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.

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 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.

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.

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.