Taurine attenuates lipopolysaccharide-induced disfunction in mouse mammary epithelial cells.

The intent of this study was to evaluate the active defense reaction of mouse mammary epithelial cells and the cytoprotective and anti-inflammatory properties of taurine to lipopolysaccharide (LPS)-induced disfunction in mouse mammary epithelial cells. (1) Primary cultured mouse mammary epithelial cells were stimulated with LPS for 24 h (final concentration=0, 5, 10, 20 µg/mL). Western blotting demonstrated a significant decrease in the secretion of ß-casein in the 20 µg/mL LPS treatment group (P<0.05), while nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), lactoferrin (LF) and N-acetyl-ß-D-glucosaminidase (NAGase) were all significantly increased following LPS treatment (P<0.01). Furthermore, cell survival was significantly inhibited after treatment with 20 µg/mL LPS; however, neither 5 µg/mL nor 10 µg/mL LPS had any effect on cell survival. Therefore, a level of 10 µg/mL LPS was selected to test the protective effect of taurine on mouse mammary epithelial cells. (2) Primary cultured mouse mammary epithelial cells were treated with 0, 5, 15 or 45 mmol/L taurine for 3 h, followed by 10 µg/mL LPS for 24 h. Taurine significantly attenuated the LPS-induced increase in NAGase activity, NO concentrations and the level of TNF-α, IL-1ß, IL-6 and LF. Taurine at 45 mmol/L markedly increased ß-casein secretion in response to LPS-induced disfunction. This study demonstrated that the addition of taurine to a culture medium significantly inhibited the LPS-induced release of inflammatory factors and increased ß-casein secretion from mammary epithelial cells, thereby providing a possible explanation for the protective effect proposed for taurine in the prevention of LPS-induced disfunction in mammary epithelial cells.

Taurine attenuates Streptococcus uberis-induced mastitis in rats by increasing T regulatory cells.

Taurine (Tau) is reported to have a key role in the regulation of the innate immune response and thus reduce tissue damage induced by bacterial infection. In this study, the effects of Tau on a rat model of mastitis induced by Streptococcus uberis (S. uberis) and the changes of T regulatory cells (Tregs) were assessed. Starting on gestation day 14 and continuing until parturition, 100 mg/kg of taurine (group TS) or an equal volume of physiological saline (group CS) was administered daily, per os. Seventy-two hours after parturition, rats were infused with approximately 100 cfu of S. uberis into each of two mammary glands. The results showed that the resultant inflammation, evidenced by swelling, secretory epithelial cell degeneration, increased adipose tissue and neutrophil (PMN) infiltration were evident in mammary tissue following injection with S. uberis. Pre-treatment with Tau attenuated these morphologic changes, the expression of interleukin (IL)-2, interferon (INF)-γ mRNA, myeloperoxidase (MPO) activity and N-acetyl-ß-D-glucosaminidase (NAGase) in mammary tissue. The percentages of Foxp3+CD25+CD4+/lymphocytes (Tregs) were dramatically increased after the S. uberis challenge. Significant differences (P<0.05) were observed at 24, and 72 h post S. uberis-injection (PI) in CS. Pre-treatment further increased the percentage of Tregs and a significant difference between CS and TS (P<0.05) was apparent at 24 h PI. Our data indicate that in rats, Tau can be used to regulate the immune response following infection by S. uberis and consequently prevent mammary tissue damage by increasing Tregs.

Effect of dehydroepiandrosterone on cell growth and mitochondrial function in TM-3 cells.

Dehydroepiandrosterone (DHEA), a major steroid hormone, decreases with age, and this reduction has been shown to be associated with physical health. In the present study, the effect of DHEA on cell growth and mitochondrial function was investigated using TM-3 cells, a Leydig cell line. The growth of TM-3 cells exposed to 100 µM DHEA for 24h was inhibited due to cell cycle arrest, primarily in the S and G2/M phases, and this effect was caused by decreased activity of glucose-6-phosphate dehydrogenase (G6PD) and reduced expression of cyclinA and cyclinB mRNA. A novel finding was that DHEA improved TM-3 cell viability in a markedly time-dependent manner. Although no differences were observed in the configuration or number of TM-3 cell mitochondria following DHEA treatment, mitochondrial membrane permeability and the activity of succinate dehydrogenase (SDH) increased subsequent to 24h treatment of cells with 100 µM DHEA. Overall, the data demonstrate that DHEA inhibited TM-3 cell growth by decreasing G6PD activity and the expression of cyclin mRNAs, whereas it improved TM-3 cell viability by increasing mitochondrial membrane permeability and the activity of SDH. This could be one of mechanisms of DHEA exerts its biological function.

Dectin1 activation of ß-(1-3)/(1-6)-D-glucan produces an anti-mastitis effect in rats.

OBJECTIVE: A lipopolysaccharide (LPS)-induced mastitis model in rats was used to study the protective effect of ß-glucan. MATERIALS AND METHODS: On gestation day 10, ß-glucan was administered to rats daily by gavage at either 0 (control), 0.1 mg/kg BW, 1 mg/kg BW and 10 mg/kg BW until parturition. LPS or pyrogen-free physiological saline was inoculated into the mammary gland 72 h after parturition and the rats were euthanized at 12 h post-infection. RESULTS: LPS increased dectin1 and toll-like receptor 4 (TLR4) mRNA and protein expression significantly in mammary tissues. Tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, N-acetyl-ß-D-glucosaminidase (NAGase), inducible nitric oxide synthase (iNOs) in mammary tissues and serum, and myeloperoxidase (MPO) in mammary tissues were significantly increased 12 h after LPS infusion. ß-glucan administration could enhance dectin1 mRNA and protein expression while downregulating the expression of TLR4. Level of TNF-α, IL-1ß in mammary tissues and serum, MPO, NAGase and iNOs activity in mammary tissues was decreased, but the level of IL-2 in serum was increased by ß-glucan. CONCLUSION: The results indicate that ß-glucan may protect mammary tissue against LPS-induced rat acute mastitis.

Dehydroepiandrosterone activates cyclic adenosine 3',5'-monophosphate/protein kinase A signalling and suppresses sterol regulatory element-binding protein-1 expression in cultured primary chicken hepatocytes.

Dehydroepiandrosterone (DHEA), a steroid hormone that is secreted by the adrenal cortex in mammals, has an array of biological actions, including inhibition of fat synthesis, decreasing the number of adipocytes, and a reduction in mammalian metabolic efficiency. Recent studies showed that DHEA may decrease fat deposition in poultry, but the mechanism of action is unclear. In the present study, we demonstrate that DHEA stimulates intracellular cyclic adenosine 3',5'-monophosphate (cAMP) accumulation in chicken hepatocytes during a 30 min incubation period. Increases in intracellular cAMP are evoked by as low as 0.1 microm-DHEA. The cAMP induced by DHEA, while suppressing cAMP-specific phosphodiesterase activity, also activates cAMP-dependent protein kinase A (PKA) in chicken hepatocytes. In addition, the activation of PKA leads to down-regulation of sterol regulatory element-binding protein-1 (SREBP-1). These findings demonstrate that direct action by DHEA leads to activation of the cAMP/PKA signalling system in the modulation of lipid metabolism by repressing SREBP-1, thereby providing a novel explanation for some of the underlying effects proposed for DHEA in the prevention of fat deposition in poultry.

Reduced prevalence of genotype 3 HEV in Shanghai pig farms and hypothetical homeostasis of porcine HEV reservoir.

A total of 493 fecal samples collected from local Shanghai pig farms were examined for Hepatitis E virus (HEV) after the introduction of stricter sanitary measures following outbreaks of a high fever-associated pig disease during 2006 and 2007. Our investigation revealed that, while the overall occurrence of HEV RNA positives decreased by only 3.7%, the incidence of HEV genotype 4 increased from 9.8% to 20.6% whereas the incidence of HEV genotype 3 decreased from 16.2% to 1.6%. As well as demonstrating that HEV genotype 3 was more sensitive than genotype 4 to the stricter sanitation procedures, our data also suggested that a homeostasis mechanism, whereby the overall incidence of HEV is maintained at a specific population level, might exist in the porcine HEV reservoir. Furthermore, in one case, we encountered the coexistence of HEV genotypes 3 and 4 within the same sample, indicating the possibility of future HEV infections of increased severity and even the occurrence of a HEV pandemic due to genetic recombination and species evolution.

Expression of the nuclear gene TaF(A)d is under mitochondrial retrograde regulation in anthers of male sterile wheat plants with timopheevii cytoplasm.

Alterations of mitochondrial-encoded subunits of the F(o)F(1)-ATP synthase are frequently associated with cytoplasmic male sterility (CMS) in plants; however, little is known about the relationship of the nuclear encoded subunits of this enzyme with CMS. In the present study, the full cDNA of the gene TaF(A)d that encodes the putative F(A)d subunit of the F(o)F(1)-ATP synthase was isolated from the wheat (Triticum aestivum) fertility restorer '2114' for timopheevii cytoplasm-based CMS. The deduced 238 amino acid polypeptide is highly similar to its counterparts in dicots and other monocots but has low homology to its mammalian equivalents. TaF(A)d is a single copy gene in wheat and maps to the short arm of the group 6 chromosomes. Transient expression of the TaF(A)d-GFP fusion in onion epidermal cells demonstrated TaF(A)d's mitochondrial location. TaF(A)d was expressed abundantly in stem, leaf, anther, and ovary tissues of 2114. Nevertheless, its expression was repressed in anthers of CMS plants with timopheevii cytoplasm. Genic male sterility did not affect its expression in anthers. The expression of the nuclear gene encoding the 20 kDa subunit of F(o) was down-regulated in a manner similar to TaF(A)d in the T-CMS anthers while that of genes encoding the 6 kDa subunit of F(o) and the gamma subunit of F(1) was unaffected. These observations implied that TaF(A)d is under mitochondrial retrograde regulation in the anthers of CMS plants with timopheevii cytoplasm.

Protective effect of CpG-DNA against mastitis induced by Escherichia coli infection in a rat model.

A mastitis model in rats, induced by Escherichia coli infection, was established and the protective effect of Cytosine-phosphate-Guanosine (CpG)-DNA was determined. An E. coli suspension containing either 2 x 10(3) colony forming units (CFU)mL(-1)(EL group), 2 x 10(5)CFU mL(-1) (EH group), or (as controls) 100 microL phosphate buffer saline (CON group), was inoculated into the mammary glands 72 h after parturition. The rats were euthanased 24 h post-infection. The histopathological changes in mammary tissue in the EL group were mild, whereas the structural changes in the EH group were severe and polymorphonuclear leukocytes (PMNs) had accumulated in the mammary alveoli. Interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha and N-acetyl-beta-d-glucosaminidase (NAGase) were significantly increased in the mammary tissue from the EH group but not significantly changed in the EL group. On the basis of these findings, the potential protective effect of CpG-DNA on mammary glands was tested using a 2 x 10(5)CFU mL(-1) suspension. An intramuscular injection of either CpG-DNA (200 microg) or PBS (100 microL) was given immediately after parturition. At 72 h post-partum, 2 x 10(5)CFU mL(-1)E. coli (100 microL) were inoculated into the mammary glands of all rats. At pre-infection (0 h), and 8, 16, 24, 48 and 72 h after inoculation six rats were euthanased. CpG-DNA induced more rapid migration of PMNs from the blood to mammary tissue at the initial stage of infection, stimulated the secretion of IL-6 and TNF-alpha at different time points, reduced viable E. coli in mammary tissues and decreased the activity of NAGase. CpG-DNA also promoted the expression of its specific receptor TLR-9 mRNA in mammary tissue. The study showed that CpG-DNA protected against E. coli mastitis in this rat model.

Evaluation of the changes of immune cells during lipopolysaccharide-induced mastitis in rats.

The aim of this study was to evaluate in rats, changes in peripheral blood immune cells and mammary tissue after an intramammary infusion of lipopolysaccharide (LPS). The results of the study showed that infusion of LPS induced a rapid migration of neutrophils (PMNs) from the blood to mammary alveoli, increased the activity of myeloperoxidase (MPO) and the concentration of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6) in mammary tissues, decreased the activity of myeloperoxidase in serum and reduced the CD4+/CD8+ ratio. This is the first report of changes in peripheral blood immune cells and mammary tissue in rat mastitis.

Protective effect of CpG-DNA against mastitis induced by Staphylococcus aureus infection in a rat model.

A mastitis model in rats, induced by Staphylococcus aureus infection, was established and the protective effect of CpG-DNA on this model was determined. A S. aureus suspension containing 2 x 10(3) CFU.mL(-1) (SL group), 2 x 10(5) CFU.mL(-1) (SH group) or 100 microL PBS (CON group) was inoculated into the mammary glands of rats 72 h after parturition. The rats were euthanized at 24 h post-infection. The histopathologic changes in mammary tissue from SL were mild, whereas the structural changes of the mammary gland from SH were severe and polymorphonuclear leukocytes (PMNs) accumulated in mammary alveoli. Interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and N-acetyl-beta-d-Glucosaminidase (NAGase) in mammary tissue from SH were significantly increased, however, those from SL were not significantly changed. Therefore, 2 x 10(5) CFU.mL(-1) was selected to test the potential protective effect of CpG-DNA on mammary glands. CpG-DNA (200 microg) or PBS (100 microL) controls were intramuscularly injected right after parturition of rats. At 72 h post-partum, 2 x 10(5) CFU.mL(-1)of S. aureus (100 microL) were inoculated into the mammary gland of all rats and at pre-infection (0 h), 8, 16, 24, 48 and 72 h after inoculation six rats were euthanatized. CpG-DNA induced more rapid migration of PMNs from blood to mammary tissue at the initial stage of infection, stimulated the secretion of IL-6 and TNF-alpha at different time points, reduced viable S. aureus in mammary tissue and decreased the activity of NAGase. CpG-DNA also promoted the expression of its specific receptor TLR-9 mRNA in mammary tissue. In conclusion, CpG-DNA protected against S. aureus mastitis in a rat model.

Effects of intra-gastric beta-casomorphin-7 on somatostatin and gastrin gene expression in rat gastric mucosa.

AIM: To investigate the in vivo effect of beta-casomorphin-7 on the regulation of gastric somatostatin and gastrin messenger RNA in rat gastric mucosa. METHODS: Somatostatin and gastrin mRNA were quantified by RT-PCR and in situ hybridization (ISH) in 24 rats. The rats were divided into three treatment groups: basal diet + physiological saline (n=8), basal diet + beta-casomorphin-7 (7.5 x 10(-7)mol) (n=8), and basal diet + poly-Gly-7 (containing equal mol of N with 7.5 x 10(-7) mol beta-casomorphin-7) (n=8). After oral administration for 30 days, rats were killed by exsanguinations. RESULTS: After intra-gastric administration of beta-casomorphin-7 for 30 d, gastrin mRNA increased by 52.8% (P<0.05, n=8), and somatostatin mRNA levels decreased by 30.7% compared with the controls (P<0.01, n=8). No significant differences in the expression of the two genes were observed in the poly-Gly-treated group, although gastrin mRNA expression was elevated by 35.6% as against the control group (P=0.15, n=8). The long-term oral administration of a casomorphin solution significantly decreased the even gray of D-cells, but did not lower the number of D-cells both in the antrum and fundus. Interestingly, the number of G-cells increased in the antrum and fundus, but its average density was augmented only in the antrum. CONCLUSION: Beta-casomorphin-7 is capable of modulating gene expression of the regulatory peptides from G and D cells. Data from in situ hybridization studies indicate that beta-casomorphin-7 affects gastrin gene expression indirectly by means of the paracrine action of somatostatin, and depends on its intrinsic molecular function.

CpG-ODN enhances mammary gland defense during mastitis induced by Escherichia coli infection in goats.

Seven healthy native goats in early lactation, weighing 30-40 kg, were used in this study. The right mammary gland of the seven does were infused with CpG-ODN at a dosage of 100 microg kg(-1) body weight on the day 5 postpartum (PP). The left glands were used as controls and infused with sterile phosphate-buffered saline (PBS). On day 8 PP, the same dosage of CpG-ODN or PBS was again infused. On day 9 PP, the mammary glands (both right and left) of the seven does were infused with 6 x 10(6) colony-forming units (CFU) Escherichia coli and, at 0, 8, 16, 24, 48 and 72 h postinfection (PI), milk samples were collected from all glands. Goats were euthanized at 72 h PI and the mammary tissue harvested. Infusion with 6 x 10(6)CFU ml(-1)E. coli induced acute mastitis. Histopathological evaluations showed that polymorphonuclear neutrophils (PMNs) were still present in alveoli at 72 h PI, but PMNs in the CpG-ODN-treated glands has disappeared. Bacteria counts in milk peaked at 16 h PI and CpG-ODN induced a significant decrease in viable bacteria from 16 h PI until the end of the experiment. This study showed that CpG-ODN promoted the expression of its specific receptor (TLR-9 mRNA) in mammary tissue, stimulated IL-6 production, reduced bacteria counts in milk, attenuated the impact of inflammation mediators on cells and significantly shortened the inflammation course. These results suggest that the CpG-ODN improved mammary gland defense and, thereby, had a beneficial effects against mastitis caused by E. coli infection in goats.

Effects of in ovo administration of DHEA on lipid metabolism and hepatic lipogenetic genes expression in broiler chickens during embryonic development.

In order to study the mechanism of DHEA (Dehydroepiandrosterone) in reducing fat in broiler chickens during embryonic development, fertilized eggs were administrated with DHEA before incubation and its effect on lipid metabolism and expression of hepatic lipogenetic genes was investigated. The mRNA levels of acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), malic enzyme (ME), apolipoprotein B100 (apoB100) and sterol regulator element binding protein-1c (SREBP-1c) were determined using real time quantitative PCR. Samples of livers were collected from the chickens on days 9, 14, and 19 of embryonic development as well as at hatching. Blood samples were extracted on days 14, 19 of incubation and at hatching. The results showed that DHEA decreased the concentration of triacyglycerol in the blood and the content in liver, and the mRNA levels of ACC, FAS, ME, SREBP-1c and apoB. This suggested that DHEA decreased the expression of hepatic lipogenetic genes and suppressed triglycerols transport, by which it reduced the deposition of fat in adipose tissue in broiler chickens during embryonic development and hatching.

Effects of dehydroepiandrosterone (DHEA) on hepatic lipid metabolism parameters and lipogenic gene mRNA expression in broiler chickens.

The aim of the present study was to identify the effects of dehydroepiandrosterone (DHEA) on hepatic lipid metabolism parameters and lipogenic gene mRNA expression in broiler chickens. A total of 72 1-day-old broiler chicks received a common basal diet with DHEA added at either 0 (control), 5 or 20 mg/kg feed. In the present study, the hepatic triglyceride (TG) concentration was significantly lower in male and female broilers that had bed administered DHEA than in control birds. In contrast, DHEA administration caused a marked rise in the hepatic non-esterified fatty acid (NEFA) concentration in both male and female broilers and also increased lipase (HL) activity in male broilers, while in female birds, no significant differences were observed in HL activity. The expression of peroxisome proliferators-activated receptor alpha (PPARalpha) and carnitine palmitoyl transferase I (CPTI) mRNA was decidedly enhanced following treatment with DHEA, and a similar tendency was also observed in the expression of acyl-Coenzyme A oxidase 1 (ACOX1). However, no significant differences were observed in the expression of either sterol regulatory element binding protein-1c (SREBP-1c) or acetyl CoA carboxylase (ACC) mRNA, except for a decline in the expression of ACC in females treated with 5 mg DHEA/kg. Numerous peroxisomes without a core and an increased number of peroxisomes were evident during morphological observations of broiler livers, in animals that had been treated with DHEA. Overall, the results of the present study indicated that DHEA accelerated lipid catabolism by direct regulation of hepatic lipid metabolism and by induction of relevant gene expression.

Sodium butyrate protects against oxidative stress in HepG2 cells through modulating Nrf2 pathway and mitochondrial function.

Sodium butyrate (NaBu) is a by-product of microbial fermentation of dietary fiber in the gastrointestinal tract and has been shown to increase the activity of antioxidant enzymes, such as catalase or heme oxidase-1, in vivo. However, the mechanism of this effect is still unclear. This study investigated the antioxidant effect of NaBu on HepG2 cells under H2O2-induced oxidative stress. NaBu (0.3 mM) attenuated cell death and accumulation of reactive oxygen species and improved multiple antioxidant parameters in H2O2-injured HepG2 cells. NaBu inhibited glycogen synthase kinase-3 beta (GSK-3ß) by increasing the p-GSK-3ß (Ser9) level and promoted nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2), which increased the expression of downstream antioxidant enzymes. Together with promotion of peroxisome proliferator-activated receptor gamma coactivator 1-alpha and mitochondrial DNA copy number, NaBu modulated energy metabolism and mitochondrial function, decreasing glycolysis, increasing ß-oxidation, and enhancing the tricarboxylic acid cycle and oxidative phosphorylation. NaBu increased mitochondrial manganese-superoxide dismutase and glutathione peroxidase activity. In conclusion, NaBu protected HepG2 cells against oxidative stress by modulating Nrf2 pathway activity and mitochondrial function.

Separation of growth-stimulating peptides for Bifidobacterium from soybean conglycinin.

AIM: To isolate and identify the soybean conglycinin peptides that selectively stimulates the growth of bifidobacteria in vitro, and to investigate the effect of soybean conglycinin peptides on intestinal ecosystem in vivo. METHODS: Soybean conglycinin was purified from soybean seeds by gel filtration (Sepharose-CL-6B). These proteins were submitted to hydrolysis by pepsin. Several growth-stimulating peptides for bifidobacteria were isolated chromatographically from pepsin hydrolysis of soybean conglycinin and identified by means of matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Parallel to in vitro study, in vivo experiments with soybean conglycinin peptides were performed in mice. Ninety male KM mice were randomly assigned into five groups of 16 mice each, and each group was administered for 21d intragastrically with physiological saline (control), conglycinin, pepsin-treated conglycinin (PTC), the most active fraction which isolated from pepsin-treated conglycinin (P2-PTC) and HCl-full hydrolysis of conglycinin (HCl-FHC), respectively. Intestinal microflora were evaluated by standard microbiologic methods and biochemical assays of cecal content samples after treatment. RESULTS: The results showed that the peptides which were isolated from soybean conglycinin could stimulate the growth of bifidobacteria in vitro, and the molecular mass of purified peptides with MALDI-TOF-MS ranged from 693.32 to 1829.55. Compared with control group, in vivo experiments showed that P2-PTC group decreased cecal pH (7.08+/-0.08 vs 7.21+/-0.09, P<0.05) and enterococci counts (5.38+/-0.26 log10CFU/g vs 5.78+/-0.19 log10CFU/g, P<0.05), significantly increased sIgA level (172.08+/-35.40 ng/g vs 118.27+/-33.93 ng/g, P<0.01) and beta-galactosidase activity (1.28+/-0.23 U/g vs 1.82+/-0.58 U/g, P<0.05). CONCLUSION: The results have shown that conglycinin is good source for enzyme-mediated production of peptides which stimulate the growth of bifidobacteria. These peptides are inactive within the sequence of the parent protein but can be released during enzymatic hydrolysis, and in vivo experiments demonstrate that conglycinin peptides may be beneficial for improving gastrointestinal health.

[Effect of soy isoflavones on cAMP/PKA pathway in breast cancer cells of the rat.].

Soy isoflavones have been reported to be natural chemopreventive in several types of human cancer. Daidzein and genistein are two main components of soy isoflavones. In our previous study, they were shown to be anti-proliferative and induce cell cycle arrest at S phase of SHZ-88 rat breast cancer cells. We hypothesized that soy isoflavones might exert its anticancer effect by activating cAMP/PKA pathway. The present study was designed to analyze the effect of soy isoflavones on the cAMP/PKA pathway in SHZ-88 cells. Daidzein and genistein were dissolved in DMSO. Cells were treated with 50 mug/ml daidzein and 15 mug/ml genistein, respectively, and with only equal DMSO in the culture medium as control. The cellular cAMP content was tested by radioimmunoassay (RIA). The activity of adenylate cyclase (AC), phosphodiesterase (PDE) and PKA were measured by RIA and (gamma-(32)P) ATP incorporation. Reverse transcript-polymerase chain reaction (RT-PCR) was used to analyze the expression of cAMP response element binding protein (CREB) mRNA of the cells. The results showed that the concentration of cAMP in the cells treated with 50 mug/ml daidzein and 15 mug/ml genistein was significantly increased by 9.5%and 11.0%, respectively, 5 min later (P<0.05), then increased by 31.0%and 40.3%, respectively, 10 min later (P<0.01), compared with that of the control group cells. The activity of AC was not affected during the course of experiment, but that of PDE was decreased to 71.8%and 71.6%, respectively, in the control group 5 min later (P<0.05). The PKA activity was increased to 125.8%and 122.3%, respectively, in the control group 20 min after the cells were treated with daidzein and genistein (P<0.05), and kept at high level till 40 min after treatment. CREB mRNA of the cells treated with daidzein and genistein was increased by 31.6%and 51.1%, respectively, 3 h later (P<0.05), then began to decrease 6 h after treatment. The current study suggests that soy isoflavones activate the cAMP/PKA pathway in SHZ-88 rat breast cancer cells by inhibiting the activity of phosphodiesterase.

The crosstalk between Dectin1 and TLR4 via NF-κB subunits p65/RelB in mammary epithelial cells.

Mammary epithelial cells (MECs), as part of the functional unit of the udder, are not only responsible for the synthesis of many components in milk that provide necessary nutritional and immunological support to the offspring, but also playing essential roles in the reaction to mastitis pathogens and the initiation of the immune signaling pathway. There are contributions of MECs to the signaling and production of pathogen associated molecular patterns (PAMPs) such as LPS, lipoteichoic acid (LTA), and ß-glucans, but the crosstalk of different PAMPs induces signalings and productions in rat MEC that need further study. In the present study, we have demonstrated that ß-glucan up-regulates Dectin1 and LPS up-regulates TLR4 directly, as confirmed by generation of siDectin1 and siTLR4 in rat MECs. Then our results have described that either ß-glucan or LPS can activate RelB and/or p65 in rat MECs. Furthermore, the association of p65 and RelB has been analyzed that collaboration of ß-glucan and LPS promotes p65/RelB heterodimers, producing inflammatory responses in rat MECs. In conclusion, summary of our present results suggests that ß-glucan can be considered as a potential immuno-modulator, which s with TLR4 via NF-κB subunits to initiate and regulate the innate immunity in rat MECs.

ß-Glucan modulates the lipopolysaccharide-induced innate immune response in rat mammary epithelial cells.

Mastitis, caused by mammary pathogenic bacteria which are frequent implications of Escherichia coli, is an important disease affecting women and dairy animals worldwide. The ß-glucan binding of dectin-1 can induce its own intracellular signaling and can mediate a variety of cellular responses. This work was to investigate the effect of ß-glucan on the lipopolysaccharide (LPS)-induced inflammatory response and related innate immune signaling in primary rat mammary epithelial cells. Cells were treated with serum-free medium added with a DMSO solution containing ß-glucans at concentrations of 0, 1, 5, 25 µmol/L for 12h, and then exposed to 10 µg/mL LPS for 40 min. Moreover, cells were pretreated with BAY 11-7082 to inhibit NF-κB and then successively exposed to 5 µmol/L ß-glucan, 10 µg/mL LPS, 5 µmol/L ß-glucan and 10 µg/mL LPS, according to the specific experimental design. Normal control cultures contained an equal volume of DMSO, which was collected at the same time. After incubating rat mammary epithelial cells for 40 min with 10 µg/mL LPS, TLR4, MyD88 and NF-κB expression all increased (P<0.05), as did the secretion of TNF-α and IL-1ß (P<0.05), but IκB and ß-casein expression both decreased (P<0.05). Treatment with different concentrations of ß-glucan for 12h activated Dectin1/Syk, which subsequently suppressed TLR4, MyD88 and NF-κB expression and TNF-α and IL-1ß secretion. However, it restored the IκB and ß-casein expression that had been induced by the 40 min incubation with 10 µg/mL LPS. Pretreatment with BAY 11-7082 at 10 µmol/L for 2h partially prevented NF-κB induction by LPS, but the presence of ß-glucan prevented this inactivation. BAY 11-7082 could not simultaneously inhibit LPS induction of TLR4, MyD88 and ß-glucan activation of Dectin1/Syk in rat mammary epithelial cells. These findings demonstrated that ß-glucan activation of Dectin1/Syk attenuated LPS induction of TLR4/MyD88/NF-κB and inhibited the LPS-induced inflammation factors in mammary epithelial cells, thereby providing a possibly protective effect of ß-glucan in the prevention of LPS-induced dysfunction in mammary epithelial cells.

Use of comparative proteomics to identify the effects of creatine pyruvate on lipid and protein metabolism in broiler chickens.

Four hundred male chickens were selected to study the effects of pyruvate (Pyr), creatine pyruvate (CrPyr) and creatine (Cr) on the expression of hepatic mitochondrial and cytoplasm proteins associated with lipid and protein metabolism. Mitochondrial purification was accomplished using the two-step differential centrifugation and density gradient method, and the activities of organelle-specific marker enzymes were determined to assess the purity of the mitochondria. Proteins were extracted and fractionated by two-dimensional electrophoresis and the differential protein spots were assessed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. CrPyr reduced fatty acid accumulation by down-regulating adipose differentiation-related protein, inhibited ATP synthase expression, and reduced cholesteryl ester transfer protein (CETP) expression, thus reducing the levels of high density lipoprotein and triglycerol (TG) levels (thereby lowering fat and cholesterol deposition). CrPyr increased the expression of eukaryotic translation initiation factor (eIF) 2B, calreticulin (CRT) and eIF3a, thus promoting protein synthesis. CrPyr up-regulated the expression of fatty acid-binding proteins, CETP and apolipoprotein A-IV in cytoplasmic extracts, and these proteins accelerated the decomposition of fatty acids and TG, thus reducing fat deposition. In conclusion, CrPyr plays an important role in lipolysis and protein synthesis, and this effect was more pronounced than was the effect of Pyr and Cr.