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.

Developmental and Reproductive Toxicity of Soybean Isoflavones to Immature SD Rats

Objective To investigate the dose-dependent toxic effect of soybean isoflavone extracts （SIE） on reproductive development in immature rats. Methods Growing male and female rats （n=50 each, 4 weeks） were divided into five groups fed with a standard cereal-based diet and gastrogavaged daily with 0, 30, 150, 300, and 600 mg SIE / kg body weight, respectively, for 12 weeks. Body weight, organ weights, and serum level of estrogen and testosterone were measured. Results Oral administration of SIE had no effect on food intake but decreased food efficiency ratio （P〈0.01）. Suppression on body weight gain by SIE was dose-dependent and the effect was greater on male than on female rats （P〈0.01）. SIE at high doses exhibited hepatotoxicity by increasing a relative liver weight, and also caused a smaller uterus but a greater relative ovary in female rats, while leading to larger relative testis and epididymis in male rats. SIE could decrease progesterone concentrations in female rats, whereas in male rats it reduced not only total testosterone level but also sperm count compared with the control group （P〈0.05）. Conclusion SIE at a range of 50-1000 times of human intake level affects not only growth but also development of reproductive system in growing rats.

Genetic Analysis of Combining Abilities and Heterosis for the Contents of Soybean Isoflavone and Its Components Among the Soybean Varieties [Glycine max (L.) Merr.]

The genetic analysis of soybean isoflavone content and its components were carried out based on the NC Ⅱ matingdesign in eight soybean varieties. The results showed that the isoflavone contents and its components of soybean seedare quite differences among the tested materials, the contents of isoflavone and daidzein are controlled not only byadditive effects and but also by non-additive effects, while the content of genistin is dominated by non-additive effects,and genistein, glycitin and daidzin are mainly controlled by additive effects. There are significant differences in thecontents of isoflavone and its components among the combinations derived from different parents. Results also indicatedthat the tested traits are negatively heterosis except for the contens of daidzein and daidzin are positively heterosis basedon the data of the GCA and SCA in average heterosis values. In this research we have a suggestion that soybean varietywith high isoflavone should be used as one of the parents in the breeding program, and it is the best choice that thecombinations crossed between two high isoflavone varieties or a high variety and a low one.

Identification of novel QTL associated with soybean isoflavone content

Soybean isoflavones are essential secondary metabolites synthesized in the phenylpropanoid pathway and benefit human health. In the present study, highresolution QTL mapping for isoflavone components was performed using specific-locus amplified fragment sequencing(SLAF-seq) with a recombinant inbred line(RIL) population(F5:7) derived from a cross between two cultivated soybean varieties, Luheidou 2(LHD2) and Nanhuizao(NHZ). Using a high-density genetic map comprising 3541 SLAF markers and the isoflavone contents of soybean seeds in the 200 lines in four environments, 24 stable QTL were identified for isoflavone components, explaining 4.2%–21.2% of phenotypic variation.Of these QTL, four novel stable QTL(qG8, qMD19, qMG18, and qTIF19) were identified for genistin, malonyldaidzin, malonylgenistin, and total isoflavones, respectively. Gene annotation revealed three genes involved in isoflavone biosynthesis(Gm4CL, GmIFR, and GmCHR) and 13 MYB-like genes within genomic regions corresponding to stable QTL intervals, suggesting candidate genes underlying these loci. Nine epistatic QTL were identified for isoflavone components, explaining 4.7%–15.6% of phenotypic variation. These results will facilitate understanding the genetic basis of isoflavone accumulation in soybean seeds. The stable QTL and tightly linked SLAF markers may be used for markerassisted selection in soybean breeding programs.

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.

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.

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.

QTL Mapping of Isoflavone,Oil and Protein Contents in Soybean (Glycine max L.Merr.)

Soybean （Glycine max L. Merr.） is the world＇s foremost source of edible plant oil and proteins, meantime, the biologically active secondary metabolites such as saponins and isoflavones are benefit to human health. The objective of this study was to identify quantitative trait loci （QTL） and epistatic interactions associated with isoflavone, protein, and oil contents in soybean seeds. An F13 recombinant inbred line （RIL） comprising 474 lines was derived from a cross between Jindou 23 and Huibuzhi cultivars. SSR technique was employed for mapping of the QTLs. The QTLs for isoflavone, protein, and oil contents were analyzed and 23 QTLs were detected based on the constructed linkage map. Six QTLs for isoflavone content were localized in linkage groups J, N, D2, and G, eleven QTLs for oil content were localized in the linkage groups A1, A2, B2, C2, and D2, and six QTLs for protein content were localized in linkage groups B2, C2, G, and H1. The correlative analysis demonstrated that the isoflavone content had significant correlation with protein content, while significantly negative correlations was existed between oil and protein content, and significantly positive correlations was existed between protein and oil content. All these findings have laid an important basis for the marker assisted breeding in soybean. The phenotypic correlations of quantitative traits may be resulted from the correlation of the QTL controlling those traits.

Evaluations and Selections for High Isoflavone Black Soybean Mutants Induced by NaN₃Treatment

Sodium azide (NaN3) (2 mM) was used to induce mutation in black soybean variety CRWD for improvement of isoflavone content. The individual selections were made in M6, M7 and M8 generations, respectively. Both high levels of isoflavones and medium seed size traits were used as selection criteria. Across three growth seasons, the selected five NaN3-induced mutants accumulated more total isoflavones (average increases of 5% to 25%) than their wild type variety CRWD. Four mutants yielded more (average increases of 17% to 25%) than CRWD. Both mutants SA-M-03 (yellow cotyledon) and SA-M-05 (green cotyledon) are rich in isoflavones and may be useful in food and other applications.

Rapid HPLC Method for Determination of 12 Isoflavone Components in Soybean Seeds

It is important to determine the isoflavone components by high-performance liquid chromatography （HPLC） for the molecular assistant selection of isoflavone in soybean. Based on the standard samples of 12 isoflavone components, the isoflavone components were analyzed using the determination of absorbance peaks method by HPLC. The results showed that there were different maximum ultraviolet （UV） absorbance for the aglycones of daidzein, glycitein, and genistein, which were at 250, 257, and 260 nm, respectively. A linear gradient elution of acetonitrile （13-30%） containing 0. 1% acetic acid as a mobile phase was applied on a YMC-C18 column at 35℃. The 12 isoflavone components were determined using the UV detector by HPLC. We concluded that this is a rapid and precise method which adapted to determine the large numbers of samples with microanalysis.

Genetic Analysis on Isoflavone Content in Soybean Seeds

Fifteen combinations with six soybean cultivars of different isoflavone content were formulated and planted in a randomized complete-block design model; genetic factors of isoflavone quantity were analyzed. Results indicated that genetic factors of isoflavone contents in F2 population inherited quantitatively. Isoflavone content of F1 F2 seeds normally trended. There were heterosis in F1, F2 of most combinations, and also heterobeltiosis in part of the crosses. The broad sense heritability of F2 was higher in parts of the crosses. It predicted the selection might be carried out preliminarily in F2 hybrids. There was significant positive correlation between hybrids and mid-parent.

Perform Stability of Isoflavones of Soybean Cultivar Evaluated by Genotype-genotype×environment(GGE) Biplot

As one of the secondary metabolites,the isoflavones formed during the development of soybean[Glycine max(L.)Merr.]seeds.The total and individual isoflavone contents,a typical quantitative trait,were affected by significant genotypes of environments(GE)interaction and controlled by many genes with main or minor effects.In the present study,99 soybean cultivars,collected from northeastern China,were used to analyze the isoflavone performances.Genotype-genotype×environment(GGE)biplot software demonstrated an ability to provide information on genetic main effects than solely on phenotypic perform.Highperformance liquid chromatography(HPLC)system was used to extract and determine the isoflavone contents.The results indicated that most genotypes significantly varied among six tested environments.P40(Xiaolimoshidou)was the best-performed genotype with mean performance and stability for glycitein content across six different environments.P88(L-59Peking)was the super genotype with mean performance and stability on each tested environment for daidzein,genistein and the total isoflavone.E5(Gongzhuling in 2016)was the best environment for optimal environmental factor mining.P70(Charleston),P67(Baichengmoshidou)and P50(Jiunong 20)were the optimal genotypes with the highest field among 99 cultivars on each tested environment for genistein.P70(Charleston),P67(Baichengmoshidou)and P14(Hefeng 25)were the optimal genotypes with the highest field among 99 cultivars on each tested environment for daidzein.P40(Xiaolimoshidou),P45(Jinshanchamodou),P33(Dongnong 48)and P56(L-5)were the optimal genotypes with the highest field among 99 cultivars on each tested environment for glycitein.P70(Charleston)and P67(Baichengmoshidou)were the optimal genotypes with the highest field among 99 cultivars on each tested environment for the total isoflavone.GGE biplot was a rational method for stability and adaptation evaluation of soybean isoflavones,and could assist soybean breeder to select a good culture and a suitable tested site.It provided a scientific basis for the establishment of a breeding site and a selection site of soybean isoflavones.This study was valuable to identify genotypes with stable performances of isoflavones of these 99 cultivars for developing new cultivars.

Soybean Seed Phenol, Lignin, and Isoflavones and Sugars Composition Altered by Foliar Boron Application in Soybean under Water Stress

Previous research showed that foliar boron (B) application at flowering or seed-fill growth stages altered seed protein, oil, and fatty acids. The objective of this research was to investigate the effects of foliar B fertilizer on seed phenolics (phenol, lignin, and isoflavones) and sugars concentrations. A repeated greenhouse experiment was conducted on soybean [(Glycine max(L.) Merr.)] under watered and water-stressed conditions. Soybean plants were divided into different sets, and each set was subjected to one of the following treatments: W = plants were watered with no foliar B;WB = plants were watered and received foliar B;WS = plants were water-stressed with no foliar B;WSB = plants were waterstressed and received foliar B. Foliar B was applied at rate of 0.45 kg/ha twice at flowering and twice at seed-fill stages. The results showed that total phenol and lignin concentrations were higher in seed collected from water-stressed plants compared with those collected from watered plants whether B was applied or not. The higher total phenol and lignin concentration in seed collected of water-stressed plants may be due to B-deficiency in plant tissues. Application of B resulted in higher concentrations of total seed B and isoflavones under watered and water-stressed plants. Higher cell wall B was higher in water-stressed plants than in watered plants, having an opposite trend to total B. Application of B resulted in higher seed sucrose in watered and water-stressed plants, but raffinose and stachyose were significantly higher under water-stressed plants. The research demonstrated that foliar B fertilizer altered seed phenol, lignin, isoflavones, and sugars, suggesting that B involved in phenolics and sugar metabolism. The higher cell wall B in waterstressed plants than in watered plants supports previous research that B has mainly a structural role. The higher sucrose resulting from foliar B in watered plants is desirable as sucrose contributes to seed quality. The increase of raffinose and stachyose concentrations in seed of water-stressed plants is undesirable as raffinose, and especially stachyose may be involved in water stress/drought tolerance. The current knowledge would help soybean breeders select for higher phenolic compounds and desirable sugars for higher seed qualities under drought conditions.

Studies on the Correlations Between Isoflavone Contents in Soybean Seed and the Eco-Physiological Factors

The soybean cultivar Yudou25 was sown at 5 locations of Henan Province on 13 differentdates in 2001 and 2002. The data of isoflavone contents for the 109 samples of soybeanseed and 33 eco-physiological factors including meteorological factors, soil nutritionand altitudes were received and used for statistical analysis. The step-wise regressionwas used to screen the correlated factors, which significantly effected isoflavonecontents. Results showed that 9 eco-physiological factors were highly correlated withisoflavones. Low mean temperature, high diurnal temperature range at seed filling andmaturity, more sunlight hours and low mean temperature at emergence were favorable toisoflavone accumulation. The rainfall at emergence showed a nonlinear relationship withisoflavone content and its optimum value was 75 mm for isoflavone formation. Low diurnaltemperature range at branching, high organic matter and low sulfur content in soil weresuitable for the formation of isoflavones. The isoflavone contents would not be affectedby other eco-physiological factors in this study.

Genetic Analysis of Embryo, Cytoplasm and Maternal Effects and Their Environment Interactions for Isoflavone Content in Soybean [Glycine max (L.) Merr.]

Soybean seed products contain isoflavones （genistein, daidzein, and glycitein） that display biological effects when ingested by humans and animals. These effects are species, dose and age dependent. Therefore, the content and quality of isoflavones in soybeans is a key factor to the biological effect. Our objective was to identify the genetic effects that underlie the isoflavone content in soybean seeds. A genetic model for quantitative traits of seeds in diploid plants was applied to estimate the genetic main effects and genotype x environment （GE） interaction effects for the isoflavone content （IC） of soybean seeds by using two years experimental data with an incomplete diallel mating design of six parents. Results showed that the IC of soybean seeds was simultaneously controlled by the genetic effects of maternal, embryo, and cytoplasm, of which maternal genetic effects were most important, followed by embryo and cytoplasmic genetic effects. The main effects of different genetic systems on IC trait were more important than environment interaction effects. The strong dominance effects on isoflavone from residual was made easily by environment conditions. Therefore, the improvement of the IC of soybean seeds would be more efficient when selection is based on maternal plants than that on the single seed. Maternal heritability （65.73%） was most important for IC, followed by embryo heritability （25.87%） and cytoplasmic heritability （8.39%）. Based on predicated genetic effects, Yudou 29 and Zheng 90007 were better than other parents for increasing IC in the progeny and improving the quality of soybean, The significant effects of maternal and embryo dominance effects in variance show that the embryo heterosis and maternal heterosis are existent and uninfluenced by environment interaction effects.

Phytoestrogen Enriched Tofu from Soybean Meal

Isoflavone, a group of phytoestrogen, reduces postmenopausal symptoms and the risk of osteoporosis of women. Glycosidic forms of isoflavones are presented in non-fermented soyfoods such as tofu and they are less bioavailable than the aglycone isoflavones. Aglycone forms of isoflavones or more bioavailable forms can be increased by acid hydrolysis during tofu processing. The present study investigated the possibility of increasing the aglycone forms of isoflavones by acid hydrolysis. We used five types of tofu in this study: soybean tofu with hydrolysis, soybean meal tofu with hydrolysis, soybean tofu in general process, soybean meal tofu in general process, and commercial tofu. Defatted soybean meal was used as the major ingredient in the tofu which was made by using the new method—acid hydrolysis. To identify the isoflavone quantities in all five types of tofu, high performance liquid chromatography with diode array detection (HPLC-DAD) analysis was employed. The genistein ratio between hydrolyzed tofu and standard tofu was 1:1-8, and the daidzein ratio between hydrolyzed tofu and standard tofu was 1:6-12. The five types of tofu were analyzed for the crude protein and micronutrients such as calcium (Ca), magnesium (Mg), potassium (K), iron (Fe), zinc (Zn), and selenium (Se) by the modified Kjeldahl method and inductively coupled plasma emission spectroscopy (ICP-ES), respectively. The mean crude protein concentration of hydrolyzed tofu from soybean meal was 40.8%. In addition, especially the hydrolyzed soybean meal tofu showed the higher concentration of Ca (27,307 mg/kg) and K (25,553 mg/kg). By and large, soybean meal tofu with acid hydrolysis is a rich source of isoflavone aglycone compared with other types of tofu.

大豆异黄酮对坝上长尾鸡生长性能、免疫功能、睾丸发育及血浆生殖激素的影响

试验旨在研究大豆异黄酮对坝上长尾鸡生长性能、免疫功能、睾丸发育及生殖激素的影响。选取750只40周龄坝上长尾鸡,随机分为5组,每组3个重复,每个重复50只鸡。Ⅰ组、Ⅱ组、Ⅲ组、Ⅳ组和Ⅴ组饲粮中分别添加5、10、15、20、25 mg/kg大豆异黄酮。预试期7 d,正式试验期63 d。结果显示,Ⅱ组、Ⅲ组、Ⅳ组和Ⅴ组坝上长尾鸡增重显著高于Ⅰ组(P<0.05)。Ⅲ组坝上长尾鸡的胸腺指数显著高于其他组(P<0.05);Ⅲ组坝上长尾鸡的法氏囊指数分别比Ⅰ组、Ⅱ组和Ⅴ组显著提高16.09%、14.70%和22.67%(P<0.05);Ⅲ组坝上长尾鸡的脾脏指数比Ⅰ组显著提高17.98%(P<0.05)。Ⅲ组坝上长尾鸡的睾丸指数最高,显著高于Ⅰ组(P<0.05),提高了11.74%。Ⅲ组坝上长尾鸡的采精量、精子密度和有效精子数显著高于Ⅰ组(P<0.05),分别提高了9.52%、4.79%和8.97%。Ⅲ组坝上长尾鸡的睾酮(TESTO)含量分别比Ⅰ组、Ⅱ组和Ⅴ组显著提高10.42%、4.72%和6.42%(P<0.05);Ⅲ组坝上长尾鸡的黄体生成素(LH)含量分别比Ⅰ组、Ⅱ组和Ⅴ组显著提高7.63%、5.83%和10.12%(P<0.05)。研究表明,大豆异黄酮能够改善坝上长尾鸡的生长性能、免疫功能、睾丸发育、精液品质及血浆生殖激素的分泌水平,且最适添加量为15 mg/kg。

淡豆豉异黄酮调控TGF-β1/SnoN通路对糖尿病肾病小鼠肾组织的保护作用研究

[目的]观察淡豆豉异黄酮对糖尿病肾病(DKD)小鼠肾组织的保护作用,并探讨其对转化生长因子-β1(TGF-β1)/Smad核转录共抑制因子(SnoN)通路的调控作用。[方法]50只清洁级db/db小鼠和10只清洁级db/m小鼠,前者验证DKD建模成功后随机分5组,后者记为对照组。依那普利组予以依那普利10 mg/kg溶于0.1 mL/kg生理盐水中灌胃,淡豆豉异黄酮低、中、高剂量组分别予以12.5、25、50 mg/kg淡豆豉异黄酮溶于生理盐水中灌胃,其余组均予以生理盐水灌胃,均每日1次,共干预8周。己糖激酶法检测各组干预前、4周后、干预后空腹血糖(FBG),蛋白质定量(BCA)法检测24 h尿蛋白;检测血肌酐(Scr)、血尿素氮(BUN)、TGF-β1水平;干预后处死小鼠并取肾组织,观察肾组织病理;实时-逆转录聚合酶链反应(RT-qPCR)检测各组肾组织TGF-β1、Smad2/3、Smad7、SnoN信使核糖核酸(mRNA)表达;检测肾组织TGF-β1、Smad2/3、Smad7、SnoN蛋白表达及磷酸化-Smad2/3(p-Smad2/3)。[结果]模型组4周后、干预后FBG、24 h尿蛋白、Scr、BUN、TGF-β1水平均高于正常组(P<0.05),依那普利组24 h尿蛋白、Scr、BUN、TGF-β1水平和淡豆豉异黄酮低、中、高剂量组FBG、24 h尿蛋白、Scr、BUN、TGF-β1水平均低于模型组(P<0.05);模型组肾组织呈显著病理改变,依那普利组和淡豆豉异黄酮低、中、高剂量组病理改变和胶原纤维沉积均减轻;模型组肾组织TGF-β1 mRNA与蛋白表达、Smad2/3 mRNA表达与p-Smad2/3升高(P<0.05),依那普利组和淡豆豉异黄酮低、中、高剂量组均低于模型组(P<0.05);模型组肾组织Smad7、SnoN表达降低(P<0.05),依那普利组和淡豆豉异黄酮低、中、高剂量组均高于模型组(P<0.05)。淡豆豉异黄酮组的作用呈剂量依赖性,除FBG外淡豆豉异黄酮中剂量组上述定量指标与依那普利组差异均无统计学意义(P>0.05)。[结论]淡豆豉异黄酮可降低DKD小鼠血糖,减少24 h尿蛋白,保护肾功能,降低血清TGF-β1水平,减轻肾病变,下调TGF-β1、Smad2/3表达,降低p-Smad2/3水平,上调Smad7、SnoN表达。