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.

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.

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.

The effect of taurine on the toll-like receptors/nuclear factor kappa B (TLRs/NF-κB) signaling pathway in Streptococcus uberis-induced mastitis in rats.

To investigate whether taurine ameliorates mammary damage in a rat model of S. uberis mastitis by suppressing inflammation related to the toll-like receptors/nuclear factor kappa B (TLRs/NF-κB) signaling pathway. 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 to rats. Seventy-two hours after parturition, rats were infused with 100 cfu of S. uberis into each of 2 mammary glands. The resultant inflammation, evidenced by swelling, degeneration of secretory epithelium, increased tissue loss and neutrophil (PMN) infiltration was observed. Pretreatment with taurine attenuated inflammatory changes and significantly decreased mRNA expression of TLR-2 (8 h post S. uberis-injection, PI), NF-κB p65 (16 h and 24 h PI), and NF-κB DNA binding activity (16 h PI). Tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS) levels were also decreased. Significant differences (P<0.05) were present at 24 h and 48 h PI for TNF-α and at 16 h PI for iNOS. TLR-4 mRNA expression was increased by taurine administration and significant differences were observed at 8h, 16 h and 24 h PI. These results suggest that the in vivo relationship of immunomodulatory reagents with TLRs is complex. Taurine may modulate inflammatory injury induced by S. uberis in mammary glands though TLR-2 and TLR-4. Suppression of inflammation may be related to TLRs/NF-κB and may be one mechanism of taurine action in controlling S. uberis mastitis.

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.

Retinoic acid attenuates lipopolysaccharide-induced inflammatory responses by suppressing TLR4/NF-kappaB expression in rat mammary tissue.

The retinoids, a group of natural or synthetic derivatives of vitamin A, exert various anti-neoplastic and immunomodulatory actions. Recent studies have demonstrated that retinoic acid protects rats against lipopolysaccharide (LPS)-induced mastitis, but the mechanism of action is unclear. In the present study, an LPS-induced rat mastitis model and primary cultures of rat mammary epithelial cells were used to investigate the effect of retinoic acid on the TLR4/NF-kappaB signaling pathway. The data indicated that toll-like receptor 4 (TLR4) gene expression reached its peak value earlier in retinoic acid-treated rats than in the control group, and that retinoic acid significantly decreased NF-kappaB DNA binding activity and the level of IL-1beta in the mammary gland. The animal study result was confirmed by an in vitro cell culture system trial. TLR4 protein expression and NF-kappaB DNA binding activity were significantly decreased in primary rat mammary epithelial cells pretreated with 1mumol/l retinoic acid at 1h post-LPS stimulation. IL-1beta gene expression was also significantly decreased at 2, 4 and 8h post-LPS stimulation. These findings demonstrate that direct action by retinoic acid leads to attenuation of the LPS-induced inflammatory response by suppression of the TLR4/NF-kappaB signalling system, thereby providing a novel explanation for the underlying effect proposed for retinoic acid in the protection of mammary tissue during LPS-induced 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.

Retinoid protects rats against neutrophil-induced oxidative stress in acute experimental mastitis.

Activated polymorphonuclear neutrophilic leukocytes (PMN) are able to produce large quantities of bactericidal molecules such as reactive oxygen species (ROS) that are associated with tissue damage in models of inflammatory mastitis. In this study, the putative protective effect of retinoid was evaluated in a lipopolysaccharide (LPS) induced mastitis model in rats. Commencing at 10 d of gestation, retinoid (dissolved in olive oil) or an equal volume of olive oil were administered daily by gavage to pregnant rats until parturition. LPS or pyrogen-free physiological saline were infused into the mammary gland 72 h after parturition. At pre-infusion (defined as 0 h) and at 2, 4, 8, 16 and 24 h post-infusion, six rats from each group were euthanized. Retinoid administration decreased PMN accumulation in mammary alveoli, significantly decreased the level of TNF-alpha in mammary tissues and IL-8 in serum at the different time points. ROS release was significantly increased after LPS infusion and was reduced by retinoid at 16 h PI. Retinoid reduced N-acetyl-beta-D-glucosaminidase (NAGase) activity in both serum and mammary tissue at 8 h PI. Intercellular adhesion molecule 1 (ICAM-1) mRNA expression reached its peak value earlier in retinoid treated rats than in the control group. Overall, the results suggest that activated PMN play an important role in the pathogenesis of acute mastitis and retinoid administration may be an effective tool for protecting mammary tissue against PMN-induced oxidative stress during LPS-induced acute mastitis.

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.

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.

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.