金雀异黄素对大鼠骨代谢的影响

目的 探讨金雀异黄素对大鼠骨代谢的影响。方法 取SD大鼠32只，随机分成4组，灌胃给药4周，测体重、血清碱性磷酸酶（AKP）、抗酒石酸酸性磷酸酶（ACP）活性和骨钙素（BGP）、雌二醇（E2）水平。结果 金雀异黄素40mg／kg可以增加大鼠体重、AKP活性和骨钙素水平；金雀异黄素20、30mg／kg对大鼠体重及AKP、ACP活性无明显影响。结论 金雀异黄素对骨代谢有一定的调节作用。     

金雀异黄素对缺氧诱导的人ARPE-19细胞VEGF表达的抑制作用

目的:研究长期缺氧对体外培养的人ARPE鄄19细胞血管内皮生长因子(VEGF)表达的诱导作用,以及金雀异黄素(genistein,Gen)对其表达的影响初步研究。方法:使用半定量RT鄄PCR和酶联免疫吸附试验(ELISA)检测ARPE鄄19细胞中VEGFmRNA和蛋白的表达,分析不同浓度Gen以及PD98059、TyrphostinA25对ARPE鄄19细胞缺氧24h诱导的VEGF表达的影响。结果:①正常对照组有少量VEGF表达,缺氧24h可明显提高ARPE鄄19细胞VEGFmRNA和蛋白的表达,分别为9.566±1.528和2.636±0.499倍;②Gen可明显抑制缺氧诱导ARPE鄄19细胞中VEGFmRNA和蛋白的表达,且呈浓度依赖性(P<0.05);③PD98059、TyrphostinA25也可抑制低氧诱导的VEGF的产生,但抑制作用没有Gen(200μmol/L组)明显。结论:Gen可抑制长期缺氧诱导的ARPE鄄19细胞VEGF表达,并可能是通过抑制p42/p44MAPK途径和HIF鄄1途径共同抑制VEGF的产生,提示Gen对视网膜新生血管的形成具有预防和潜在的临床治疗价值。     

不同剂量染料木黄酮及其代谢产物在大鼠尿中的排泄

目的:研究不同剂量染料木黄酮及其葡萄糖醛酸化代谢产物在大鼠尿中的排泄动力学。方法:将染料木黄酮制成混悬液,按6.25、12.5、50mg.kg-1给大鼠灌胃,于灌胃后不同时间收集尿液,用葡萄糖醛酸酶溶液处理尿液。采用高效液相色谱法测定尿液中染料木黄酮及其葡萄糖醛酸化代谢产物的浓度。结果:6.25、12.5、50 mg.kg-1时,累积以原形经尿液排泄的药物分别为34.79±10.83、187.30±69.96和213.56±30.58μg,累积经尿液排泄的总药物(原形药物+葡萄糖醛酸化药物)分别为217.79±52.06、583.05±106.92和1108.37±88.14μg,累积经尿液排泄的葡萄糖醛酸化代谢产物分别占尿液排泄总量的84.03%、67.88%和80.73%。结论:染料木黄酮在大鼠尿液中主要以葡萄糖醛酸结合形式排泄,原形及其葡萄糖醛酸化代谢产物的排泄呈现明显的非线性剂量依赖性特征。     

不同剂量染料木黄酮及其代谢产物在大鼠胆汁中的排泄动力学

目的研究不同剂量染料木黄酮及其葡糖醛酸化代谢产物在大鼠胆汁内的排泄动力学。方法将染料木黄酮制成混悬液，分别按6.25，12.5和50 mg·kg^-1给大鼠灌胃，于灌胃后不同时间收集胆汁，用葡糖醛酸酶溶液处理胆汁。采用高效液相色谱法测定胆汁中染料木黄酮及其葡糖醛酸化代谢产物的浓度。结果3种剂量6.25，12.5和50 mg·kg^-1分别给药后，累积以原形从胆汁排泄的药物分别为(42.56±6.54)，(75.17±18.87)和(126.60±34.78) μg，累积经胆汁排泄的总药物(原形药物+葡糖醛酸化药物)分别为(108.46±35.23)，(423.46±158.31)和(853.74±320.84) μg，葡糖醛酸化代谢产物分别占胆汁排泄总量(原形药物+葡糖醛酸化药物)的60.76%，82.25%和85.17%。结论染料木黄酮在大鼠胆汁中主要以葡糖醛酸结合形式排泄，原形药物及其葡糖醛酸化代谢产物在大鼠胆汁中的排泄呈现明显的非线性剂量依赖性特征。     

Inhibition of motility of hamster spermatozoa by protein tyrosine kinase inhibitors

Genistein, tyrphostin and piceatannol, which are specific inhibitors of protein tyrosine kinase, were screened for their effects on the motility of intact and demembranated hamster spermatozoa. Of the three inhibitors only piceatannol inhibited the motility of intact spermatozoa. None of the inhibitors had any inhibitory effect on the reactivation of motility of demembranated hamster spermatozoa. Taken together these results indicated that a protein tyrosine kinase associated with the membrane of hamster spermatozoa was probably involved in sustenance of hamster sperm motility. Therefore in the present study a membrane-associated protein tyrosine kinase was purified from a detergent-soluble extract of plasma membranes of mature hamster spermatozoa. The purification involved cation exchange chromatography on fast protein liquid chromatography (FPLC) followed by affinity chromatography either on an antiphosphotyrosine antibody agarose or poly glu-tyr agarose column. The pure protein tyrosine kinase had an apparent molecular mass of 45 kDa. The enzyme was not inhibited by genistein or herbimycin but was inhibited by piceatannol. This is the first report on the purification of a sperm plasma membrane-associated protein tyrosine kinase, an enzyme which has also been implicated in hamster sperm motility.     

Genistein对高钾和化学缺氧所致人视网膜色素上皮细胞损伤的保护作用

目的:研究Genistein(gen)对高钾和化学缺氧所致人视网膜色素上皮(retinalpigmentepithelium,RPE)细胞损伤的保护作用。方法:采用体外培养人RPE细胞,MTT法测定细胞存活率以及在倒置相差显微镜下观察细胞形态改变。结果:200mmol/LKCl作用12h可降低细胞存活率至(43.97±3.43)%,gen50、100、200μmol/L可明显提高细胞存活率且呈浓度依赖性。相差显微镜观察细胞形态发现,200mmol/LKCl作用2h细胞膜开始皱缩,4h胞膜皱缩更加明显,胞核也开始浓缩,8h后仅见细胞核和断裂呈絮状的细胞膜,12h仅可见固缩的细胞核,而200μmol/Lgen可以减轻细胞损伤,4h后方见细胞膜开始皱缩;CoCl2损伤模型中,500μmol/LCoCl2作用12h可降低细胞存活率至(57.81±17.19)%,gen50、100、200μmol/L时也可浓度依赖性升高细胞存活率。结论:Gen对高钾和化学缺氧所致人RPE细胞损伤具有保护作用。     

H-Ras oncogene counteracts the growth-inhibitory effect of genistein in T24 bladder carcinoma cells

Among eight human bladder cancer cell lines we examined, only T24 cells were resistant to the growth inhibition effect of genistein, an isoflavone and potent anticancer drug. Since the T24 cell line was the only cell line known to overexpress oncogenic H-Ras(val 12), we investigated the role of H-Ras(val 12) in mediating drug resistance. Herein, we demonstrate that the phenotype of T24 cells could be dramatically reversed and became relatively susceptible to growth inhibition by genistein if the synthesis of H-Ras(val 12) or its downstream effector c-Fos had been suppressed. The inhibition of Ras-mediated signalling with protein kinase inhibitors, such as PD58059 and U0126 which inhibited MEK and ERK, in T24 cells also rendered the identical phenotypic reversion. However, this reversion was not observed when an inhibitor was used to suppress the protein phosphorylation function of PI3 K or PKC. These results suggest that the signal mediated by H-Ras(val 12) is predominantly responsible for the resistance of the cells to the anticancer drug genistein.     

In vitro exposure to quercetin and genistein alters lipid peroxides and prevents the loss of glutathione in human progenitor mononuclear (U937) cells

The effects of flavonoids quercetin and genistein were investigated according to their potency to inhibit the oxidation of U937 cells via Fenton's pathway through the analysis of lipid peroxides and glutathione (GSH) levels. Human leukemia (U937) cells from the American Type Culture Collection were maintained at 37 degrees C for 24 h under 5% CO2 tension in RPMI-1640 medium containing 10% fetal bovine serum and 50 units ml(-1) each of penicillin and streptomycin. Cells were oxidized with iron 50 microM) or copper (50 microM) in H2O2 (0.01 mM) without or with a flavonoid sample (10 or 20 microM) for the lipid peroxidation studies. The GSH levels were measured (GSH Kit) before and after oxidation as above with different concentrations of flavonoids (0-40 microM). Lipid peroxide was measured by the thiobarbituric acid assay. Both quercetin and genistein at either the 10 or 20 microM level decreased lipid peroxidation significantly compared with their respective controls (P < 0.01). Lipid peroxides by Fe compared to the Cu-treated samples did not differ significantly from each other. However, the combination of flavonoids at the doses tested significantly (P < 0.001) decreased lipid peroxides, the effect being the same for both metal ions. The GSH levels increased significantly before exposure to the metal ions (for the different doses for the differences between the flavonoid samples and their respective untreated levels). For quercetin and genistein the increases in GSH above their untreated levels were 4.5, 8.3, 11.7 and 15 and 3.8, 7.9, 12.5 and 14.6 nmol 10(-6) cells, respectively, for the 5-40 microM levels tested for each flavonoid. Following the exposure to the metal ions, GSH levels remained almost the same for the different concentrations for each of the flavonoids tested but significantly above all of the controls and same for those of the untreated samples. The results indicate that both flavonoids inhibited lipid peroxides and the inhibition may be attributed to the prevention of loss of intracellular GSH levels in U937 cells.     

The role of tyrosine kinase-mediated pathways in diabetes-induced alterations in responsiveness of rat carotid artery

1 G-protein-coupled receptor signalling, including transactivation of receptor tyrosine kinases (RTKs), has been implicated in vascular pathology. However, the role of specific RTKs in the development of diabetes-induced cardiovascular complications is not known. 2 We investigated the ability of a chronic administration of genistein, a broad-spectrum inhibitor of tyrosine kinases (TKs), AG1478, a specific inhibitor of epidermal growth factor receptor (EGFR) TK activity, and AG825, a specific inhibitor of Erb2, to modulate the altered vasoreactivity of isolated carotid artery ring segments to common vasoconstrictors and vasodilators in streptozotocin (STZ)-induced diabetes. 3 In diabetic carotid artery, the vasoconstrictor responses induced by noradrenaline (NE), endothelin-1 (ET-1), and angiotensin II (Ang II), were significantly increased whereas vasodilator responses to carbachol and histamine were significantly reduced. Inhibition of TKs, EGFR or Erb2 pathway did not affect the body weight or agonist-induced vasoconstrictor and vasodilator responses in the non-diabetic control animals. However, inhibition of TKs by genistein, EGFR TK by AG1478 or Erb2 by AG825 treatment produced a significant normalization of the altered agonist-induced vasoconstrictor responses without affecting blood glucose levels. Treatment with diadzein, an inactive analogue of genistein, did not affect the vasoconstrictor and vasodilator responses in the diabetic animals. 4 Treatment with genistein, AG1478 or AG825 resulted in a significant improvement in diabetes-induced impairment in endothelium-dependent relaxation to carbachol and histamine. 5 These data suggest that activation of TK-mediated pathways, including EGFR TK signalling and Erb2 pathway, are involved in the development of diabetic vascular dysfunction in the carotid artery.     

The chemopreventive properties of soy isoflavonoids in animal models of breast cancer

Genistein (5,7,4-trihydroxyisoflavone), one of two major isoflavonoids in soy, has anti-proliferative effects on mitogen-stimulated cell growth of human breast cancer cells in culture and is a candidate for use in the prevention of breast cancer. Soy protein preparations containing isoflavonoid conjugates have chemopreventive activity in carcinogen-induced rat models of breast cancer. Recent experiments in these models with purified genistein have revealed that the timing of the exposure of rats to this isoflavonoid is critical. Rats treated neonatally or prepuberally with genistein have a longer latency before the appearance of 7,12-dimethylbenz[a]anthracene-induced mammary tumors and a marked reduction in tumor number. The mechanism of genistein's preventive action is in part dependent on its estrogenic activity, which causes a more rapid differentiation of the cells of the mammary gland, analogous to the effects of an early pregnancy. Rats administered genistein after 35 days of age have smaller alterations in breast cancer risk, with a maximum reduction in mammary tumor number of 27%. In ovariectomized nude mice, dietary genistein increases cell proliferation of human breast cancer MCF-7 cell xenografts compared with a control diet. This estrogen-like effect of genistein is not observed in non-ovariectomized rats. Future studies on the anticancer potential of soy isoflavonoids should examine their interaction with other phytochemical components of soybeans and exploit newly developed animal models of breast cancer in which specific genes have been activated or inactivated.