染料木黄酮抑制3T3-L1细胞成脂分化机制

目的：研究染料木黄酮是否通过雌激素受体介导影响3T3-L1前脂肪细胞成脂分化,并探讨其可能的机制。方法：以不同浓度染料木黄酮处理3T3-L1细胞,四甲基偶氮唑蓝（methyl thiazolyl tetrazolium,MTT）法测定细胞存活率;在3T3-L1前脂肪细胞成脂分化过程中分别加入不同浓度染料木黄酮、染料木黄酮和ICI182780（一种雌激素受体抑制剂）混合物及不同浓度雌二醇,油红染色观察分化结果,测定细胞甘油三酯（triglyceride,TG）含量;染料木黄酮、染料木黄酮和ICI182780混合物及不同浓度雌二醇处理诱导成熟后的脂肪细胞,测定培养液甘油含量;分别用染料木黄酮（30 μmol/L）、染料木黄酮（30 μmol/L）和ICI182780混合物干预细胞成脂分化,Western blotting法检测细胞外信号调节激酶（extracellular signal-regulated kinase,ERK）、p-ERK、腺苷酸活化蛋白激酶（adenosine monophosphate activated protein kinase,AMPK）、p-AMPK、激素敏感性脂肪酶（hormone sensitive lipase,HSL）、脂肪酸合成酶（fatty acid synthetase,FAS）蛋白表达量。结果：3T3-L1细胞存活率随染料木黄酮浓度的升高而降低,50～200 μmol/L染料木黄酮能抑制细胞生长（P＜0.01）;染料木黄酮能负向调节成脂分化后细胞胞浆内TG含量,在0～50 μmol/L浓度范围内呈剂量依赖关系,高剂量雌二醇（100 nmol/L）能下调TG含量;染料木黄酮能促进成熟脂肪细胞脂解,提高细胞培养液甘油浓度;染料木黄酮（30 μmol/L）能上调p-AMPK、HSL蛋白表达,下调FAS蛋白表达（P＜0.01）,对ERK、p-ERK、AMPK表达量无明显影响（P＞0.05）。ICI182780（1 μmol/L）能部分阻断染料木黄酮（30 μmol/L）对p-AMPK的调节作用（P＜0.05）。结论：染料木黄酮能抑制3T3-L1细胞成脂分化,其机制可能是染料木黄酮一方面下调FAS蛋白表达,抑制脂肪合成,另一方面激活AMPK-HSL途径促进脂肪分解;染料木黄酮还可能通过非雌激素受体途径抑制3T3-L1细胞成脂分化。     

水豆豉发酵前后理化特性变化及其提取物对前列腺癌细胞抑制作用的初探

目的:为研究水豆豉发酵前后理化特性变化,并初步探讨水豆豉提取物对去势抵抗前列腺癌细胞（CRPC）的抑制作用。方法:使用三株高产蛋白酶菌株发酵制备水豆豉,参照国家标准方法检测水豆豉发酵前后氨基酸态氮、总酸、pH、还原糖及大豆异黄酮的含量,化学法检测总酚及总黄酮的含量,并测定水豆豉提取物的抗氧化能力。最后,观察最优发酵菌株制备的水豆豉提取物对CRPC细胞的抑制作用。结果:高产蛋白酶菌株发酵水豆豉后,其氨基酸态氮、还原糖、大豆异黄酮含量及抗氧化能力指标均比蒸熟黄豆有所提高（P<0.05）,其中B19菌株发酵水豆豉的效果最佳。使用B19菌株发酵制备的水豆豉提取物对CRPC细胞有选择性杀伤作用,并能促进其凋亡,同时对前列腺特异性抗原（PSA）、雄激素睾酮（T）和二氢睾酮（DHT）分泌有抑制作用。结论:高产蛋白酶菌株发酵水豆豉后能改变其理化特性,并增强其营养活性。且B19菌株发酵的水豆豉对CRPC细胞有抑制作用。本研究为水豆豉相关保健功能食品的研发提供了数据参考及理论支撑。     

染料木黄酮对慢性缺氧大鼠血管活性物质的调节作用

目的：探讨染料木黄酮对大鼠血管活性物质的调节作用，以了解其抑制缺氧性肺动脉高压及肺血管结构改建的作用机制。方法：将雄性Wistar大鼠随机分为3组，常氧对照组（C）、慢性缺氧组（H）和慢性缺氧＋染料木黄酮组（H＋G），C组在半原，H组和H＋G组置于减压舱，模拟海拔5000m高原，8h／d，持续21d，分别检测血中一氧化氮（NO）、内皮素（ET-1）、前列环素（PGl2）、雌二醇（E2）、超氧化物歧化酶（SOD）及丙二醛（MDA）的含量。结果：染料木黄酮可以显著抑制ET-1和MDA的产生，促进NO和前列环素的生成，提高SOD活性。与平原对照组和单纯缺氧组比较，染料木黄酮组大鼠血清雌激素水平无显著差异。结论：染料木黄酮可以调节内皮依赖性舒血管／缩血管活性物质的平衡，加强血管舒张作用，抑制血管平滑肌细胞增殖，从而实现对慢性缺氧性肺动脉高压和肺血管结构改建防治作用。染料木黄酮对雄性大鼠体内雌激素水平无显著影响，可以避免长期使用雌激素产生的女性乳腺和了宫的癌变及男性雌化等不良反应。     

槲皮素对前列腺癌PC-3细胞凋亡和HSP27表达的影响

目的：评价槲皮素对前列腺癌PC-3细胞凋亡和热休克蛋白27（HSP27）表达的影响,探讨槲皮素抗前列腺癌的可能作用机制。方法：体外培养人前列腺癌RWPE-1、LNCa P、PC-3和TSU-Pr1细胞株,分别随机分为4组：空白对照组;阴性对照组：给予二甲亚砜（DMSO）;给药高剂量组：给予0.3mg/m L槲皮素150μL;给药低剂量组：给予0.6mg/m L槲皮素150μL。以TUNEL法检测给予槲皮素后PC-3细胞凋亡、RT-PCR检测HSP27的表达情况。结果：给药组可见大量PC-3细胞凋亡,且呈剂量依赖性,与空白对照组比较,差异具有统计学意义（P〈0.05）;给药组PC-3细胞HSP27的表达明显降低,且呈剂量依赖性,与空白对照组比较,差异具有统计学意义（P〈0.05）。结论：槲皮素可诱导前列腺癌PC-3细胞凋亡和抑制HSP27的表达,可能是其抗前列腺癌的机制之一。     

类黄酮化合物抑制NDM-1活性的构效关系

选择食物来源相对丰富且分子结构具有一定差异的3种类黄酮化合物(槲皮素、木犀草素、染料木黄酮),通过碘量法比较它们对新德里金属β内酰胺酶-1(NDM-1)的抑制活性。通过分子对接软件分析化合物对NDM-1的理论结合能力,建立类黄酮化合物抑制NDM-1活性的构效关系。碘量法分析结果表明,3种类黄酮化合物对NDM-1抑制活性差异表现为染料木黄酮木犀草素槲皮素,这一结果与分子对接理论分析结果一致。由此,3种化合物抑制NDM-1的构效关系为:B环处于C环3位(染料木黄酮),是抑制NDM-1的相对优势结构;而C环3位羟基(槲皮素)对抑制NDM-1活性具有一定削弱作用。     

豆浆热处理过程中3种大豆异黄酮苷原的热降解比较

将大豆加工成豆浆并分别用95、121和140℃处理不同时间，以高效液相色谱（HPLC）法检测其中的3种大豆异黄酮皆原，染料木黄酮（genistein）、黄豆苷原（daidzein）和大豆黄素（glycitein）的含量变化，与原粒大豆、生豆浆进行比较。结果发现，染料木黄酮、黄豆苷原和大豆黄素的热稳定性存在较大差异，在95℃下，染料木黄酮在60min的处理时间内稳定，而黄豆苷原和大豆黄素的T（loss0.5）值（损失50％含量的时间）分别为1442s和453s，表明95℃下3种大豆异黄酮的热稳定性表现为：染料木黄酮〉黄豆苷原〉大豆黄素。而在121℃和140℃的处理条件下，3种大豆异黄酮苷原均随着处理时间的延长而出现不同的热降解，黄豆苷原、大豆黄素和染料木黄酮在121℃的T（loss0.5）值分别为26．36、37．88和1015s，而在140℃下，黄豆苷原、大豆黄素和染料木黄酮的T（loss0.5）值则分别缩短为6．94、8．47和369s，结果表明在121℃和140℃下，3种大豆异黄酮的热稳定性表现为：染料木黄酮〉大豆黄素〉黄豆苷原。     

苦荞清蛋白酶解物对高糖诱导的HepG2细胞胰岛素抵抗的保护作用

目的：建立胰岛素抵抗HepG2细胞模型,观察苦荞清蛋白酶解物（tartary buckwheat albumin hydrolysate,TBAH）对HepG2细胞胰岛素抵抗的影响。方法：采用碱性蛋白酶水解苦荞清蛋白制备TBAH,超滤法截留分子质量小于3 kDa的TBAH;利用高糖高胰岛素诱导HepG2细胞36 h建立胰岛素抵抗模型,以2.5、5、10 μg/mL的TBAH培养细胞24 h。观察TBAH对HepG2细胞葡萄糖代谢的影响;检测细胞氧化损伤指标（一氧化氮（nitric oxide,NO）、丙二醛（malondialdehyde,MDA）、乳酸脱氢酶（lactic dehydrogenase,LDH）、超氧化物歧化酶（superoxide dismutase,SOD）和活性氧（reactive oxygen species,ROS））水平;Western blot检测胰岛素受体底物1（insulin receptor substrate 1,IRS-1）/磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase,PI3K）/蛋白激酶B（protein kinase B,Akt）信号通路中相关蛋白表达情况。结果：与IR模型组相比,TBAH组葡萄糖消耗量显著增多（P＜0.05）,且呈剂量依赖性,同时MDA、NO和ROS水平显著降低（P＜0.01,P＜0.05）,SOD活力显著升高（P＜0.05）,LDH活力显著下降（P＜0.05）;p-IRS-1蛋白表达水平显著降低（P＜0.05）,磷酸化Akt、PI3K、葡萄糖转运蛋白4表达水平极显著上升（P＜0.01）。结论：TBAH通过抑制氧化应激改善胰岛素抵抗。     

库尔勒香梨多糖基于抑制焦亡关键蛋白NLRP3、GSDMD的表达发挥抗炎作用

目的：筛选库尔勒香梨多糖抑制焦亡的活性组分及初步探索抑制细胞焦亡分子机制。方法：库尔勒香梨脱脂后从中提取分离获得库尔勒香梨粗多糖（Pyrus sinkiangensis Yu polysaccharide,PSP）、中性多糖（Pyrus sinkiangensis Yu neutral polysaccharide,PSN）、酸性多糖（Pyrus sinkiangensis Yu acid polysaccharide,PSA）；脂多糖（lipopolysaccharide,LPS）及三磷酸腺苷（Adenosine triphosphate,ATP）刺激RAW264.7细胞，建立焦亡模型后进行PSP、PSA、PSN干预，检测细胞存活率、白介素-1β(IL-1β)及白介素-18(IL-18)的mRNA表达量、NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)及消皮素D(GasderminD,GSDMD)蛋白的表达水平。结果：PSP、PSN、PSA的总多糖含量分别为7....     

去甲基化法制备黄豆苷元和染料木黄酮及抗氧化性分析

以KBr-H3PO4或KI-H3PO4为反应介质,对芒柄花黄素和鸡豆黄素进行去甲基化反应转化成黄豆苷元和染料木黄酮,并对产物的抗氧化性进行分析。结果表明：以KBr-H3PO4为反应介质,制备黄豆苷元最佳条件为反应时间5?h、反应温度120?℃、料液比1∶45（g/mL）、最佳饱和度75%,产率为89.42%;制备染料木黄酮最佳条件为反应时间5?h、反应温度120?℃、料液比1∶45（g/mL）、最佳饱和度100%,产率为85.98%;以KI-H3PO4为反应介质,制备黄豆苷元最佳条件为反应时间4?h、反应温度100?℃、料液比1∶15（g/mL）、最佳饱和度75%,产率可达87.03%;制备染料木黄酮最佳条件分别为反应时间5?h、反应温度100?℃和料液比1∶45（g/mL）、最佳饱和度75%,产率可达88.51%。与已有合成方法相比,本实验方法具有反应过程简单、反应条件温和、反应时间较短、产率较高等优点。所制备黄豆苷元和染料木黄酮具有一定的1,1-二苯基-2-三硝基苯肼自由基和羟自由基清除能力,以及较强的抗亚油酸氧化能力,表现出较好的抗氧化性。     

p-辛弗林通过激活AMPK-FoxO1信号通路抑制肝细胞葡萄糖生成

目的：探讨p-辛弗林对肝细胞葡萄糖生成的影响及其作用机制。方法：采用MTS法检测p-辛弗林对HepG2肝细胞株的细胞活力的影响,确定受试物的作用浓度后,比色法测定葡萄糖的生成、葡萄糖-6-磷酸酶（glucose-6-phosphatase,G6Pase）和磷酸烯醇丙酮酸羧基激酶（phosphoenolpyruvate carboxykinase,PEPCK）活力,Western blot蛋白印迹法检测腺苷酸活化蛋白激酶（adenosine 5’-monophosphate-activated protein kinase,AMPK）、磷酸化AMPK（p-AMPK）、乙酰辅酶A羧化酶（acetyl coenzyme A synthetase,ACC）、磷酸化ACC（p-ACC）、叉头框转录因子1（forkhead box class O1,FoxO1）以及磷酸化FoxO1（p-FoxO1）的表达;利用AMPK选择性抑制剂Compound C和AMPK siRNA作用HepG2肝细胞株后,检测p-辛弗林对HepG2肝细胞株葡萄糖的生成、G6Pase和PEPCK活力的影响。结果：p-辛弗林能剂量依赖性地抑制肝细胞葡萄糖的生成,激活AMPK信号通路,促进p-AMPK、p-ACC和p-FoxO1水平增加,极显著抑制G6Pase和PEPCK的活性（P＜0.01）。p-辛弗林的这些影响部分地被Compound C和AMPK siRNA所抑制（P＜0.01）。结论：p-辛弗林能够通过激活AMPK-FoxO1信号通路抑制肝细胞葡萄糖生成。     

三羟异黄酮对人乳腺癌细胞株MCF-7增殖的影响机制

研究了三羟异黄酮(geinstein，Gen)对乳腺癌细胞株MCF-7增殖的影响机制。结果发现Gen对MCF-7细胞生长有显著抑制作用，使细胞生长阻滞于G2／M期，并使cyclin B蛋白表达增加，且呈剂量．效应关系；对cyclin E蛋白表达无影响。Gen降低了bcl-2蛋白的表达，促进了bax蛋白的表达并且降低了bcl-2／bax比值。     

Curcumin provides potential protection against the activation of hypoxia and prolyl 4-hydroxylase inhibitors on prostate-specific antigen expression in human prostate carcinoma cells

Scope: Prostate‐specific antigen (PSA) is a well‐known marker for diagnosing and monitoring prostate cancer. Curcumin, a yellow curry pigment, has been reported to enhance androgen receptor (AR) degradation. We examined the effects of curcumin on increasing PSA expression by hypoxia and prolyl hydroxylase inhibitors, L ‐mimosine and dimethyloxalylglycine (DMOG), in human prostate carcinoma LNCaP cells. Methods and results: The ³H‐thymidine incorporation assay revealed that either L ‐mimosine or DMOG treatments attenuated cell proliferation. Immunoblot and enzyme‐linked immunosorbent assays (ELISA) indicated that both L ‐mimosine and DMOG have an effect similar to hypoxia, which stabilized hypoxia‐inducible factor‐1α (HIF‐1α) and induced PSA gene expression. The results of the immunoblot and transient gene expression assays indicated that induction of the PSA expression by hypoxia is both HIF‐1α‐ and AR‐dependent. Immunoblot assays revealed that a curcumin treatment (10 μM) decreased the protein abundance of AR but did not significantly affect the protein levels of HIF‐1α and vascular endothelial growth factor, which were induced by hypoxia. ELISA and transient gene expression assays indicated that curcumin blocked the activation of L ‐mimosine or DMOG treatment on PSA expression. Conclusions: These results indicate that curcumin blocked the enhanced effect of PSA expression by L ‐mimosine and DMOG that induce hypoxia condition.     

Critical role of plectin in anti-migration potential of curcumin

Plectin, a linker protein that organizes the cytoskeleton, is critical for cell migration and wound healing. It is specifically expressed in epithelial cells, muscles, and other tissues. Numerous studies have shown that curcumin (diferuloylmethane) has anti-cancer potential. Curcumin can inhibit cancer cell migration and invasion through various signaling pathways. In this study, the down-regulation of plectin expression by siRNA was observed to induce the migration and invasion of human breast and lung cancer cell lines. Interestingly, the down-regulation of plectin expression by siRNA reversed the curcumin-induced anti-migration and anti-invasion effects in both cancer cell lines while α-tocopherol and genistein inhibited knockdown of plectin induced cancer cell migration. Curcumin did not affect the expression of plectin; however, α-tocopherol and genistein inhibited migration in plectin down-regulated cancer cells. These findings support the novel role of plectin and its effect on the anti-migration and anti-invasion potential of curcumin.     

Two pathways for prostaglandin F2 alpha synthesis by the primate periovulatory follicle

Prostaglandin E2 (PGE2) has been identified as a PG necessary for ovulation, but the ovulatory gonadotropin surge also increases PGF2 alpha levels in primate periovulatory follicles. To better understand the role of PGF2 alpha in ovulation, pathways utilized for PGF2 alpha synthesis by the primate follicle were examined. Monkeys were treated with gonadotropins to stimulate multiple follicular development; follicular aspirates and whole ovaries were removed before and at specific times after administration of an ovulatory dose of hCG to span the 40 h periovulatory interval. Human granulosa cells were also obtained (typically 34-36 h after hCG) from in vitro fertilization patients. PGF2 alpha can be synthesized from PGH2 via the aldo-keto reductase (AKR) 1C3. AKR1C3 mRNA and protein levels in monkey granulosa cells were low before hCG and peaked 24-36 h after hCG administration. Human granulosa cells converted PGD2 into 11 beta-PGF2 alpha, confirming that these cells possess AKR1C3 activity. PGF2 alpha can also be synthesized from PGE2 via the enzymes AKR1C1 and AKR1C2. Monkey granulosa cell levels of AKR1C1/AKR1C2 mRNA was low 0-12 h, peaked at 24 h, and returned to low levels by 36 h after hCG administration. Human granulosa cell conversion of [(3)H]PGE2 into [(3)H]PGF2 alpha was reduced by an AKR1C2-selective inhibitor, supporting the concept that granulosa cells preferentially express AKR1C2 over AKR1C1. In summary, the ovulatory gonadotropin surge increases granulosa cell expression of AKR1C1/AKR1C2 and AKR1C3. Both of these enzyme activities are present in periovulatory granulosa cells. These data support the concept that follicular PGF2 alpha can be synthesized via two pathways during the periovulatory interval.     

Combination of low dose of genistein and daidzein has synergistic preventive effects on isogenic human prostate cancer cells when compared with individual soy isoflavone

The reduced incidence of prostate cancer (PCa) in Asia countries has been attributed to high soy diets, and major soy isoflavones, in particular daidzein and genistein, are thought to be the source of the beneficial and anti-cancer effects of soy foods. However, attention has been drawn to the safety of using high levels of soy isoflavones in humans, which is especially the concern for consumers taking regular soy isoflavone dietary supplements. The main objective of this study is thus to identify a soy isoflavone combination with lower levels of daidzein and genistein to be a more efficacious and safer chemo-preventive agent for PCa. The anticancer effects of daidzein and genistein, and their combinations on early-stage androgen-dependent PCa cells (LNCaP) and bone metastatic LNCaP-derivative PCa cells (C4-2B) were compared. Cells were treated with varying concentrations of daidzein, genistein (25–200 μM) or their combinations (25 or 50 μM) and cell proliferation, apoptosis, cell cycles and cellular uptakes of the isoflavones were measured after 48 h. Daidzein and genistein showed a synergistic effect on inhibiting cell proliferation and inducing apoptosis of both PCa cells. Twenty-five μM daidzein/50 μM genistein and 50 μM daidzein/50 μM genistein significantly increased the apoptotic effects on C4-2B cells although they did not show any effect when used individually. Except 50 μM daidzein/50 μM genistein, all other combinations had no impacts on cell cycles. For treatment with soy isoflavone combination, genistein was always better taken up than daidzein by both LNCaP and C4-2B cells.     

Soy isoflavone genistein induces cell death in breast cancer cells through mobilization of endogenous copper ions and generation of reactive oxygen species

Scope: Worldwide geographical variation in cancer incidence indicates a correlation between dietary habits and cancer risk. Epidemiological studies have suggested that populations with high isoflavone intake through soy consumption have lower rates of breast, prostate, and colon cancer. Isoflavone genistein in soybean is considered a potent chemopreventive agent against cancer. Although several mechanisms have been proposed, a clear anticancer action mechanism of genistein is still not known. Methods and results: Here, we show that the cytotoxic action of genistein against breast cancer cells involves mobilization of endogenous copper. Further, whereas the copper specific chelator neocuproine is able to inhibit the apoptotic potential of genistein, the molecules which specifically bind iron (desferroxamine mesylate) and zinc (histidine) are relatively ineffective in causing such inhibition. Also, genistein‐induced apoptosis in these cells is inhibited by scavengers of reactive oxygen species (ROS) implicating ROS as effector elements leading to cell death. Conclusions: As copper levels are known to be considerably elevated in almost all types of cancers, in this proof‐of‐concept study we show that genistein is able to target endogenous copper leading to prooxidant signaling and consequent cell death. We believe that such a mechanism explains the anticancer effect of genistein as also its preferential cytotoxicity towards cancer cells.     

Enrichment of Genistein in Soy Protein Concentrate with b-glucosidase

Genistein has been reported to have the most potent anticarcinogenic effect among isoflavones. Conversion of genistin in the glucoside form to genistein in the aglucone form by a b-glucosidase enzyme hydrolytic activity during soy protein concentrate (SPC) preparation was evaluated. The optimum conditions for the conversion of genistin to genistein were pH 5.0, 50 °C incubation temperature, 2 units of enzyme/g of soy flour, 1 h incubation period, and 1:10 (w/v) defatted soy flour to water ratio. Under these conditions, 1.214 mg/g genistein were obtained in SPC and is significantly (p < 0.05) higher than the genistein content in SPC prepared under similar conditions without enzyme addition (0.845 mg/g).