马齿苋脂肪油对动脉粥样硬化大鼠胆固醇逆向转运相关基因表达的影响

目的 探讨马齿苋脂肪油有效成分对肝脏卵磷脂胆固醇酯酰转移酶(LCAT)、载脂蛋白AI (apoAI)和高密度脂蛋白受体(SR-BI)基因表达的干预作用,分析马齿苋调节胆固醇逆向转运(RCT)通路的分子机制.方法 健康雄性SD大鼠24只,随机分为模型组、多烯康组、马齿苋组、正常组,每组6只.应用高脂高胆固醇膳食诱导建立动脉粥样硬化(AS)大鼠模型,用定量反转录聚合酶链式反应(RT-PCR)技术测定LCAT、apoAI及SR-BI mRNA表达量,以免疫比浊法、沉淀法测定血清apoAI、高密度脂蛋白胆固醇(HDL-C)含量.结果 模型组大鼠肝脏LCAT mRNA和apoAI mRNA的表达量明显下降,分别为0.43±0.20、2.33±0.35,与正常组(分别为1.49±0.32、6.30±0.82)、多烯康组(分别为1.15±0.16、4.32±0.69)、马齿苋组(分别为1.12±0.12、4.21±0.76)比较,差异均有统计学意义(P＜0.05).模型组大鼠血清apoAI[(0.15±0.03)g/L]和HDL-C水平[(0.53-±0.25) mmol/L]明显下降,与马齿苋组[(0.39±0.09) g/L、(0.86±0.04) mmol/L]比较,差异有统计学意义(P＜0.05).除正常组外,多烯康组、马齿苋组、模型组肝脏LCAT mRNA表达量与HDL-C水平均呈明显的正相关(r值分别为0.935、0.927、0.892,P＜0.01).模型组大鼠肝脏SR-BI mRNA水平(1.33±0.57)下降,马齿苋组(3.20±0.41)高于模型组,差异有统计学意义(P＜0.01).结论 在转录水平阻抗高脂高胆固醇负荷下调大鼠肝脏LCAT和apoAI mRNA以及SR-BI mRNA表达的作用,促进RCT过程是马齿苋改善高脂血症、抗动脉粥样硬化的重要分子机制.     

Cytotoxicity of oxidation derivatives of cholesterol on cultured aortic smooth muscle cells and their effect on cholesterol biosynthesis.

Aortic smooth muscle cell death is an important initial lesion of atherosclerosis. A number of autooxidation products of cholesterol which has been recognized recently has the capability of inducing rabbits' aortic smooth cell death in vitro. Twelve oxidation derivatives of cholesterol, available commercially, were dissolved in small amounts of ethanol, then added to the culture medium at levels not exceeding 0.8%. The medium contained 10% fetal calf's serum which served as an in situ vehicle for the sterols. The degrees of cytotoxicity were graded and measured as percentage of dying and dead cells in the cultures within 24 hr. 25-Hydroxycholesterol and cholesthan-3 beta, 5 alpha, 6 beta-triol, were the most toxic compounds among the sterols tested. When these oxidation derivatives of cholesterol were added to these cultured cells, they significantly depressed activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a regulatory enzyme of cholesterol biosynthesis (up to 83% inhibition by 25 hydroxycholesterol at a 3 microgram/ml concentration in culture medium) but the sequence of degree of inhibition was not exactly correlated with that of cytotoxicity. Various mechanisms are speculated. Purified cholesterol showed no cytotoxic effect and minimal inhibition of cholesterol biosynthesis.     

Fish oil attenuates the cholesterol induced rise in lipoprotein cholesterol

Fish oils rich in n-3 fatty acids lower plasma triglyceride profoundly but the effect on plasma cholesterol is not clear. This study tested the capacity of MaxEPA oil to modify the rise in lipoprotein cholesterol during cholesterol-rich diets. Six subjects were tested with three diets: 1) habitual (P/S 0.47, cholesterol 710 mg/d); 2) fish oil (40 g/d MaxEPA, P/S 1.62, cholesterol 190 mg/d); 3) fish oil + egg yolk (P/S 1.62, cholesterol 940 mg/d). Changing from habitual to fish oil significantly lowered cholesterol and triglyceride levels in plasma, VLDL, LDL, and HDL and in plasma apo-A1 and apo-B. However the addition of 750 mg/d cholesterol to the fish oil failed to raise lipoprotein cholesterol or apoprotein levels significantly, although plasma cholesterol rose slightly; n-3 fatty acids are therefore capable of lowering lipoprotein cholesterol even when the intake of cholesterol is high.     

Fish oil supplementation modulates immune function in healthy infants

(n-3) PUFA influence immune function in adults and may also affect immune maturation during development. This randomized trial is, to our knowledge, the first to investigate whether fish oil supplementation in late infancy modifies immune responses. The study was a 2 x 2 intervention in 64 healthy Danish infants, who received cow's milk or infant formula alone or with fish oil (FO) (3.4 +/- 1.1 mL/d) from 9 to 12 mo of age. Before and after the intervention, fatty acid composition of erythrocyte membranes, plasma IgE, C-reactive protein, and soluble IL-2 receptor concentrations were measured. TNF-alpha, INF-gamma, and IL-10 concentrations in whole-blood cultures, stimulated for 22 h with LPS+phytohemaglutinin (PHA) or Lactobacillus paracasei, were also determined. IgA was measured in feces when infants were 10 mo of age. FO supplementation effectively raised erythrocyte (n-3) PUFA (P < 0.001), increased L. paracasei-induced INF-gamma (P = 0.05) and tended to reduce LPS+PHA-induced IL-10 (P = 0.08). The FO intervention did not affect any of the other analyzed immune variables. The erythrocyte content of eicosapentanoic acid was negatively associated with LPS+PHA-induced IL-10 (r = -0.38, P = 0.02). Feeding milk rather than formula did not affect cytokine production, but plasma soluble IL-2 receptor concentration was greater in the formula group than in the cow's milk group (P = 0.03). Since the capacity to produce INF-gamma has been proposed as a maturation marker for the immune system in early life, this study suggests a faster immune maturation with FO supplementation with no apparent reduction in immune activation. The implications for later health need further investigation.     

Naringin suppresses the mitogenic effect of lysophosphatidylcholine on vascular smooth muscle cells

Naringin, a flavonoid found in citrus species and grapes, has anti-inflammatory, anti-oxidative properties. In this study, we investigated whether naringin protects vascular smooth muscle cells (VSMCs) from the mitogenic effect of lysophosphatidylcholine. Lysophosphatidylcholine, an atherogenic lysophospholipid of oxidized low density lipoprotein (LDL), is known to impair endothelial release of nitric oxide, up-regulate the expression of adhesion molecules, and promote the proliferation of VSMCs. VSMCs were prepared from the aortas of Sprague-Dawley rats, and cultured in DMEM/F12 media. Near-confluent cells were incubated with 0, 10, or 100 μM of naringin for 24 hours, and then incubated with 0, 10, 20, or 100 μM of lysophosphatidylcholine. Lysophosphatidylcholine promoted the growth of VSMCs, whereas naringin suppressed its mitogenic effect on VSMCs. In an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay, lysophosphatidylcholine promoted cell proliferation by 63 ± 24% and 89 ± 17% at 10 μM and 20 μM respectively, when compared with the control group (p < 0.01). [H³]-thymidine incorporation assay showed that the degree of DNA replication also increased by 61 ± 25% and 92 ± 25% at 10 μM and 20 μM of lysophosphatidylcholine (p < 0.01). Naringin at 100 μM significantly suppressed the mitogenic effect of lysophosphatidylcholine on VSMCs by 34 ± 5% (MTT assay), and 35 ± 5% ([H³]-thymidine incorporation assay) (p = 0.01). This study demonstrates that naringin suppresses the mitogenic effect of lysophosphatidylcholine on VSMCs, whereas lysophosphatidylcholine promotes the proliferation of VSMCs.     

Fish oil supplementation does not alter energy efficiency in healthy males

BACKGROUND AND AIMS: Fish oil (FO) supplementation prevents the development of obesity and insulin resistance, and upregulate the expression of UCP3 in skeletal muscle in rodents. This may represent indirect evidence that FO promotes fat oxidation and/or alter energy efficiency. The aim of this study was to evaluate whether such effects can be observed in humans. The metabolic effects of FO were assessed during exercise in order to obtain a direct measurement of energy efficiency. METHODS: Eight healthy male volunteers were studied with and without supplementation with 7.2 g/day FO (including 1.1 g/day eicosopentaenoic acid and 0.7 g/day decosahexaenoic acid) during 14 days. Their VO(2 max) was measured on cycle ergometer. Thereafter, energy metabolism (substrate oxidation, energy expenditure and energy efficiency) was assessed during a 30 min cycling exercise at 50% VO(2 max) performed 2 h 30 after a standardized, high carbohydrate breakfast. RESULTS: VO(2 max) was 38.6+/-2.2 after FO and 38.4+/-2.0 (mL x kg(-1) x min(-1)) in control conditions (NS). Basal plasma glucose, insulin and NEFA concentrations, and energy metabolism were similar with FO and in controls. During exercise, the increases in plasma NEFA concentrations, energy expenditure, glucose and lipid oxidation, and the decreases in glycaemia and insulinemia were not altered by FO intake. Energy efficiency was 22.4+/-0.6% after FO vs 21.8+/-0.7% in controls. In order to ascertain that the absence of effects of FO was not due to consumption of a carbohydrate meal immediately before exercise, 4 of the 8 subjects were re-studied in fasting conditions, FO also failed to alter energy efficiency in this subset of studies. CONCLUSION: FO supplementation did not significantly alter energy metabolism and energy efficiency during exercise in healthy humans.     

Transcriptome-based identification of antioxidative gene expression after fish oil supplementation in normo- and dyslipidemic men

Background

The beneficial effects of omega-3 polyunsaturated fatty acids (n-3 PUFAs), especially in dyslipidemic subjects with a high risk of cardiovascular disease, are widely described in the literature. A lot of effects of n-3 PUFAs and their oxidized metabolites are triggered by regulating the expression of genes. Currently, it is uncertain if the administration of n-3 PUFAs results in different expression changes of genes related to antioxidative mechanisms in normo- and dyslipidemic subjects, which may partly explain their cardioprotective effects. The aim of this study was to investigate the effects of n-3 PUFA supplementation on expression changes of genes involved in oxidative processes.

Methods

Ten normo- and ten dyslipidemic men were supplemented for twelve weeks with fish oil capsules, providing 1.14?g docosahexaenoic acid and 1.56?g eicosapentaenoic acid. Gene expression levels were determined by whole genome microarray analysis and quantitative real-time polymerase chain reaction (qRT-PCR).

Results

Using microarrays, we discovered an increased expression of antioxidative enzymes and a decreased expression of pro-oxidative and tissue enzymes, such as cytochrome P450 enzymes and matrix metalloproteinases, in both normo- and dyslipidemic men. An up-regulation of catalase and heme oxigenase 2 in both normo- and dyslipidemic subjects and an up-regulation of cytochrome P450 enzyme 1A2 only in dyslipidemic subjects could be observed by qRT-PCR analysis.

Conclusions

Supplementation of normo- and dyslipidemic subjects with n-3 PUFAs changed the expression of genes related to oxidative processes, which may suggest antioxidative and potential cardioprotective effects of n-3 PUFAs. Further studies combining genetic and metabolic endpoints are needed to verify the regulative effects of n-3 PUFAs in antioxidative gene expression to better understand their beneficial effects in health and disease prevention.

Trial registration

ClinicalTrials.gov (ID: NCT01089231)     

同型半胱氨酸对人血管平滑肌细胞5,10-亚甲基四氢叶酸还原酶基因启动子甲基化修饰及mRNA表达的影响

目的探讨同型半胱氨酸(Hcy)对人血管平滑肌细胞(HVSMCs)5,10-亚甲基四氢叶酸还原酶(MTHFR)基因启动子甲基化修饰及其mRNA表达的影响。方法正常人脐动脉血管平滑肌细胞体外培养,在培养基中加入临床高Hcy血症相关浓度及极高浓度的Hcy(0、30、100、200、500和1000μmol/L),并孵育72h,以MS-PCR分析MTHFR启动子区域甲基化状态,采用RT-PCR测定MTHFR mRNA的表达水平。结果不同浓度Hcy处理人血管平滑肌细胞后,其MTHFR基因启动子区域出现去甲基化,甲基化水平明显降低。RT-PCR结果显示MTHFR mRNA表达增加。结论Hcy可促进人血管平滑肌细胞MTHFR启动子区域去甲基化,并使MTHFR mRNA表达增加。     

Cyclic regulation of apoptotic gene expression in the mouse oviduct

The oviduct is a dynamic structure whose function relies upon cyclic changes in the morphology of both ciliated and secretory luminal epithelial cells. Unfortunately, infection of these epithelial cells by sexually transmitted pathogens can lead to pelvic inflammatory disease, ectopic pregnancies and infertility. The disruption of normal, cyclic apoptosis in the oviducal epithelium appears to be a causal factor of oviducal pathology and therefore, these pathways represent a potential target for diagnosis and therapeutic intervention. The objective of this study was to determine the pattern of expression for apoptotic genes in the oviduct of the naturally cycling mouse, generating fundamental information that can be applied to the development of animal models for research and the identification of targets for disease intervention. Whole oviducts were collected from regular cycling mice killed at 1p.m. on each day of the oestrous cycle and the expression of 84 apoptotic genes determined by targeted PCR super-array. Intact and cleaved caspases were then evaluated by western blotting. The expression of mRNA for genes classified as pro-apoptotic (Bad, Bak1 and Bok) and anti-apoptotic (Bag3, Bnip2 and Xiap) was regulated by day (P < 0.05). Differences in the temporal expression of several p53-related genes (Trp53bp2, Trp53inp1 and Trp73), those specific to the TNF superfamily (Tnfrsf10 and Tnfsf10b) and one caspase (Casp14) were also observed (P < 0.05). The cleaved forms of Caspases-3, -6 and -12 were all detected throughout the oestrous cycle. These results represent the first pathway-wide analysis of apoptotic gene expression in the murine oviduct.     

葛根素对低氧性肺动脉平滑肌细胞增殖与凋亡及Kv1.5表达的影响

目的 观察葛根素对低氧性肺动脉平滑肌细胞( PASMCs)增殖与凋亡及电压门控型钾离子通道亚型(Kv1.5)表达的影响,探讨葛根素在改善低氧性肺动脉高压与抑制肺血管重建中可能的作用机制.方法 原代培养大鼠PASMCs,随机分为正常对照组(5％CO2常氧),低氧组(5％O2、5％CO2、90％N2三气培养),3个浓度葛根素干预组(在低氧组基础上分别加入终浓度为1×104、1×10-4、1×10-3 mol/L葛根素,37℃培养24 h).采用CCK-8法和流式细胞仪检测细胞增殖情况,分光光度法检测caspase-3活力,蛋白印迹及实时定量聚合酶链反应(PCR)法分别检测Kv1.5蛋白和mRNA表达.结果 与正常对照组(细胞活性:0.940±0.045,S期细胞比例:9.67％±1.28％,Caspase-3活力:0.1073±0.0113,Kv 1.5蛋白:0.886±0.038,Kv 1.5 mRNA 0.0377±0.0031)比较,低氧组细胞活性(1.296±0.034)、S期细胞比例(18.19％±1.19％)升高,Caspase-3活力(0.0664±0.0049)下降,Kv1.5蛋白(0.602±0.064)及mRNA (0.0108±0.0014)表达下降,差异均有统计学意义(P＜0.05);与低氧组比较,各剂量葛根素干预组细胞活性和S期细胞比例下降,Caspase-3活力升高,Kv 1.5蛋白及mRNA表达上调,差异均有统计学意义(P＜0.05).结论 葛根素可抑制低氧性PASMCs增殖,促进其凋亡,其作用机制可能通过上调Kv 1.5表达抑制PASMCs的增殖.     

骨形成蛋白-2对低氧状态下的人肺动脉平滑肌细胞PTEN表达的作用

目的:研究骨形成蛋白-2(BMP-2)对低氧状态下人肺动脉平滑肌细胞(Pu lmonary artery smooth musc le cells,PASMCs)的PTEN(Phosohatase and tensin homolog deleted on chromosom e 10)表达调节作用。方法:将培养的人PASMCSs分为对照组(1%的氧浓度,5%的CO2和94%N2条件下培养)和BMP-2组(另加入BMP-2),培养48 h后通过CyQUANT细胞增殖检测试剂盒测定PASMCs的增殖,定量RT-PCR方法检测PTEN基因表达,W estern b lot方法检测PTEN蛋白的表达。结果:BMP-2组与对照组比较细胞增殖明显降低(P<0.05);定量RT-PCR结果显示,与对照组比较,BMP-2组细胞培养4 h、8 h、24 h后PTEN相对表达量(2-△△ct)均明显升高(P<0.05);进一步的western b lot证实BMP-2组PTEN蛋白表达也明显升高。结论:BMP-2能抑制缺氧状态下的PASMCs的增殖,通过上调PTEN表达可能是其机制之一。     

Fish oil supplementation of rats during pregnancy reduces adult disease risks in their offspring

Metabolic programming in utero due to maternal undernutrition is considered to increase the risk of adult diseases in offspring. It is therefore of relevance to investigate how dietary supplementation of specific nutrients can ameliorate the negative effects of maternal malnutrition. We examined the effects of supplementing fish oil or folic acid, both of which are conventional supplements in maternal intervention, on risk factors in the offspring as adults. Pregnant female rats from 4 groups (n = 6/group) were fed casein diets with 18 g/100 g protein (control diet), 12 g/100 g protein supplemented with 8 mg folic acid/kg diet (0.08 mg/kg diet) (FAS), 12 g/100 g protein without folic acid (FAD) or 12 g/100 g protein supplemented with 7 g/100 g fish oil (FOIL). Pups were weaned to a standard laboratory diet with 18 g/100 g protein. Serum glucose, insulin and cholesterol and plasma homocysteine levels were measured in the offspring at 6 and 11 mo of age. Serum glucose in 11-mo-old male and female pups was greater (P < 0.05) in both the FAS (males 2.46 +/- 0.51, females 2.49 +/- 0.29 mmol/L) and FAD groups (2.48 +/- 0.28 and 2.67 +/- 0.41 mmol/L) than in controls (2.03 +/- 0.15 and 2.02 +/- 0.18 mmol/L). Serum insulin concentrations were higher (P < 0.05) in the FAD group (males 1476 +/- 317, females 1441 +/- 220 pmol/L) but were lower in males from the FAS group (483 +/- 165 pmol/L) compared with controls (males 917 +/- 373, females 981 +/- 264 pmol/L). Glucose and insulin concentrations did not differ between the control and FOIL groups. Plasma homocysteine levels were lower (P < 0.05) only in 11-mo-old folate-deficient males; none of the other groups differed from the controls. Maternal supplementation of fish oil to a diet containing marginal protein was beneficial in maintaining circulating glucose, insulin, cholesterol and homocysteine levels in the offspring as adults.     

RAS拮抗剂调节血管平滑肌细胞增殖、凋亡因子表达

目的 探讨血管紧张素转化酶抑制剂卡托普利及血管紧张素Ⅱ(AngⅡ)受体拮抗剂氯沙坦对AngⅡ诱导的肿瘤坏死因子-α(TNF-α)、转化生长因子- β1(TGF- β1)表达的干预作用.方法 体外原代培养Wistar大鼠主动脉平滑肌细胞,收集不同AngⅡ浓度和作用时间下的细胞和卡托普利组、氯沙坦组和二者联合作用组的细胞,以逆转录(RT)-PCR检测细胞中TNF-α和TGF-β1mRNA的表达.结果 TNF-αmRNA表达量随AngⅡ浓度和作用时间增加而增加,AngⅡ浓度为10-7、10-6、10-5、10-4mol/L时表达量分别为(0.43±0.05)、(0.81±0.13)、(0.97±0.17)、(1.09±0.19),与对照组(0.11±0.03)比较,差异有统计学意义(P<0.05或0.01);一定浓度卡托普利(5×10-6mmol/L)、氯沙坦(5×10-6mmol/L)可明显抑制AngⅡ这一作用;TGF- β1mRNA表达量随AngⅡ浓度和作用时间增加而明显增加,具有浓度依赖性;在AngⅡ10-7、10-6、10-5和10-4mol/L时,卡托普利组mRNA表达量分别为(0.19±0.03)、(0.23±0.05)、(0.38±0.14)、(0.34±0.07),较AngⅡ组分别降低了59.57%、73.26%、54.44%和66.67%,差异均有统计学意义(P<0.05或0.01),氯沙坦组分别为(0.17±0.03)、(0.39±0.11)、(0.41±0.12)和(0.39±0.08),较AngⅡ组分别下降了63.82%、54.65%、54.44%和61.76%,差异均有统计学意义(P<0.05).结论 AngⅡ可促进血管平滑肌细胞TNF-α、TGF- β1mRNA表达,且有时间和浓度依赖关系;卡托普利、氯沙坦对AngⅡ诱导的血管平滑肌细胞TNF-α、TGF- β1mRNA表达均有抑制作用,二者联合作用时抑制作用最明显.     

Fish oil supplementation during chemotherapy increases posterior time to tumor progression in colorectal cancer

The authors evaluated clinical outcomes during and after chemotherapy in colorectal cancer patients supplemented with fish oil during the first 9 wk of treatment. Thirty individuals never submitted to chemotherapy were randomized into supplemented group (SG), which received 2 g/day of fish oil (0.6 g/day of EPA and DHA) for 9 wk or control group (CG), which received neither fish oil nor placebo. Outcomes assessed were number of chemotherapy cycles administered; days undergoing chemotherapy; number of delays and interruptions in the administration of chemotherapy; number of hospitalizations during chemotherapy; tumor progression; values of carcinoembryonic antigen (CEA); days until events (death and progression); and 3 yr survival. Time to tumor progression was significantly longer in SG [S593 days (±211.5)] vs. CG [330 days (± 135.1); P = 0.04], other outcomes did not differ between groups. Subjects with advanced cancer who received fish oil presented longer time to tumor progression and lower CEA values after chemotherapy; however these differences were not statistically significant. Supplementation with 2 g/day of fish oil for the first 9 wk of chemotherapy may contribute to delay in tumor progression in colorectal patients, possibly by enhancing the antineoplastic action of the chemotherapeutic drug.