-α (CEBP/α) and adipocyte fatty acid-binding protein (FABP4). Our results showed that duck primary preadipocytes began to adhere 12 h after seeding as short spindle shapes or litter triangles, which grew quickly 3 days post attachment and maintained stable after day 7. After 8 days the preadipocytes were induced to differentiate into mature adipocytes, which were stained red by oil red O. Additionally, it showed that during preadipocyte differentiation PPARγ mRNA was highly expressed at day 3, while CEBP/α and FABP4 mRNA peaked at day 5 and 8, respectively. These results indicate that we have successfully isolated and cultured Peking duck preadipocytes and successfully induced them to differentiate into mature adipocytes. This work could lay a foundation for further research into waterfowl adipogenesis.

Gene expression patterns, and protein metabolic and histological analyses for muscle development in Peking duck.

In this study, we aimed to use duck breast muscle and leg muscle, the 2 main productive muscle organs, as a model to elucidate the molecular mechanism controlling how the 2 muscles have different deposition capabilities, and to analyze the mechanisms facilitating duck muscle development posthatching. Peking duck breast muscle and leg muscle were collected 3, 7, and 16 wk posthatching. The morphology of the myofibers was observed by paraffin sectioning the muscles. The expression of genes involved in protein metabolism [mammalian target of rapamycin (mTOR), RPS6-p70-protein kinase (S6K), forkhead box O1 (FoxO1), muscle RING finger 1 (MuRF1), and atrogin-1 (MAFbx)] was detected using real-time quantitative PCR and Western blot assays, and the results indicated that breast muscle had a stronger capacity for both protein synthesis and protein degradation compared with leg muscle. Satellite cell frequency declined during muscle development in both tissues, and the expression of Pax3/7, satellite cell marker genes, was not significantly different between breast muscle and leg muscle. No notable apoptosis was observed in either tissue. The results of this study suggest that protein metabolism signaling is the main reason promoting duck skeletal muscle mass gain.

Role of leptin in the regulation of sterol/steroid biosynthesis in goose granulosa cells.

Leptin is critical for reproductive endocrinology. The aim of this study is to assess the expression patterns of leptin receptor (Lepr) during ovarian follicle development and to reveal the mechanism by which leptin affects steroid hormone secretion in goose granulosa cells. Transcripts of Lepr were ubiquitous in all tested tissues, with pituitary and adrenal glands being the predominant sites. Goose ovarian follicles were divided into several groups by diameter including prehierarchical (4 to 6, 6 to 8, and 8 to 10 mm) and hierarchical (F5-F1) follicles. Lepr gene expression was significantly higher in granulosa cells than in theca cells from follicles of 4 to 8 mm in diameter. Expression of Lepr in granulosa cells decreased gradually as follicles developed, with fluctuating expression in F5 and F3 follicles. Lepr mRNA in theca cells underwent a slight decrease from the 6- to 8-mm cohorts to F5 follicle and then exhibited a transient increase and declined later. In vitro experiments in cultured goose granulosa cells showed that estradiol release was significantly stimulated, whereas progesterone increased slightly and testosterone decreased dramatically after leptin treatment. In accordance with the data for steroids, expression of Lepr, Srebp1, Cyp51, StAR, and Cyp19a1 were induced by the addition of leptin, and the concomitant changes in Hmgcs1, Dhcr24, Cyp11a1, 17ß-hsd, Cyp17, and 3ß-hsd gene expression were seen. These results suggested that leptin is involved in the development of goose ovarian follicles, and leptin's effect on steroid hormone secretion could be due to altered sterol/steroidogenic gene expression via interaction with its receptor.