刺五加提取物对人脐静脉内皮细胞紫外线辐射损伤的防护作用

研究刺五加提取物对紫外线辐射损伤人脐静脉内皮细胞(Human umbilical vein endothelial cell,HUVEC)的影响。将进入对数生长期的HUVEC细胞分为空白对照组、辐射组、给药预防组(低、中、高剂量)和给药修复组(低、中、高剂量)。各组均接受相同辐射,给药预防组提前加入不同质量浓度的刺五加提取物孵化12 h。采用MTT法检测细胞存活率,TBA法检测丙二醛(MDA)含量,WST法检测超氧化物歧化酶(SOD)水平,通过DNA-Ladder检测细胞凋亡。结果表明:经辐射后,给药预防组和给药修复组细胞存活率均有所提高,且呈质量浓度依赖性。与空白对照组相比,辐射组MDA含量升高,SOD水平下降;与辐射组比较,实验组给药后MDA含量降低,SOD水平升高。DNA-Ladder可以得到明显DNA凋亡条带。刺五加提取物能有效预防和修复辐射损伤的HUVEC细胞,且上述试验中给药预防组效果均优于给药修复组。     

Effective suppression of azoxymethane‐induced aberrant crypt foci formation in mice with citrus peel flavonoids

Citrus peel or its extract has been reported to exhibit a broad spectrum of biological activity. Herein, we report the first investigation of inhibitory effects of a formulated product from citrus peel extract, gold lotion (GL), on azoxymethane‐induced colonic tumorigenesis. We have demonstrated that oral feeding of GL decreased the number of aberrant crypt foci (ACF), particularly large size of ACF in colonic tissues of mice. Both gene and protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) were suppressed by GL treatment. The in vivo data have revealed for the first time that the citrus peel extract–GL–is an effective antitumor agent mechanistically downregulating the protein levels of iNOS, COX‐2, ornithine decarboxylase, vascular endothelial growth factor, and matrix metallopeptidase 9 in colonic tissues of mice, suggesting that GL is a novel functional natural product capable of preventing inflammation‐associated colon tumorigenesis.     

Glabridin,an isoflavan from licorice root,inhibits migration,invasion and angiogenesis of MDA‐MB‐231 human breast adenocarcinoma cells by inhibiting focal adhesion kinase/Rho signaling pathway

Scope: In this study we first report the antimigration, antiinvasive effect of glabridin, a flavonoid obtained from licorice, in MDA‐MB‐231 human breast adenocarcinoma cells. Methods and results: Glabridin exhibited effective inhibition of cell metastasis by decreasing cancer cell migration and invasion of MDA‐MB‐231 cells. In addition, glabridin also blocked human umbilical vein endothelial cells (HUVEC) migration and decreased MDA‐MB‐231‐mediated angiogenesis. Further investigation revealed that the inhibition of cancer angiogenesis by glabridin was also evident in a nude mice model. Blockade of MDA‐MB‐231 cells and HUVEC migration was associated with an increase of αγβ3 integrin proteosome degradation. Glabridin also decreased the active forms of FAK and Src, and enhanced levels of inactivated phosphorylated Src (Tyr 416), decreasing the interaction of FAK and Src. Inhibition of the FAK/Src complex by glabridin also blocked AKT and ERK1/2 activation, resulting in reduced activation of RhoA as well as myosin light chain phosphorylation. Conclusion: This study demonstrates that glabridin may be a novel anticancer agent for the treatment of breast cancer in three different ways: inhibition of migration, invasion and angiogenesis.     

Regulation of endothelial proliferation by the renin-angiotensin system in human umbilical vein endothelial cells

This study was performed in order to evaluate the role of angiotensin II in physiological angiogenesis. Human umbilical vein endothelial cells (HUVEC) were stained for angiotensin II type 1 receptor (AGTR1) immunocytochemically and for gene expression of renin-angiotensin system (RAS) components. The regulation of the angiogenesis-associated genes vascular endothelial growth factor (VEGF) and angiopoietins (ANGPT1 and ANGPT2) were studied using quantitative RT-PCR. Furthermore, we examined the effect of angiotensin II on the proliferation of HUVEC using Ki-67 as well as BrdU immunocytochemistry and investigated whether the administration of the AGTR1 blocker candesartan or the VEGF antagonist FLT1-Fc could suppress the observed angiotensin II-dependent proangiogenic effect. AGTR1 was expressed in HUVEC and the administration of angiotensin II significantly increased the gene expression of VEGF and decreased the gene expression of ANGPT1. Since the expression of ANGPT2 was not affected significantly the ratio of ANGPT1/ANGPT2 was decreased. In addition, a significantly increased endothelial cell proliferation was observed after stimulation with angiotensin II, which was suppressed by the simultaneous administration of candesartan or the VEGF antagonist FLT1-Fc. These results indicate the potential capacity of angiotensin II in influencing angiogenesis by the regulation of angiogenesis-associated genes via AGTR1. Since VEGF blockade opposed the effect of angiotensin II on cell proliferation, it is hypothesised that VEGF mediates the angiotensin II-dependent effect in concert with the changes in angiopoietin expression. This is the first report of the RAS on the regulation of angiogenesis-associated genes in physiology.     

桑葚提取物体外抗炎作用及机制的研究

本文采用脂多糖(LPS)刺激小鼠腹腔巨噬细胞RAW264.7,建立细胞体外的炎症模型,研究桑葚提取物对LPS诱导巨噬细胞RAW264.7分泌功能的影响及其作用机制。实验用1μg/mL LPS刺激RAW264.7细胞,在不同浓度样品的干预下,用MTT法检测不同浓度的样品对RAW264.7细胞的作用;用Griess法检测细胞液中NO的含量;用酶联免疫吸附法(ELISA)检测细胞液中PGE2含量;用免疫印迹法(Western Blot)和RT-PCR法检测桑葚提取物对细胞iNOS和COX-2表达的影响;用HPLC法检测桑葚提取物中白藜芦醇的含量。结果表明桑葚提取物浓度在0.5~2 mg/mL范围内对细胞生长无明显影响;在1~2 mg/mL范围内能有效抑制NO和PGE2的分泌并能有效抑制iNOS和COX-2的表达;桑葚提取物中白藜芦醇的含量为107.44±0.48μg/g。这表明桑葚提取物抑制炎症相关因子表达量,从而减弱促炎症反应,发挥抗炎功效,其抗炎活性可能与桑葚中含有较高的白藜芦醇相关。     

Antihypertensive properties of flavonoid-rich apple peel extract

Hypertension is a major public health problem rising across the globe. Inhibition of angiotensin converting enzyme (ACE) is identified as a main therapeutic target in controlling high blood pressure. The present study investigated the ACE inhibitory property of a flavonoid-rich apple peel extract (FAE), its constituents, selected flavonoids and some quercetin metabolites using a biochemical assay of ACE inhibition and a human umbilical vein endothelial cell (HUVEC) model. FAE, all the tested flavonoids except genistein, and two quercetin metabolites (quercetin-3-O-glucuronic acid and quercetin-3-O-sulfate) significantly (p < 0.05) inhibited ACE. Enzyme kinetic analysis revealed that flavonoids are competitive inhibitors of ACE. In the HUVEC model, FAE, quercetin-3-O-glucoside and quercetin-3-O-glucuronic acid inhibited significantly (p < 0.05) ACE activity. Overall, FAE and most of the flavonoids tested showed ACE inhibition in vitro which needs further investigations using animal and human clinical trials.     

榆干离褶伞溶栓酶对脂多糖诱导的血管内皮细胞损伤的保护作用

目的：探讨榆干离褶伞溶栓酶对脂多糖（lipopolysaccharide,LPS）诱导的血管内皮细胞炎性损伤的保护作用。方法：以LPS诱导人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVEC）炎性损伤。将HUVEC分为空白对照组、模型组和榆干离褶伞溶栓酶（Lyophyllum ulmarium fibrinolytic enzyme,LUFE）低、中、高剂量组。采用噻唑蓝法测定HUVEC存活率,通过酶联免疫吸附测试法检测细胞上清液乳酸脱氢酶（lactate dehydrogenase,LDH）、肿瘤坏死因子α（tumor necrosis factor α,TNF-α）、白介素6（interleukin 6,IL-6）、E-选择素和单核细胞趋化因子1（monocyte chemoattractant protein 1,MCP-1）水平。流式细胞术检测细胞间黏附分子1（intercellular cell adhesion molecule 1,ICAM-1）表达水平,采用Hoechst染色法观察HUVEC与人急性单核细胞白血病细胞系（human acute monocytic leukemia cell line-1,THP-1）的黏附作用。用蛋白印迹实验检测HUVEC的Toll样受体4（toll-like receptor 4,TLR4）、丝裂原活化蛋白激酶（mitogen-activated protein kinase,MAPK）以及核因子-κB（nuclear factor κB,NF-κB）通路中主要蛋白（髓样分化因子88（myeloid differentiation factor 88,MyD88）、转化生长因子β激活激酶1（transforming growth factor β activated kinase 1,TAK1）、磷酸化TAK1（phosphorylated TAK1,p-TAK1））的表达和活化情况。结果：LUFE能够抑制LPS所诱导的HUVEC培养上清液LDH、TNF-α、IL-6、E-选择素和MCP-1水平的升高,降低细胞ICAM-1表达水平并减弱HUVEC与THP-1的黏附作用。与模型组比较,LUFE各剂量组TLR4、MyD88、p-TAK1/TAK1、磷酸化c-Jun氨基末端激酶（phosphorylated c-Jun N-terminal kinase,p-JNK）/JNK、p-p38/p38、p-NF-κB/NF-κB水平显著降低（P＜0.05）。结论：LUFE对血管内皮细胞的炎性损伤具有保护作用,其作用机制可能是通过抑制TLR4/MyD88/TAK1/NF-κB信号通路及MAPK通路,进而降低炎症因子水平,从而保护血管内皮细胞。     

Effects of raspberry phytochemical extract on cell proliferation, apoptosis, and serum proteomics in a rat model

The red raspberry extract possesses potent antioxidant capacity and anticancerous activity in vitro and in vivo. The objective of this study was to determine whether red raspberry extract affected the cell cycle, angiogenesis, and apoptosis in hepatic lesion tissues from a rat model induced by diethylnitrosamine (DEN) as well as changes of serum proteomics. Rats were treated with red raspberry extract (0.75, 1.5, or 3.0 g/kg of body weight) by gavage starting 2 h after DEN administration and continued for 20 wk. Red raspberry extract inhibited cell proliferation, vascular endothelial growth factor VEGF expression, and induced apoptosis in the hepatic lesion tissues. In addition, 2 protein peaks (2597.93 and 4513.88 m/z) were identified to differentially express in the 3.0 g/kg body weight and positive control groups by serum proteomics. These results suggest that a dietary supplement with red raspberry effectively protects against chemically induced hepatic lesions in rats.     

Modulation of cancer cell proliferation by cell survival signal Akt and tumor suppressive energy sensor AMP-activated protein kinase in colon cancer cells treated with resveratrol

It has been well known that resveratrol inhibits the proliferation of various cancer cells. AMP-activated protein kinase (AMPK), a sensor of cellular energy status, has emerged as a potent target for cancer prevention and/or treatment. It has been found that the activation of AMPK by resveratrol was crucial for the inhibition of HT-29 colon cancer cells. Resveratrol strongly inhibited phosphorylation of Akt. The possibility whether AMPK activation was essential to the inhibition of p-Akt was investigated in resveratrol-treated cancer cells. The inhibitory effect of resveratrol on Akt was not observed when AMPK activities were blocked by the treatment with AMPK siRNA at a relatively lower level of resveratrol. However, the higher concentrations of resveratrol inhibited Akt without the activation of AMPK. Therefore, it was concluded that resveratrol could modulate Akt AMPK-dependently or AMPK-independently. The inhibition of Akt along with the activation of AMPK may contribute to the unraveling anti-cancer mechanism of resveratrol.     

Resveratrol protects RINm5F pancreatic cells from methylglyoxal-induced apoptosis

The effects of resveratrol on the prevention of apoptosis as well as insulin production are unclear. To examine the effects, we treated insulin-secreting beta cells with methylglyoxal (MG), a metabolite known to induce apoptosis and diabetes in the presence or absence of resveratrol. The MG treatment induced the expression of apoptotic signaling molecules and decreased insulin production. Pretreatment with resveratrol resulted in an increase in insulin synthesis via upregulation of peroxisome proliferator-activated receptor gamma (PPARgamma) and pancreatic-duodenal homeobox-1 (PDX-1). Resveratrol also inhibited MG-mediated expression of CCAAT/enhancer-binding protein beta (C/EBPbeta), a negative regulator of insulin production. Moreover, resveratrol strongly activated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), which resulted in the attenuation of oxidative stress. These results suggest that resveratrol can attenuate MG-induced oxidative stress in pancreatic cells and increase insulin levels.     

绿豆皮黄酮对H2O2诱导人脐静脉血管内皮细胞损伤的保护作用

目的:研究绿豆皮黄酮对H2O2诱导损伤人脐静脉血管内皮细胞的保护作用及其机制。方法:用体积分数70%的乙醇提取绿豆皮中黄酮,采用高效液相色谱法测定其有效成分;通过H2O2诱导HUVEC细胞损伤,建立细胞氧化损伤模型。试验被分为正常对照组、H2O2组、维生素C组、绿豆皮黄酮低、中、高剂量组(20,60,80μg/m L),通过MTT法测定细胞增殖能力,生化比色法检测各组细胞及培养液中的超氧化物酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、乳酸脱氢酶(LDH)活性以及谷胱甘肽(GSH)和丙二醛(MDA)含量。结果:H2O2呈剂量依赖性降低内皮细胞活力,并引起细胞凋亡。其中1 000μmol/L H2O2处理HUVEC后,与正常组相比,细胞存活率明显受到抑制,而绿豆皮各剂量组能显著提高细胞和细胞培养液中的GSH-Px、SOD、GSH活性,降低MDA含量,同时降低细胞中LDH活性并增强细胞培养液中的LDH含量。结论:绿豆皮黄酮能抑制H2O2诱导的内皮细胞凋亡,减轻H2O2对内皮细胞的损伤作用,其机制是:绿豆皮黄酮抑制损伤细胞脂质过氧化,提高机体抗氧化酶活性,清除自由基。     

黑蒜促进血管生成作用

目的:通过转基因斑马鱼体内模型和人脐静脉内皮细胞体外模型,研究黑蒜对血管生成的促进作用。方法:采用绿色荧光蛋白标记血管的转基因flk-1:GFP系斑马鱼作为体内评价模型,在胚胎发育过程中,同时加入血管生成抑制剂(PTK787)与黑蒜提取液(0.2~3.2 μL/mL),观察斑马鱼胚胎节间血管和肠下静脉的生成情况;利用人脐静脉内皮细胞作为体外模型,借助细胞增殖检测实验,通过检测细胞活性,研究黑蒜提取液对人脐静脉内皮细胞增殖的影响。结果:黑蒜提取液可显著(p<0.05)改善由PTK787引起的胚胎节间血管和肠下静脉紊乱,增加斑马鱼胚胎节间血管和肠下静脉数量,并呈现出剂量依赖性,当黑蒜提取液浓度提高至3.2 μL/mL时,由PTK787抑制的胚胎节间血管和肠下静脉数目恢复至正常水平。另一方面,黑蒜提取液可剂量依赖性地提高人脐静脉内皮细胞的活性,促进人脐静脉内皮细胞的生长。结论:黑蒜在体内和体外模型上均体现出显著的促进血管生长的作用。     

Butterfly pea (Clitoria ternatea) seed and petal extracts decreased HEp‐2 carcinoma cell viability

The hydrophilic phenolics, lipophilic tocopherols, phytosterols and fatty acids in butterfly pea seeds and petals were determined. The seeds had fifteen phenolics; of them, sinapic acid, epicatechin and hydroxycinnamic acid derivative concentrations were above 0.5 mg g^?1. The petals contained a group of ternatins, flavone glycosides and delphinidin derivatives. Both the seeds and petals had four phytosterols and α‐ and γ‐tocopherols. However, the level of β‐sitosterol or γ‐tocopherol in the seeds was much higher than in the petals. Linoleic acid was the most abundant fatty acid in the seeds and petals, while phytanic acid was found in the petals. The effect of lipophilic and hydrophilic extracts of the seeds [lipophilic extract of the butterfly pea seeds (LBS) and hydrophilic extract of butterfly pea seeds (HBS)] and petals [lipophilic extract of the butterfly pea petals (LBP) and hydrophilic extract of butterfly pea petals (HBP)] on decreased HEp‐2 human carcinoma cell viability was evaluated. The effect of HBS or HBP on decreased cancer cell viability was much higher than that of either LBS or LBP, while HBS showed significantly higher effect than HBP. The results indicated that butterfly pea seed and petal extracts could have the potential in functional food development.     

Citrus-derived auraptene stimulates angiogenesis by activating the Erk- and PI3K/Akt/eNOS-dependent signaling pathways in human umbilical vein endothelial cells

Auraptene is a citrus-derived natural monoterpene that has been shown to exert anti-cancer, anti-bacterial, anti-inflammatory, and antioxidant roles. Since little is known about other biological functions of auraptene, we examined the efficacy of auraptene for stimulating angiogenesis in human umbilical vein endothelial cells (HUVECs). Treatment with low concentrations of auraptene stimulated endothelial cell proliferation, migration, and tube formation. Furthermore, auraptene activated Erk, Akt, and endothelial nitric oxide synthase (eNOS), and increased NO production. Auraptene also partially induced the phosphorylation of VEGFR2. Furthermore, auraptene-induced activation of Erk, Akt, and eNOS were significantly inhibited by the inhibitors PD98059, LY294002, and l-NIO dihydrochloride. These results suggest that auraptene stimulates angiogenesis by regulating the VEGFR2, Erk, and PI3K/Akt-eNOS signaling pathways.     

Differential effects of resveratrol and its naturally occurring methylether analogs on cell cycle and apoptosis in human androgen‐responsive LNCaP cancer cells

Stilbenes are phytoalexins that become activated when plants are stressed. These compounds exist in foods and are widely consumed. Resveratrol is a grape‐derived stilbene, which possesses a wide range of health‐promoting activities, including anticancer properties. Several other stilbenes structurally similar to resveratrol are also available in food, but their biological activities remain largely unknown. In this study, we compared the effects of resveratrol and its natural derivatives pterostilbene, trans‐resveratrol trimethylether, trans‐pinostilbene and trans‐desoxyrhapontigenin on androgen‐responsive human prostate cancer LNCaP cells. We found that these compounds exert differential effects on LNCaP cell growth, cell cycle and apoptosis. Trans‐resveratrol trimethylether appeared to be the most potent compound among the stilbenes tested. Treatment of LNCaP cells with trans‐resveratrol trimethylether resulted in G2/M blockage while other compounds, including resveratrol, induced G1/S arrest. Moreover, different from other compounds, trans‐resveratrol trimethylether induced apoptosis. At the molecular level, the effects of these compounds on cell cycle correlated with induction of the cyclin‐dependent kinase inhibitor 1A and B mRNA levels. Additionally, these compounds also inhibited both androgen‐ as well as estrogen‐mediated pathways. These results provide mechanistic information on how resveratrol and its methylether analogs may act to contribute to potential antiprostate cancer activity.     

Inhibitory effects of trans-resveratrol analogs molecules on the proliferation and the cell cycle progression of human colon tumoral cells

Resveratrol may function as a cancer chemopreventive agent. However, few data are available on the antitumoral activities of its dimer, epsilon-viniferin, also present in human diet. So, the effects of resveratrol, epsilon-viniferin, of their acetylated forms (resveratrol triacetate, epsilon-viniferin pentaacetate) and of vineatrol (a wine grape extract) were compared on human adenocarcinoma colon cells. Resveratrol and resveratrol triacetate inhibit cell proliferation and arrest cell cycle. epsilon-Viniferin and epsilon-viniferin pentaacetate slightly reduce cell proliferation. Vineatrol inhibits cell proliferation and favors an accumulation in the S phase of the cell cycle. Consequently, resveratrol triacetate and vineatrol could constitute new putative anticancer agents on colon carcinoma.     

牡丹花蕊醇提物对H2O2诱导HUVEC细胞损伤的保护作用

采用高效液相色谱分析牡丹花蕊醇提物的主要成分,并通过H_2O_2诱导HUVEC细胞建立损伤模型,研究牡丹花蕊醇提物对其保护作用。结果表明:牡丹花蕊醇提物主要成分为芦丁、槲皮素和芍药苷3种单体,含量分别为44.25%,15.50%,17.00%;牡丹花蕊醇提物能够降低细胞及其培养液中MDA含量,提高细胞内SOD和GPX活性以及GSH的含量。牡丹花蕊醇提物能够提高HUVEC细胞的抗氧化能力。     

圣草次苷的合成及其对过氧化氢诱导人脐静脉内皮细胞氧化损伤的保护作用

以橙皮苷为原料,经三氯化铝-吡啶去甲基反应合成药用天然产物圣草次苷,产物通过1H核磁共振（nuclear magnetic resonance,NMR）和13C NMR进行结构确证,并探究其对过氧化氢（H2O2）诱导氧化应激损伤的人脐静脉内皮细胞系（human umbilical vein endothelial cells,HUVEC）细胞的保护作用机制。结果表明：圣草次苷对HUVEC细胞无毒性作用,能显著提高氧化损伤HUVEC细胞的存活率（P＜0.05）,但无显著剂量效应;显著降低细胞中活性氧水平和丙二醛含量（P＜0.05）,显著提高超氧化物歧化酶、谷胱甘肽过氧化物酶、过氧化氢酶等抗氧化酶的活力（P＜0.05）;显著上调Bcl-2/Bax蛋白比值和下调p53蛋白表达（P＜0.05）。结论：圣草次苷能有效阻止H2O2所致HUVEC细胞损伤,该作用与圣草次苷的抗氧化活性和抑制细胞死亡有关。     

The role of progesterone in endometrial angiogenesis in pregnant and ovariectomised mice

The role of progesterone (and oestrogen) in endometrial angiogenesis remains controversial. The aims of this study were to quantify endometrial angiogenesis in pregnant mice and to investigate the role of progesterone in promoting endothelial cell proliferation in ovariectomized mice. Uteri were collected on days 1 to 4 of pregnancy when circulating progesterone concentrations were increasing, prior to implantation. Before dissection, mice were injected with bromodeoxyuridine (BrdU) enabling proliferating endothelial cells to be quantified with CD31/BrdU double-immunohistochemistry. There was a significant increase in proliferating endothelial cells on day 3 of pregnancy when plasma progesterone also increased. To determine if this endothelial cell proliferation was due to progesterone, an experiment was performed on ovariectomised mice. One group was treated with a single oestradiol injection on day 8 after ovariectomy, followed by a no-treatment day and three consecutive daily injections of progesterone. Other groups were treated with either the vehicle, oestradiol or progesterone injections only; all were dissected on day 13 following ovariectomy. Unexpectedly, mice treated with progesterone-only had the highest amount of endothelial cell proliferation and oestrogen priming was found to significantly reduce this progesterone-induced endothelial cell proliferation. To determine if this proliferation is mediated by vascular endothelial growth factor (VEGF), a further experiment in which VEGF anti-serum was administered concurrently with the progesterone injections was performed. Endothelial cell proliferation was reduced but not abolished suggesting progesterone-induced endometrial angiogenesis is only partly mediated by VEGF. Results indicate that oestrogen priming is not required for progesterone to stimulate endometrial endothelial cell proliferation and that oestrogen inhibits progesterone-induced angiogenesis in ovariectomised mice.