Effects of Soybean Isoflavones on In vitro Antioxidative Capacity of Satellite Cells of Porcine Skeletal Muscles

A synthetic isoflavone （ISO-S） or genistein was added in culture medium at different concentrations （0, 10, 20, 30, 40, and 80 p.mol L^-1） to investigate the effects of soybean isoflavones on antioxidative capacity of porcine skeletal muscle satellite cells. After 48 h incubation, the suspension was cryopreserved for the determination of superoxide dismutase （SOD）, catalase （CAT）, glutathione peroxidase （GSH-Px） activities, and malondialdehyde （MDA） content. The mRNA levels of SOD, CAT, and GSH-Px gene in cells were detected with Taqman fluorescent probe method. The results showed that the content of MDA and the activities and the mRNA levels of SOD of porcine skeletal muscle satellite cells were influenced by supplemented soybean isoflavone （P〈0.05） when adding 10-80 μmol L^-1 ISO-S or genistein in the medium. The MDA contents, SOD and CAT activities and their mRNA expression levels of porcine skeletal muscle cells responded quadratically （P〈 0.05） as the level of ISO-S or genistein increased. Pre-incubation of porcine skeletal muscle satellite cells with ISO-S or genistein at 10-40 pmol L-1 elevated the activities and the mRNA expression levels of SOD and CAT in cells concurrently and decreased the cellular content of MDA （P〈 0.05）. The results indicated that pre-incubation of ISO-S or genistein at 10- 40μmol L^-1 could improve the antioxidative capacity of porcine skeletal muscle satellite cells.

Expression of estrogen receptor alpha,nerve growth factor,interleukin-2,and androgen receptor in the cerebellum of ovariectomized rats following soybean isoflavone treatment

BACKGROUND： Studies have shown that estrogen receptor alpha （ERα）, nerve growth factor （NGF）, interleukin-2 （IL-2）, and androgen receptor （AR） expression in the cerebellum decreases when estrogen levels decrease in vivo. Soybean isoflavone, a type of non-steroid estrogen with similar molecular structure and function to estradiol, exhibits estrogen-like characteristics. OBJECTIVE： To investigate the effects of various doses of soybean isoflavone on expression of ERa, NGF, IL-2, and AR in the cerebellum of ovariectomized rat, and to determine whether there is a dose-dependent effect.DESIGN, TIME AND SETTING： Controlled trial at the cellular and molecular level. The study was performed at the Experimental Animal Engineering Center, College of Veterinary Medicine, Sichuan Agricultural University from July 2006 to May 2008. MATERIALS： Soybean isoflavone, comprised of daidzin, genistein and isoflavone, was provided by Taiyuan Yuantai Biochemical Industry, China. The ERα, NGF, IL-2, and AR in situ hybridization kit, rabbit anti-rat ERa, NGF, IL-2, and AR monoclonal antibodies, and SABC kit were purchased from Wuhan Boster Biological Technology, China. METHODS： A total of 50 female, Sprague Dawley rats, aged 3 months, were randomly assigned to 5 groups, with 10 animals in each group. With the exception of the sham-operation group （abdominal cavity opening alone）, all rats underwent bilateral ovariectomy. At 14 days after surgery, rats in the high-, middle-, and low-dose soybean isoflavone groups were subcutaneously injected with 1.5, 1.0, and 0.5 mg/kg soybean isoflavone, respectively, every 2 days for 6 consecutive weeks. Rats in the sham-operation and ovariectomized groups were subcutaneously injected with absolute alcohol （0.5 mL/kg）. MAIN OUTCOME MEASURES： Expression levels and distribution of ERα, NGF, IL-2, and AR in the cerebellum were detected by immunohistochemistry and in situ hybridization. RESULTS： Compared with the sham-operation group, immunoreactive products and hybridization signals of ERa, NGF, IL-2, and AR were significantly decreased in the cerebellar cortex and nuclei of ovariectomized rats （P 〈 0.05 or P 〈 0.01）, but increased following soybean isoflavone treatment. In particular, levels of the high-dose soybean isoflavone group were almost restored to levels of the sham-operation group （P 〉 0.05）. The immunoreactive products were primarily located in the cytoplasm and neurites, and rarely in the cell membrane and nuclei. However, the hybridization signals were predominantly located in the nuclei, but rarely in the cytoplasm, cell membrane, or neurites. CONCLUSION： Soybean isoflavone upregulated ERα, NGF, IL-2, and AR protein and gene expression in a dose-dependent manner, and played an important role in sustaining and protecting structure and function of cerebellar neurons. Moreover, the similarity of expression patterns of these molecules indicated that they were mutually interactive during the regulation of soybean isoflavone to the cerebellum.

Effects of soybean isoflavone on intestinal antioxidant capacity and cytokines in young piglets fed oxidized fish oil

To investigate the effect of glycitein, a synthetic soybean isoflavone（ISF）, on the intestinal antioxidant capacity, morphology, and cytokine content in young piglets fed oxidized fish oil, 72 4-d-old male piglets were assigned to three treatments. The control group was fed a basal diet containing fresh fish oil, and the other two groups received the same diet except for the substitution with the same dosage of oxidized fish oil alone or with ISF（oxidized fish oil plus ISF）. After 21 d of feeding, supplementation of oxidized fish oil increased the levels of malondialdehyde（MDA）, oxidized glutathione（GSSG）, interleukin-1β（IL-1β）, tumor necrosis factor-α（TNF-α）, interleukin-2（IL-2）, nuclear factor κ B（NF-κB）, inducible nitric oxide synthase（iN OS）, NO, and Caspase-3 in jejunal mucosa, and decreased the villous height in duodenum and the levels of secretory immunoglobulin A（sI gA） and IL-4 in the jejunal mucosa compared with supplementation with fresh oil. The addition of oxidized fish oil plus ISF partially alleviated this negative effect. The addition of oxidized fish oil plus ISF increased the villous height and levels of sI gA and IL-4 in jejunal mucosa, but decreased the levels of IL-1β and IL-2 in jejunal mucosa（P0.05） compared with oxidized fish oil. Collectively, these results show that dietary supplementation of ISF could partly alleviate the negative effect of oxidized fish oil by improving the intestinal morphology as well as the antioxidant capacity and immune function in young piglets.

Apoptosis induction of colorectal cancer cells HTL-9 in vitro by the transformed products of soybean isoflavones by Ganoderma lucidum

Soybean isoflavones have been one of the potential preventive candidates for antitumor research in recent years. In this paper, we first studied the transformation of soybean isoflavones with the homogenized slurry of Ganoderma lucidum. The resultant transformed products（TSI） contained（703.21±4.35） mg/g of genistein, with transformed rates of 96.63% and 87.82% of daidzein and genistein, respectively, and TSI also could enrich the bioactive metabolites of G. lucidum. The antitumor effects of TSI on human colorectal cancer cell line HTL-9, human breast cancer cell line MCF-7, and human immortalized gastric epithelial cell line GES-1 were also studied. The 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide（MTT） assay showed that TSI could dramatically reduce the viability rates of HTL-9 cells and MCF-7 cells without detectable cytotoxicity on GES-1 normal cells when the TSI concentration was lower than 100 μg/ml. With 100 μg/ml of TSI, HTL-9 cells were arrested in the G1 phase, and late-apoptosis was primarily induced, accompanied with partial early-apoptosis. TSI could induce primarily earlyapoptosis by arresting cells in the G1 phase of MCF-7 cells. For HTL-9 cells, Western-blot and reverse-transcriptase polymerase chain reaction（RT-PCR） analysis showed that TSI（100 μg/ml） can up-regulate the expression of Bax, Caspase-3, Caspase-8, and cytochrome c（Cyto-c）, indicating that TSI could induce cell apoptosis mainly through the mitochondrial pathway. In addition, the expression of p53 was up-regulated, while the expression of Survivin and nuclear factor κB（NF-κB） was down-regulated. All these results showed that TSI could induce apoptosis of HTL-9 cells by the regulation of multiple apoptosis-related genes.

Characterization of β-glucosidase from Aspergillus terreus and its application in the hydrolysis of soybean isoflavones

An extracellular β-glucosidase produced by Aspergillus terreus was identified, purified, characterized and was tested for the hydrolysis of soybean isofiavone. Matrix-assisted laser desorption/ionization with tandem time-of- flight/time-of-flight mass spectrometry （MALDI-TOF/TOF MS） revealed the protein to be a member of the glycosyl hydrolase family 3 with an apparent molecular mass of about 120 kDa. The purified 13-glucosidase showed optimal activity at pH 5.0 and 65℃ and was very stable at 50℃. Moreover, the enzyme exhibited good stability over pH 3.0-8.0 and possessed high tolerance towards pepsin and trypsin. The kinetic parameters Km （apparent Michaelis- Menten constant） and Vmax （maximal reaction velocity） for p-nitrephenyl-β-D-cjlucopyranoside （pNPG） were 1.73 mmol/L and 42.37 U/mg, respectively. The Krn and Vmax for cellobiose were 4.11 mmol/L and 5.7 U/mg, respectively. The enzyme efficiently converted isoflavone glycosides to aglycones, with a hydrolysis rate of 95.8% for daidzin, 86.7% for genistin, and 72.1% for glycitin. Meanwhile, the productivities were 1.14 mmol/（L.h） for daidzein, 0.72 mmol/（L.h） for genistein, and 0.19 mmol/（L.h） for glycitein. This is the first report on the application of A. terreus β-glucosidase for converting isoflavone glycosides to their aglycones in soybean products.