G蛋白偶联受体激酶4对氧化低密度脂蛋白诱导的血管平滑肌细胞增殖的影响

目的研究G蛋白偶联受体激酶4(GRK4)对氧化低密度脂蛋白(ox-LDL)诱导的血管平滑肌细胞增殖的影响。方法以大鼠胸主动脉平滑肌细胞株(A10细胞)为靶细胞,检测在不同浓度的ox-LDL(10、20、30、40、60、80 mg/L)刺激下A10细胞的增殖情况;观察ox-LDL(40 mg/L)刺激下A10细胞GRK4表达变化;用siRNA干扰A10细胞GRK4表达后,检测ox-LDL对A10细胞增殖的影响。Western blot检测GRK4蛋白表达,CCK-8检测细胞增殖活力。结果用不同浓度ox-LDL刺激A10细胞,发现ox-LDL(40 mg/L)刺激下细胞增殖幅度可达78.3%(P<0.05),且细胞GRK4蛋白表达增加[实验组(0.367 9±0.049 1),对照组(0.193 0±0.038 5),P<0.05];与对照组相比,用siRNA干扰A10细胞GRK4表达后ox-LDL对A10细胞增殖的影响明显减弱[对照组(1.050 1±0.302 9),ox-LDL刺激组(1.929 1±0.390 0),siRNA组(1.403 2±0.164 4),P<0.05]。结论 ox-LDL可增强GRK4表达,干扰细胞GRK4表达后可减弱ox-LDL诱导的大鼠平滑肌细胞的增殖。     

脱氢表雄酮对氧化低密度脂蛋白诱导的人脐静脉内皮细胞表达VCAM-1的影响

目的探讨雄激素脱氢表雄酮（DHEA）对氧化低密度脂蛋白（ox—LDL）诱导的人脐静脉内皮细胞（HUVECs）表达血管细胞黏附分子-1（VCAM-1）的影响。方法体外培养HUVECs，采用免疫细胞化学、Western blot、原位杂交等方法，观察不同浓度DHEA（1，5，50μmol／L）对ox—LDL诱导的HUVECs表达VCAM-1的影响。结果ox—LDL诱导培养HUVECs后，HUVECs VCAM-1表达明显升高，预先用DHEA处理HUVECs可使VCAM-1的表达降低（P〈0．01），且这种降低呈浓度依赖性。结论DHEA能浓度依赖性地抑制ox-LDL诱导的HUVECs VCAM-1的表达。     

氧化型烟酰胺腺嘌呤二核苷酸抗辐射损伤作用及其机制

目的探讨氧化型烟酰胺腺嘌呤二核苷酸(NAD+)的抗辐射损伤作用及其机制。方法将培养的人正常肝细胞株L02细胞分为3组:对照组、照射组和照射+NAD+组。对照组细胞不予照射,直接加入不含NAD+的RPMI-1640培养基培养;照射组细胞照射后加入不含NAD+的RPMI-1640培养基培养;照射+NAD+组细胞照射后加入含有NAD+(终浓度1 g.L-1)的RPMI-1640培养基培养。采用四甲基偶氮唑盐(MTT)法检测NAD+对受照射L02细胞生长活性的影响;应用流式细胞仪检测各组细胞凋亡率以及凋亡相关蛋白p53、bax、bcl-2阳性表达率和各周期细胞含量;采用Caspase-3活性检测试剂盒检测细胞Caspase-3活性。结果细胞受照射后加入NAD+能够对抗X射线照射对L02细胞的生长抑制作用,细胞生长活性较照射组细胞活性显著提高;L02细胞在受照射后p53、bax蛋白表达增加,bcl-2表达下调,Caspase-3活性增加,而且细胞周期表现为G1期阻滞;受照射后加入NAD+,则p53、bax蛋白表达下调,bcl-2表达增加,Caspase-3活性下降,G2/M期细胞比例明显增加。结论 NAD+可对抗L02细胞的X线辐射损伤,其作用机制可能通过下调p53、bax与上调bcl-2表达以及抑制Caspase-3活性,抑制受照射细胞凋亡。     

Aqueous Extracts of Tribulus terrestris Protects Against Oxidized Low-Density Lipoprotein-Induced Endothelial Dysfunction

Objective:To investigate the role of aqueous extracts of Tribulus terrestris(TT) against oxidized low-density lipoprotein(ox-LDL)-induced human umbilical vein endothelial cells(HUVECs) dysfunction in vitro.Methods:HUVECs were pre-incubated for 60 min with TT(30 and 3 μg/mL respectively) or 10~(-5) mol/L valsartan(as positive controls) and then the injured endothelium model was established by applying 100 μg/mL ox-LDL for 24 h.Cell viability of HUVECs was observed by real-time cell electronic sensing assay and apoptosis rate by Annexin V/PI staining.The cell migration assay was performed with a transwell insert system.Cytoskeleton remodeling was observed by immunofluorescence assay.The content of endothelial nitric oxide synthase(eNOS) was measured by enzyme-linked immunosorbent assay.Intracellular reactive oxygen species(ROS) generation was assessed by immunofluorescence and flow cytometer.Key genes associated with the metabolism of ox-LDL were chosen for quantitative real-time polymerase chain reaction to explore the possible mechanism of TT against oxidized LDL-induced endothelial dysfunction.Results:TT suppressed ox-LDL-induced HUVEC proliferation and apoptosis rates significantly(41.1%and 43.5%after treatment for 3 and 38 h,respectively;P0.05).It also prolonged the HUVEC survival time and postponed the cell's decaying stage(from the 69 th h to over 100 h).According to the immunofluorescence and transwell insert system assay,TT improved the endothelial cytoskeletal network,and vinculin expression and increased cell migration.Additionally,TT regulated of the synthesis of endothelial nitric oxide synthase and generation of intracellular reactive oxygen species(P0.05).Both 30 and 3 μg/mL TT demonstrated similar efficacy to valsartan.TT normalized the increased mRNA expression of PI3Kα and Socs3.It also decreased mRNA expression of Akt1,AMPKα1,JAK2,LepR and STAT3 induced by ox-LDL.The most notable changes were JAK2,LepR,PI3 K α,Socs3 and STAT3.Conclusions:TT demonstrated potential lowering lipid benefits,anti-hypertension and endothelial protective effects.It also suggested that the JAK2/STAT3 and/or PI3K/AKT pathway might be a very important pathway which was involved in the pharmacological mechanism of TT as the vascular protective agent.     

巨噬细胞ABCA1对Ox-LDL诱导的炎症因子的调节及其意义

 【目的】研究在氧化低密度脂蛋白(Ox-LDL)诱导作用下,ATP结合盒转运子A1(ABCA1)对巨噬细胞中细胞间黏附分子-1(ICAM-1)、单核细胞化学趋向蛋白-1(MCP-1)mRNA和蛋白质及白介素-1β(IL-1β)蛋白质表达的影响,在细胞水平证实ABCA1对动脉粥样硬化的影响。【方法】用氟波酯(PMA)刺激THP-1细胞使之转变为巨噬细胞,Ox-LDL(30μg/mL)刺激3、6、12、24h后,以荧光定量RT-PCR和Western蛋白印迹法及酶联免疫吸附法(ELISA)检测ABCA1、ICAM-1、MCP-1及IL-1β mRNA和蛋白质表达量;用反义寡核苷酸(100nmol/L)抑制ABCA1的表达,观察Ox-LDL刺激下上述指标的改变。【结果】给予Ox-LDL刺激后,巨噬细胞ABCA1、ICAM-1、MCP-1的mRNA和蛋白及IL-1β蛋白质表达均增高;给予反义寡核苷酸转染后Ox-LDL刺激3、6h,上述指标的mRNA表达降低(P<0.01),12、24h蛋白质表达降低(P<0.01)。【结论】在巨噬细胞,ABCA1可增加Ox-LDL诱导的炎症因子表达,参与动脉粥样硬化的发生。     

Tissue factor antisense deoxyoligonucleotide prevents monocrotaline/LPS hepatotoxicity in mice

Tissue factor (TF) is a membranous glycoprotein that functions as a receptor for coagulation factor VII/VIIa and activates the coagulation system when blood vessels or tissues are damaged. TF was upregulated in our monocrotaline (MCT)/lipopolysaccharide (LPS) hepatotoxicity model. We tested the hypothesis that TF‐dependent fibrin deposition and lipid peroxidation in the form of oxidized low‐density‐lipoprotein (ox‐LDL) accumulation contribute to liver inflammation induced by MCT/LPS in mice. In the present study, we blocked TF using antisense oligodeoxynucleotides against mouse TF (TF‐ASO). TF‐ASO (5.6 mg kg^?1) was given i.v. to ND4 male mice 30 min after administration of MCT (200 mg kg^?1) p.o. followed after 3.5 h by LPS i.p. (6 mg kg^?1). Blood alanine aminotransferase (ALT), TF, ox‐LDL, platelets, hematocrit and keratinocyte‐derived chemokine (KC) levels were evaluated in different treatment groups. Fibrin deposition and ox‐LDL accumulation were also analyzed in the liver sections using immunofluorescent staining. The results showed that TF‐ASO significantly restored blood ALT, hematocrit and KC levels, distorted after MCT/LPS co‐treatment, as well as preventing the accumulation of ox‐LDL and the deposition of fibrin in the liver tissues, and thereby inhibited liver injury caused by MCT/LPS. In a separate experiment, TF‐ASO administration significantly prolonged animal survival. The current study demonstrates that TF is associated with MCT/LPS‐induced liver injury. Administration of TF‐ASO successfully prevented this type of liver injury. Copyright © 2012 John Wiley & Sons, Ltd.     

Relation of circulating oxidized LDL to obesity and insulin resistance in children

Kelly AS, Jacobs DR Jr, Sinaiko AR, Moran A, Steffen LM, Steinberger J. Relation of circulating oxidized LDL to obesity and insulin resistance in children. Introduction: Circulating oxidized low‐density lipoprotein (LDL), a marker of oxidative stress, is associated with obesity, insulin resistance, metabolic syndrome, and cardiovascular disease in adults. However, little is known about its relation to insulin resistance and cardiovascular risk factors in children. The purpose of this study was to assess the relation of oxidative stress, measured by circulating oxidized LDL, with measures of adiposity and insulin resistance in children. Methods: Oxidized LDL, measures of body fatness (body mass index: BMI, percent body fat, waist circumference, percent trunk fat, abdominal visceral and subcutaneous fat), insulin resistance with euglycemic insulin clamp (Mlbm), blood pressure, and blood lipids were obtained in 78 children. Oxidized LDL was compared between normal weight children (BMI < 85th percentile) and overweight/obese children (BMI ≥ 85th percentile) and levels were evaluated for associations with body fatness and insulin resistance. Results: Oxidized LDL levels were significantly higher in overweight/obese vs. normal weight children (p < 0.0001). Oxidized LDL was significantly correlated with BMI, percent body fat, waist circumference, percent trunk fat, abdominal visceral fat, and abdominal subcutaneous fat (all p‐values <0.0001). Moreover, oxidized LDL was negatively correlated with Mlbm, even after adjustment for adiposity (p < 0.01). Conclusions: Oxidized LDL is significantly associated with adiposity and with insulin resistance, independent of body fatness, in children. Oxidative stress may be independently related to the development of insulin resistance early in life, especially in obese youth.     

谷胱甘肽-S-转硫酶法快速测定红细胞GSH和其氧化产物

红细胞谷胱甘肽（GSH）、氧化型谷胱甘肽（GSSG）及蛋白结合谷胱甘肽（PSSG）是反映氧化溶血的指标。Ellman试剂测定GSH干扰多。高效液相层析测定GSH及其衍生物受仪器和效率限制。谷胱甘肽-S-转硫酶（GST）法测定GSH干扰少，但从未用于测定红细胞GSH或其衍生物。本尝试改进GST法并用于测定红细胞GSH及其衍生物。     

降脂通脉方治疗高脂血症的临床研究

目的 :探讨中药治疗高脂血症的疗效及其作用机理。方法 :6 1例高脂血症患者随机分为中药降脂通脉口服液治疗组 (治疗组 )和西药洛伐他丁对照组 (对照组 )。采用酶法测定治疗前后的总胆固醇 (TC)、甘油三酯 (TG)、高密度脂蛋白胆固醇 (HDL C)等的变化 ,应用酶联免疫吸附试验法测定氧化低密度脂蛋白 (oxLDL)治疗前后的变化。结果 :中药降脂通脉口服液可明显改善多种脂质成分 ,阻止低密度脂蛋白氧化。结论 :降脂通脉方对高脂血症有一定的治疗作用 ,对抗AS具有积极的意义     

Increased uracil misincorporation in lymphocytes from folate-deficient rats

The development of certain human cancers has been linked with inadequate intake of folates. The effects of folate deficiency in vivo on DNA stability (strand breakage, misincorporated uracil and oxidative base damage) in lymphocytes isolated from rats fed a diet deficient in folic acid was determined. Because the metabolic pathways of folate and other methyl donors are closely coupled, the effects of methionine and choline deficiency alone or in combination with folate deficiency were determined. Feeding male Hooded Lister rats a folate-free diet for 10 weeks created a moderate folate deficiency (25-50% (approx.) decrease in plasma, red blood cell and hepatic folate concentrations (P < 0.05) and a 20% rise in plasma homocysteine (P < 0.05)). Lymphocyte DNA strand breakage was increased successively in all groups after 4 weeks and 8 weeks on the diet (50-100% (approx.) after 8 weeks). Only low folate specifically and progressively induced uracil misincorporation throughout the study (100% (approx.) after 8 weeks). Neither folate deficiency nor choline/methionine deficiency altered oxidative DNA base damage. In summary, moderate folate deficiency in vivo is associated with a decrease in DNA stability, measured as increased DNA strand breakage and misincorporated uracil.