Tannin 1-alpha-O-galloylpunicalagin induces the calcium-dependent activation of endothelial nitric-oxide synthase via the phosphatidylinositol 3-kinase/Akt pathway in endothelial cells

Many polyphenols have been found to increase endothelial nitric oxide (NO) production. In our present study, we investigated the effects of 1-alpha-O-galloylpunicalagin upon endothelial nitric oxide synthase (eNOS) activity in endothelial cells (ECs). Both 1-alpha-O-galloylpunicalagin and punicalagin induced NO production in a dose-dependent manner in ECs. Despite having similar chemical structures, punicalagin induced lower levels of NO production than 1-alpha-O-galloylpunicalagin. After 1-alpha-O-galloylpunicalagin addition, a rise in the intracellular Ca(2+) concentration preceded NO production. The Ca(2+) ionophore A23187 stimulated eNOS phosphorylation and augmented NO production. Pretreatment with Ca(2+) chelators inhibited 1-alpha-O-galloylpunicalagin-induced eNOS phosphorylation and NO production. Treatment with 1-alpha-O-galloylpunicalagin did not alter the eNOS protein levels but, unlike punicalagin, induced a sustained activation of eNOS Ser(1179) phosphorylation. 1-alpha-O-galloylpunicalagin was also found to activate ERK1/2, JNK and Akt in ECs. Moreover, simultaneous treatment of these cells with specific phosphatidylinositol-3-kinase inhibitors significantly inhibited the observed increases in eNOS activity and phosphorylation levels. In contrast, the inhibition of (ERK)1/2, JNK and p38 had no influence on eNOS Ser(1179) phosphorylation. Our present results thus indicate that the 1-alpha-O-galloylpunicalagin-induced calcium-dependent activation of eNOS is primarily mediated via a phosphatidylinositol 3-kinase/Akt-dependent increase in eNOS activity, and occurs independently of the eNOS protein content.     

A Maillard reaction product enhances eNOS activity in human endothelial cells

Nitric oxide (NO) produced by the endothelial nitric oxide synthase (eNOS) is an important signaling molecule in the cardiovascular system. Although dietary factors can modulate eNOS activity, putative effects of processed food are barely investigated. We aimed to examine whether the model Maillard reaction product 3‐hydroxy‐2‐methyl‐1‐propyl‐4(1H)‐pyridone (HMPP), formed from maltol or starch and propylamine, affects the eNOS system. Incubation of EA.hy926 endothelial cells with 30–300 μM HMPP for 18 h enhanced endothelial NO release measured with the fluorescent probe diaminofluorescein‐2 and eNOS activity determined by the [14C]L‐arginine‐[14C]L‐citrulline conversion assay. HMPP increased NO production also in two different types of primary human endothelial cells. Protein levels of eNOS and inducible NO synthase remained unaltered by HMPP. HMPP inhibited eNOS activity within the first 2–4 h, whereas it potently increased eNOS activity after 12–24 h. Levels of eNOS phosphorylation, expression of heat‐shock protein 90, caveolin‐1 and various antioxidant enzymes were not affected. Intracellular reactive oxygen species remained unchanged by HMPP. This is the first study to demonstrate positive effects of a Maillard reaction product on eNOS activity and endothelial NO production, which is considered favourable for cardiovascular protection.     

苦荞蛋白源肽AFYRW对脂多糖诱导的人脐静脉内皮细胞损伤的影响

目的：探讨苦荞蛋白源肽AFYRW在脂多糖（lipopolysaccharide,LPS）诱导的血管内皮细胞损伤中的作用。方法：采用LPS诱导人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVECs）建立血管内皮细胞炎症损伤模型;采用CCK8试剂盒检测AFYRW对细胞增殖活力的影响;Western blot检测血管细胞黏附分子-1(vascular cell adhension molecule-1,VCAM-1)、细胞内黏附分子-1(intercellular adhension molecule-1,ICAM-1)、白细胞介素（interleukin,IL）-6、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、内皮型一氧化氮合酶（endothelial nitric oxide synthase,e NOS）、诱导型一氧化氮合酶（inducible nitricoxide synthase,iNOS）水平及核因子-κB(nuclear factor kappa-B,NF-κB)p65的磷酸化水平;酶法...     

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.     

Trans C18:1通过NOS-NO系统诱导内皮细胞损伤研究

为了观察trans C18:1对人脐静脉内皮细胞损伤的影响及其与NOS-NO系统的关系,首先将不同浓度trans C18:1 (50、100、200、400μmol/L)与人脐静脉内皮细胞共培养24或48h后,MTT法检测细胞存活率;用trans C18:1(200μmol/L)处理内皮细胞24h后,分别检测一氧化氮含量(NO)及一氧化氮合酶(NOS)活性;将trans C18:1与一氧化氮合酶阻断剂亚硝酸左旋精氨酸甲酯(LNAME)及一氧化氮供体(SNP)单一或联合处理内皮细胞,检测细胞存活率的变化。结果显示：trans C18:1以剂量和时间依赖方式导致内皮细胞的存活率下降; LNAME与trans C18:1联合处理内皮细胞后细胞存活率下降,而SNP与trans C18:1联合处理后细胞存活率上升;trans C18:1可诱导NO水平和内皮型一氧化氮合酶(eNOS)活性显著下降,而诱导型一氧化氮合酶(iNOS)活性无显著改变。表明：trans C18:1能通过抑制eNOS活性减少NO的分泌,并暗示NOS-NO系统可能是trans C18:1诱导内皮细胞损伤的作用机制之一。     

鱼肠道中抗氧化活性乳酸菌的筛选及鉴定

从海水和淡水鱼肠道的乳酸菌中筛选具有抗氧化活性的乳酸菌菌株,旨在降低和预防水产品加工过程中氧化导致的质量问题。结果表明：从48 株乳酸菌中筛选出5 株具有较强抗氧化活性的乳酸菌,进一步复筛获得来源于草鱼肠道的菌株CY1-2的活性最强。菌株CY1-2对1,1-二苯基-2-三硝基苯肼自由基、羟自由基、超氧阴离子自由基清除率和抗脂质过氧化率分别为（67.29±0.42）%、（60.67±1.44）%、（29.87±1.14）%和（40.77±0.50）%,其无细胞提取物对羟自由基、超氧阴离子自由基清除率和抗脂质过氧化率分别为（64.27±1.26）%、（21.97±1.47）%和（51.03±0.40）%。此外,菌株CY1-2对革兰氏阳性菌地衣芽孢杆菌、金黄色葡萄球菌、蜡样芽孢杆菌和革兰氏阴性菌志贺氏菌、埃希氏大肠杆菌、铜绿假单胞菌及荧光假单胞菌均有抑菌活性。经生理生化和16S rDNA序列分析,菌株CY1-2被鉴定为植物乳杆菌（Lactobacillus plantarum）。植物乳杆菌CY1-2具有较强的抗氧化活性和广谱抑菌活性,对开发新型水产品天然微生物源性抗氧化剂具有一定的应用价值。     

Characterization of endothelin-1 and nitric oxide generating systems in corpus luteum-derived endothelial cells

Endothelium-derived endothelin-1 (ET-1) and nitric oxide (NO) are pivotal regulators of corpus luteum (CL) function. To have a better insight into their synthesis and action, members of the ET system (ET-1, ET converting enzyme (ECE-1) isoforms a-d, ETA and ETB receptors) along with NO synthase (NOS) isoforms--endothelial (e)NOS and inducible (i)NOS--were quantified in CL-derived endothelial cells (CLEC). The expression of these genes in microvascular CLEC, obtained by lectin-coated magnetic beads, was compared with cells removed from the luteal microenvironment and maintained in culture for different durations, and with endothelial cells (EC) derived from a large blood vessel (i.e. bovine aortic endothelial cells, BAEC). The profile of gene expression in the different EC types was determined by quantitative real-time PCR. Freshly isolated EC from mid-cycle CL exhibited high ET-1 receptor expression (both ETA and ETB), low ET-1 synthesizing ability (both prepro (pp) ET-1 and ECE-1), but elevated iNOS - the high throughput NOS isoform. The distinct phenotype of CLEC was lost soon after an overnight culture. ETA and ETB receptor levels declined, ppET-1 levels increased while iNOS was reduced. These changes were extenuated during long-term culture of CLEC. The general pattern of gene expression in BAEC and long-term cultured CLEC was similar yet some differences, reminiscent of freshly isolated CLEC, remained: ECE-1c, ETB receptor and NOS isoforms were expressed differently in BAEC as compared with lines of CLEC. This study suggests that the luteal microenvironment is necessary to sustain the selective phenotype of its resident endothelial cells. The inverse relationship between ppET-1 and iNOS observed in freshly isolated CLEC and in cultured cells is physiologically significant and suggests that ET-1 and NO may modulate the production of each other.     

酶法降解坛紫菜多糖及其产物分析

以单因素试验为基础,通过正交试验确定了坛紫菜多糖的最佳酶解工艺条件为：酶解温度40 ℃、pH?6.0、酶解时间3 h。在此条件下,坛紫菜多糖酶解后的还原糖质量浓度为（1.42±0.12）mg/mL。利用液相色谱和质谱技术对酶解产物进行了组分分析和抗氧化活性检测。结果表明：酶解产物S1和S2均以半乳糖为主,半乳糖在两样品中分别占93.0%、90.5%,S1和S2的单糖组成相差不大。S1和S2都具有抗氧化活性,尤其对·OH的清除作用明显,且抗氧化活性大小S1＞S2。聚合度在10～16内的偶数糖比聚合度为4～6的坛紫菜寡糖清除·OH和O2 － ·的能力好,说明酶解产物的抗氧化活性与其聚合度有关,只有处于特定聚合度范围内才具有明显的抗氧化活性。     

银鲳酶解物抗氧化活性研究

选用胃蛋白酶、胰蛋白酶、碱性蛋白酶和中性蛋白酶对银鲳蛋白进行酶解以制备蛋白酶解物,以羟基自由基清除活性为指标确定银鲳最佳水解酶。结果显示,碱性蛋白酶的水解物抗氧化活性最强。实验对碱性蛋白酶水解银鲳的酶解条件(时间、温度、pH、酶添加量和固液比)进行正交实验设计,并对最佳水解条件下所获得的酶解物进行抗氧化活性测试。结果表明,银鲳蛋白碱性蛋白酶水解物对DPPH自由基和羟基自由基具有清除作用,其自由基清除效果呈现剂量依赖性,而且银鲳蛋白水解物还具有明显还原能力。所有这些体外抗氧化数据说明,银鲳蛋白水解物有明显的抗氧化效力。     

Protective effect of Cordyceps militaris against high glucose-induced oxidative stress in human umbilical vein endothelial cells

The protective effect of Cordyceps militaris against high glucose-induced oxidative stress in human umbilical vein endothelial cells (HUVECs), as compared with Cordyceps sinensis, was examined. The cytotoxicity of HUVECs induced by 40 mM glucose was ameliorated by water extracts of C. militaris (CME) and water extracts of C. sinensis (CSE). CME and CSE inhibited the increase in ROS and NO in HUVECs induced by 40 mM high glucose. Moreover, CME increased the Bcl-2/Bax ratio, modulated the mitochondrial membrane potential and reduced the caspase-3 activity in high glucose-induced HUVECs. In addition, cordycepin, a component of CME and CSE, displayed protective effects against oxidative stress, which was partly responsible for the cytoprotective effects of CME and CSE against high glucose-induced oxidative stress in HUVECs. Overall, the obtained results show C. militaris helps preventing diabetic endothelial dysfunction and related complications.     

AngⅡ诱导人脐静脉内皮细胞建立高血压损伤模型

目的:采用高血压发病因子血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)建立高血压损伤模型。方法:使用不同浓度(0.01、0.10、1.00μmol/L和10.00μmol/L)AngⅡ诱导HUVECs不同时间(3、6、9、12 h和24 h),通过考察HUVECs形态、活性、功能、微观结构及凋亡程度来评价HUVECs损伤程度,同时采用交叉设计方差分析和多重比较方法得到AngⅡ最适诱导浓度和最佳诱导时间。结果:AngⅡ呈浓度依赖式诱导HUVECs损伤,最佳诱导条件为1.00μmol/L AngⅡ诱导12 h,此时HUVECs活性为44.85%、NO含量为43.57μmol/L、总一氧化氮合酶活力为6.99 U/mg pro、内皮型一氧化氮合酶活力为1.89 U/mg pro、丙二醛含量为7.46 nmol/mL、超氧化物歧化酶活力为27.29 U/mg pro、细胞凋亡率为41.5%,微观结构显示核膜皱缩,核仁消失,胞浆内出现大量空泡状结构,线粒体或细小或肿胀,粗面内质网扩张数目较少,部分核糖体丢失,平面内质网扩张,但细胞未解体,仍然保持细胞结构。结论:1.00μmol/L AngⅡ诱导12 h可以成功诱导HUVECs建立高血压损伤模型。     

Nitric oxide fumigation stimulates flavonoid and phenolic accumulation and enhances antioxidant activity of mushroom

The effects of nitric oxide (NO) on antioxidant activity and contents of phenolics and flavonoids in mushroom Russula griseocarnosa were investigated. Freshly harvested mushrooms were fumigated with 0, 10, 20 and 30μLL(-1) NO at 20°C for 2h and then taken to examine the antioxidant activities using assays of reducing power, chelating effect on ferrous ions, scavenging effect on hydroxyl free radicals, and 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity. The results showed that the antioxidant activities of the mushrooms fumigated with NO were significantly increased when compared to the controls. Moreover, NO fumigation significantly enhanced phenolic and flavonoid contents and stimulated the activities of phenylalanine ammonia-lyase and chalcone synthase. The results indicated that NO fumigation might have potential application for enhancing the bioactive compounds and improving antioxidant activities in the mushrooms. Furthermore, the data suggested that the NO-induced phenolic and flavonoid accumulation was due to the activation of the biosynthetic pathways in the mushrooms.     

Expression of endothelial nitric oxide synthase in the ovine ovary throughout the estrous cycle

This study was conducted to evaluate the expression of endothelial nitric oxide synthase (eNOS) in ovarian follicles and corpora lutea (CL) throughout the estrous cycle in sheep. Three experiments were conducted to (1) immunolocalize eNOS protein, (2) determine expression of mRNA for eNOS and its receptor guanylate cyclase 1 soluble beta3 (GUCY1B3), and (3) co-localize eNOS and vascular endothelial growth factor (VEGF) proteins in the follicles and/or CL throughout the estrous cycle. In experiment 1, ovaries were collected from ewes treated with FSH, to induce follicular growth or atresia. In experiment 2, ovaries were collected from ewes treated with FSH and hCG to induce follicular growth and ovulation. In experiment 3, ovaries were collected from superovulated ewes to generate multiple CL on days 2, 4, 10, and 15 of the estrous cycle. In experiments 1 and 2, the expression of eNOS protein was detected in the blood vessels of the theca externa and interna of healthy ovarian follicles. However, in early and advanced atretic follicles, eNOS protein expression was absent or reduced. During the immediate postovulatory period, eNOS protein expression was detected in thecal-derived cells that appeared to be invading the granulosa layer. Expression of eNOS mRNA tended to increase in granulosa cells at 12 and 24 h, and in theca cells 48 h after hCG injection. In experiment 3, eNOS protein was located in the blood vessels of the CL during the estrous cycle. Dual localization of eNOS and VEGF proteins in the CL demonstrated that both were found in the blood vessels.     

Effect of cationic flaxseed protein hydrolysate fractions on the in vitro structure and activity of calmodulin-dependent endothelial nitric oxide synthase

The objective of this study was to determine the in vitro effects of cationic flaxseed protein hydrolysate fractions on calmodulin (CaM) structure as well as the activity of CaM-dependent endothelial nitric oxide synthase (eNOS). Flaxseed protein isolate was hydrolyzed with alcalase, and two peptide fractions were isolated by cation-exchange chromatography. Fraction I, eluted first from the column, and fraction II contained 42% and 51% contents of basic amino acids, respectively. Fractions I and II reduced the activity of CaM-dependent eNOS through a mostly mixed-type inhibition mode. Fraction II was at least ten times more effective as an eNOS inhibitor when compared to fraction I, as evident from the IC(50) (concentration of protein hydrolysate that reduced eNOS activity by 50%) values. Fluorescence spectroscopy showed that the tyrosine residues in CaM were increasingly exposed upon addition of fraction I, while the opposite was the case when fraction II was added. Circular dichroism studies showed that fractions I and II reduced the alpha-helix content but increased the rigidity of the active calcium/CaM complex. We concluded that ability of the protein hydrolysate fractions to change the secondary and tertiary structures of CaM may explain their ability to reduce activity of CaM-dependent eNOS.     

The effect of probiotic‐fermented soy milk on enhancing the NO‐mediated vascular relaxation factors

BACKGROUND: Soy milk is one of the common soy‐based foods in Asia. In this study the effects of soy milk fermented with selected probiotics on nitric oxide (NO)‐mediated vascular relaxation factors in cell model systems were investigated. RESULTS: Soy milk fermented with Lactobacillus plantarum TWK10 or Streptococcus thermophilus BCRC 14085 for 48 h showed a greater transformation of glucoside isoflavones to aglycone isoflavones (P < 0.05). An increase in aglycone isoflavones in ethanol extracts from fermented soy milk stimulated NO production and endothelial NO synthase (eNOS) activity in human umbilical vein endothelial cells. It also had a stimulating effect on superoxide anion scavenging and prostaglandin E₂ production. In addition, it enhanced mRNA expression of the E‐prostanoid 4 receptor in rat thoracic aorta smooth muscle cells. Moreover, a small amount of O induced by water extracts from fermented soy milk at low concentration (1 mg mL^?1) increased the content of calcium ions and activated eNOS, thereby promoting NO production and the coupling state of eNOS. CONCLUSION: Soy milk fermented with selected probiotics promotes the relaxation factors of vascular endothelial cells and can be applied in the development of functional foods. © 2012 Society of Chemical Industry     

Antioxidant activity of peanut seed testa and its antioxidative component,ethyl protocatechuate

The antioxidant activity of ethanolic extracts of peanut seed testa (EEPST) and its antioxidative component, ethyl protocatechuate (EP), was examined. It was found that EEPST and EP showed a dose-dependent activity on the inhibition of liposome peroxidation. EEPST and EP in the range of 50–500 mg/l were effective in protecting protein against oxidative damage. EEPST and EP at 100 mg/l showed 92.6% and 84.6% scavenging effect, respectively, on α,α-diphenyl-β-picrylhydrazyl radical, indicating that they act as a primary antioxidant. In addition, EEPST and EP, at a dose of 200 mg/l, showed 70.6% and 67.7% scavenging effect, respectively, on the hydroxyl radical. EEPST also exhibited a metal-binding ability, while EP did not. The inhibitory effect of EEPST on linoleic peroxidation correlated with their polyphenolic content. These results suggest that the antioxidant mechanism, for both EEPST and EP, could possibly be due to their scavenging effect on free radical and hydroxyl radical. In addition, its metal binding ability may contribute to antioxidant activity of EEPST.     

林檎叶提取物对Nω-硝基-L-精氨酸(L-NNA)诱导KM小鼠高血压的预防效果

该研究旨在探讨林檎叶在L-NNA诱导的小鼠高血压模型中的预防作用.林檎叶提取物可降低高血压小鼠收缩压(SBP)、平均血压(MBP)、舒张压(DBP).林檎叶提取物作用高血压小鼠后,小鼠血清、心脏、肝脏、肾脏、胃中的一氧化氮(NO)含量均高于模型组,丙二醛(MDA)含量低于模型组.林檎叶提取物作用高血压小鼠后血清内皮素-...     

鸡腿菇粗多糖的体外抗氧化性

以VC为对照,探究水提鸡腿菇粗多糖的体外抗氧化性,分别考察其对DPPH自由基,羟自由基( ·OH)、超氧阴离子自由基(O2－ ·)的清除能力及其还原力。结果表明：鸡腿菇粗多糖对DPPH自由基有着较好的清除效果;对 ·OH的清除效果也比较明显,与VC的抗氧化能力相当,清除效果随粗多糖质量浓度升高而升高;对O2－ ·也有一定的清除效果,但清除效果不明显;鸡腿菇粗多糖具有一定的还原力,其能力低于VC。说明鸡腿菇粗多糖具有较好的抗氧化活性,日常食用能够有效的起到抗氧化的作用。     

Unsaturated fatty acids liberated from VLDL cause apoptosis in endothelial cells

Certain unsaturated fatty acids (UFAs), cleaved from lipoproteins, are known to activate the serine/threonine protein phosphatase type 2C (PP2C) alpha- and beta-isoforms. To investigate the role of UFAs in apoptosis of endothelial cells, we cocultured human umbilical vein endothelial cells (HUVECs) with THP-1 monocytes. Phorbol-12-myristic-13-acetate (PMA)-treated THP-1 monocytes differentiated into macrophages and synthesized lipoprotein lipase (LPL), the major enzyme for hydrolysis of triglycerides. We demonstrated that LPL from THP-1 macrophages released UFAs from VLDL, which were capable of inducing apoptosis in HUVECs. Physiological concentrations of VLDL did not cause apoptosis in HUVECs, whereas the combination of VLDL with LPL-rich cell medium of THP-1 macrophages did. THP-1 macrophages and HUVECs in cocultivation did not interfere with each other. However, addition of VLDL to this coculture caused apoptosis in HUVECs. Furthermore, inhibition of LPL by adding orlistat to the culture medium and down-regulation of LPL by small interfering RNA (siRNA) reduced the extent of apoptosis of HUVECs. In conclusion, our results show that the amounts of UFAs liberated from lipoproteins are high enough to induce apoptosis in endothelial cells. This underlines the proatherogenic role of UFAs in hyperlipoproteinemias.