Hcy对脐静脉内皮细胞eNOS和caveolin-1表达影响

目的研究不同浓度同型半胱氨酸(Hcy)对静息和钙离子导体(A23187)激活状态下人脐静脉内皮细胞中内皮型一氧化氮合酶(eNOS)和小凹蛋白-1(caveolin-1)mRNA和蛋白表达的影响。方法人脐静脉内皮细胞(HU-VECs)随机分4组:对照组、Hcy组(20、50、100、300μmol/L)、A23187(1μmol/L)组及上述浓度Hcy分别与A23187(1μmol/L)共同孵育组,测定各组eNOS和caveolin-1蛋白及mRNA表达情况,同时检测各组一氧化氮(NO)、丙二醛(MDA)含量和eNOS、超氧化物歧化酶(SOD)活力。结果对照组caveolin-1蛋白表达为1.13,Hcy各组该蛋白为0.21～2.05,呈浓度依赖性增高,差异有统计学意义(F=30.163,P<0.05);各组eNOS mRNA表达为1.80～2.08,差异无统计学意义;对照组eNOS活力和NO含量分别为1.20 U/mL和122.41μmol/L,Hcy组eNOS活力和NO含量分别为0.65～0.74 U/mL和65.33～98.91μmol/L,均呈浓度依赖性降低,差异均有统计学意义(F=5.12、18.91,P<0.05)。结论 Hcy可能通过增强caveolin-1蛋白表达,使其与eNOS藕联增加,进而抑制了eNOS活力。     

Quercetin inhibits eNOS, microtubule polymerization, and mitotic progression in bovine aortic endothelial cells

Quercetin (QRN), one of the most abundant flavonoids in the human diet, is a known antioxidant and inhibitor of cancer cell cycle progression. Here, we provide the first evidence that QRN inhibits angiogenesis via a mechanism involving both suppression of endothelial nitric oxide synthase (eNOS) and early M-phase cell cycle arrest. Bovine aortic endothelial (BAE) cells were exposed to doses of up to 100 micromol/L QRN and assayed for eNOS activity and phosphorylation status. Phosphorylation of eNOS at Ser 617 (bovine sequence) is thought to occur in response to Akt stimulation and to be required for eNOS activity. Together with basal eNOS activity, eNOS phosphorylation at Ser 617 and Akt Ser 473 phosphorylation were dose dependently and concomitantly suppressed by QRN within 30 min. Furthermore, although the significant (P < 0.05) inhibitory effect of a single 100 micromol/L QRN dose on eNOS activity was overcome within approximately 24 h, chronic QRN exposures (24-48 h) led to early M-phase arrest and disruption of mitotic microtubule polymerization. In vivo, QRN administered i.p. to female Balb/C mice bearing both syngeneic mammary tumors and Matrigel implants suppressed angiogenesis as measured by endothelial cell immunohistochemistry and hemoglobin concentration. Taken together, these findings suggest a dual mechanism by which QRN suppresses endothelial cell proliferation, both acutely via inhibition of eNOS Ser 617 phosphorylation, and chronically via perturbation of mitotic microtubule polymerization. This novel mechanism of QRN in endothelial cells may in part explain its inhibitory action on angiogenesis and further discern a potential role of QRN as a chemopreventive agent.     

葡萄糖对内皮细胞醛糖还原酶基因表达及活性的影响

目的研究葡萄糖对人血管内皮细胞醛糖还原酶基因表达及活性的影响,为糖尿病血管并发症提供理论依据。方法体外培养的人脐静脉内皮细胞,采用RT-PCR、生化测定等方法检测醛糖还原酶(AR)活性及mRNA表达。结果经5·5、11、22mmol/L葡萄糖处理后内皮细胞AR的mRNA及其活性均高于对照组(5.5mmol/L葡萄糖)(P<0.05或P<0.01),呈浓度依赖性,但44mmol/L葡萄糖组较22mmol/L葡萄糖组低(P<0.05)。在22mmol/L葡萄糖作用下,随着作用时间的延长(0、12、24h),AR的mRNA及活性增高呈时间依赖性(P<0.05或P<0.01),但48h较24h低(P<0.05)。结论葡萄糖能诱导内皮细胞AR基因表达,增强AR活性,且在一定范围内呈时间及浓度依赖性。     

Red grape berry-cultured cells reduce blood pressure in rats with metabolic-like syndrome

Purpose

Cumulative evidence suggests that moderate red wine consumption protects the cardiovascular system. The effect of cultured cells derived from red grape berry (RGC) on blood pressure (BP) has not been investigated. We therefore studied the antihypertensive effects of oral consumption of RGC in experimental rat model of metabolic-like syndrome and assessed its effect on human umbilical vein endothelial cells (HUVECs).

Methods

Forty male Sprague–Dawley rats were fed for 5 weeks with either a high fructose diet (HFD) (n = 10) or HFD supplemented, during the last 2 weeks, with different doses (200, 400 and 800 mg/kg/day) of RGC suspended in their food (n = 30). BP, plasma triglycerides, insulin and adiponectin levels were measured at the beginning and after 3 and 5 weeks of diet. RGC effect on vasodilatation was evaluated by its ability to affect endothelin-1 (ET-1) production and endothelial nitric oxide synthase (eNOS) expression in HUVECs.

Results

BP, plasma triglycerides, insulin and adiponectin increased significantly in rats fed with a HFD. The increase in BP, plasma triglycerides and insulin was attenuated by RGC supplementation. Incubation of HUVECs with RGC demonstrated a concentration-dependent inhibition of ET-1 secretion and increase in the level of eNOS, signaling a positive effect of RGC on vasodilatation.

Conclusion

In rats with metabolic-like syndrome, RGC decreased BP and improved metabolic parameters. These beneficial effects may be mediated by the cell constituents, highly rich with polyphenols and resveratrol, reside in their natural state.     

氯化镉诱发肾上腺皮质细胞凋亡与蛋白激酶B活力的关系

目的 了解氯化镉(CdCl2)诱发肾上腺皮质细胞凋亡的发生与蛋白激酶B(protein kinase B,PKB,又称Akt)活力变化的关系.方法 体外分离培养豚鼠肾上腺皮质细胞,以蛋白因子添加素V和碘化丙啶联合标记,流式细胞仪检测,观察CdCl2诱发细胞凋亡的特征;采用免疫沉淀-化学发光法测定PKB/Akt活力.结果 6.25～100.00 μmol/L剂量CdCl2处理肾上腺皮质细胞2 h,细胞凋亡发生率随剂量增加而增加,25.00 μmol/L及更高剂量组与对照组比较,差异有统计学意义(P＜0.01);回归分析表明,CdCl2剂量与凋亡发生率呈剂量-效应关系.以50.00 μmol/L CdCl2处理细胞,细胞凋亡发生率随时间的延长而增加,CdCl2作用15 min～4h,平均凋亡率为5.58%-73.08%,其中1、2、4h时间点与对照相比,差异有统计学意义(P＜0.05);CdCl2处理时间与凋亡发生率呈时间-效应关系.6.25～200.00 μmol/L剂量CdCl2处理30 min,显示CdCl2可引起肾上腺皮质细胞PKB/Akt表达降低,在一定剂量范围内呈线性关系.结论 CdCl2诱发肾上腺皮质细胞凋亡的同时引起细胞内PKB/Akt表达水平的降低.     

氟对体外培养血管内皮细胞影响及硒干预作用研究

目的研究不同浓度氟对体外培养血管内皮细胞的影响及硒的干预作用。方法人脐静脉血管内皮细胞(HUVEC)置于37℃,5%CO2,饱和湿度条件下培养。在培养液中加入氟化钠,氟浓度分别为0、100、400、700、1000、2000μmol/L,同时加入硒,浓度为100 nmol/L。连续培养48 h后,收集细胞培养液与细胞。应用瑞氏-吉姆萨染色观察细胞形态,吖啶橙荧光染色测定细胞凋亡,四唑氮蓝(MTT)比色法检测细胞活性;检测超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性、丙二醛(MDA)含量;检测细胞培养液诱导型一氧化氮合酶(iNOS)、内皮型一氧化氮合酶(eNOS)活性、细胞iNOSmRNA、eNOSmRNA表达;检测细胞间粘附分子-1(ICAM-1)、血管细胞粘附分子-1(VCAM-1)含量。结果在氟浓度100~700μmol/L加硒100 nmol/L,可明显减轻氟对细胞生长的抑制,恢复细胞形态的改变;使SOD、GSH-Px活性升高(P<0.05)、MDA含量下降(P<0.05);降低iNOS活性和iNOSmRNA表达(P<0.05),升高eNOS的活性和eNOSmRNA表达(P<0.05);减少CAM-1和VCAM-1含量(P<0.05,P<0.01)。结论氟可致血管内皮细胞形态改变、功能异常。适宜剂量硒补充可通过提高抗氧化功能,调整NO代谢,抑制粘附分子异常表达等途径拮抗高氟所致的血管内皮损伤。     

Endothelium-Dependent Relaxation Effect of Apocynum venetum Leaf Extract via Src/PI3K/Akt Signalling Pathway

Botanical herbs are consumed globally not only as an essential diet but also as medicines or as functional/recreational food supplements. The extract of the Apocynum venetum leaves (AVLE), also known as Luobuma, exerts its antihypertensive effect via dilating the blood vessels in an endothelium- and concentration-dependent manner with optimal effect seen at as low as 10 µg/mL. A commercial Luoboma “antihypertensive tea” is available commercially in the western province of China. The present study seeks to investigate the underlying cellular mechanisms of the nitric oxide (NO)-releasing property of AVLE in rat aortas and human umbilical vein endothelial cells (HUVECs). Endothelium-dependent relaxation induced by AVLE was assessed in organ chambers in the presence or absence of polyethyleneglycol catalase (PP2, 20 µM; inhibitor of Src kinase), wortmannin (30 nM) and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (20 µM; PI3 (phosphatidylinositol3)-Kinase inhibitor), N^G-nitro-l-arginine (L-NAME, 100 µM; endothelial NO synthase inhibitor (eNOS)) and ODQ (1 µM; soluble guanylyl cyclase inhibitor). Total nitrite and nitrate (NOx) level and protein expression of p-Akt and p-eNOS were measured. AVLE-induced endothelium-dependent relaxation was reduced by PP2, wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and abolished by L-NAME and ODQ. AVLE significantly increased total NO_x level in rat aortas and in HUVECs compared to control. It also instigated phosphorylation of Akt and eNOS in cultured HUVECs in a concentration-dependent manner and this was markedly suppressed by PP2, wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. AVLE also inhibited superoxide generated from both NADPH oxidase and xanthine/xanthine oxidase system. Taken together, AVLE causes endothelium-dependent NO mediated relaxations of rat aortas through Src/PI3K/Akt dependent NO signalling pathway and possesses superoxide scavenging activity.     

Comparative effect of genistein and daidzein on the expression of MCP-1, eNOS, and cell adhesion molecules in TNF-��-stimulated HUVECs

We compared the effects of genistein and daidzein on the expression of chemokines, cell adhesion molecules (CAMs), and endothelial nitric oxide synthase (eNOS) in tumor necrosis factor (TNF)-α-stimulated human umbilical vascular endothelial cells (HUVECs). TNF-α exposure significantly increased expression of monocyte chemoattractant protein (MCP)-1, vascular adhesion molecule (VCAM)-1, and intercellular adhesion molecule-1. Genistein significantly decreased MCP-1 and VCAM-1 production in a dose-dependent manner, whereas CAM expression was not significantly lowered by genistein treatment. However, daidzein slightly decreased MCP-1 production. The effects of genistein and daidzein on MCP-1 secretion coincided with mRNA expression. Pre-treatment with either genistein or daidzein elevated eNOS expression and nitric oxide production disturbed by TNF-α exposure. A low concentration of isoflavones significantly inhibited nuclear factor (NF)κB activation, whereas a high dose slightly ameliorated these inhibitive effects. These results suggest that genistein had a stronger effect on MCP-1 and eNOS expression than that of daidzein. Additionally, NFκB transactivation might be partially related to the down-regulation of these mRNAs in TNF-α-stimulated HUVECs.     

Effect of essential fatty acids on glucose-induced cytotoxicity to retinal vascular endothelial cells

ABSTRACT: BACKGROUND: Diabetic retinopathy is a major complication of dysregulated hyperglycemia. Retinal vascular endothelial cell dysfunction is an early event in the pathogenesis of diabetic retinopathy. Studies showed that hyperglycemia-induced excess proliferation of retinal vascular endothelial cells can be abrogated by docosahexaenoic acid (DHA, 22:6 omega-3) and eicosapentaenoic acid (EPA, 20:5 omega-3). The influence of dietary omega-3 PUFA on brain zinc metabolism has been previously implied. Zn2+ is essential for the activity of Delta6 desaturase as a co-factor that, in turn, converts essential fatty acids to their respective long chain metabolites. Whether essential fatty acids (EFAs) alpha-linolenic acid and linoleic acid have similar beneficial effect remains poorly understood. METHODS: RF/6A cells were treated with different concentrations of high glucose, alpha-linolenic acid and linoleic acid and Zn2+. The alterations in mitochondrial succinate dehydrogenase enzyme activity, cell membrane fluidity, reactive oxygen species generation, SOD enzyme and vascular endothelial growth factor (VEGF) secretion were evaluated. RESULTS: Studies showed that hyperglycemia-induced excess proliferation of retinal vascular endothelial cells can be abrogated by both linoleic acid (LA) and alpha-linolenic acid (ALA), while the saturated fatty acid, palmitic acid was ineffective. A dose-response study with ALA showed that the activity of the mitochondrial succinate dehydrogenase enzyme was suppressed at all concentrations of glucose tested to a significant degree. High glucose enhanced fluorescence polarization and microviscocity reverted to normal by treatment with Zn2+ and ALA. ALA was more potent that Zn2+. Increased level of high glucose caused slightly increased ROS generation that correlated with corresponding decrease in SOD activity. ALA suppressed ROS generation to a significant degree in a dose dependent fashion and raised SOD activity significantly. ALA suppressed high-glucose-induced VEGF secretion by RF/6A cells. CONCLUSIONS: These results suggest that EFAs such as ALA and LA may have beneficial action in the prevention of high glucose-induced cellular damage.     

苯扎贝特逆转高甘油三酯血清下调内皮细胞一氧化氮合酶基因表达的机制探讨

目的研究苯扎贝特逆转高甘油三酯血清（HTS）下调原代牛主动脉内皮细胞（BVEC）一氧化氮合酶（eNOS）基因表达的可能机制。方法在前期研究证实HTS下调原代牛主动脉内皮细胞（BVEC）一氧化氮合酶（eNOS）蛋白质表达的基础上,采用Western印迹法检测苯扎贝特对HTS诱导的eNOS蛋白质表达水平下调的影响;在证实其效应的基础上研究其信号转导机制。结果发现苯扎贝特可以完全消除HTS对eNOS的抑制作用,经PPARα、PI3K、MAPKK和MAPK抑制剂预处理后,苯扎贝特逆转HTS下调eNOS的蛋白质表达的作用明显减弱。结论证实了苯扎贝特可以纠正HTS对eNOS的抑制作用,其效应的发挥既通过依赖于PPARα的方式,也可以经PI3K/MAPK/MAPKK信号通路介导。     

血管紧张素－（1—7）对血管紧张素Ⅱ诱导人脐静脉内皮细胞凋亡的影响

目的 观察血管紧张素（1-7）[Ang-（1-7）]对血管紧张素Ⅱ（AngⅡ）诱导人脐静脉内皮细胞（HUVECs）凋亡的影响.方法 采用胰蛋白酶消化法原代培养HUVECs,取2～5代用于实验,培养的HUVECs随机分为：对照组、AngⅡ组、Ang-（1-7）组、AngⅡ＋Ang-（1-7）组、AngⅡ＋Ang-（1-7）＋A-779组,用吖啶橙（AO）/溴乙锭（BE）法观察细胞凋亡的形态学变化,用流式细胞术检测内皮细胞的凋亡率.结果 （1）AngⅡ（10-6 mol/L）可以诱导HUVECs凋亡率明显增加,与对照组相比差异有统计学意义（25.60%±3.17% vs 2.32%±0.24%,P＜0.005）;不同浓度的Ang-（1-7）（10-9～10-6 mol/L）呈剂量依赖性抑制AngⅡ所诱导的内皮细胞的凋亡,与AngⅡ组相比差异有统计学意义（20.04%±2.21%,16.04%±1.32%,10.04%±2.05%,7.79%±1.50%,P＜0.05）;力Ⅱ用Ang-（1-7）特异性受体拮抗剂A-779可阻断Ang-（1-7）的上述效应,与AngⅡ＋Ang-（1-7）组比较差异有统计学意义（23.37%±0.75%vs 20.04%±2.21%,16.04%±1.32%,10.04%±2.05%,7.79%±1.50%,P＜0.05）.结论 AngⅡ可诱导内皮细胞凋亡增加,Ang-（1-7）呈浓度依赖性抑制AngⅡ的上述效应,并且是通过其特异性受体Mas发挥作用.     

Diallyl trisulfide inhibits angiogenic features of human umbilical vein endothelial cells by causing Akt inactivation and down-regulation of VEGF and VEGF-R2

We have shown recently that diallyl trisulfide (DATS), a cancer-chemopreventive constituent of garlic, inactivates Akt to trigger mitochondrial translocation of proapoptotic protein BAD in human prostate cancer cells. Because Akt activation is implicated in the promotion of endothelial cell survival and angiogenesis, we hypothesized that DATS may inhibit angiogenesis. In the present study, we tested this hypothesis using human umbilical vein endothelial cells (HUVECs) as a model. Survival of HUVECs was reduced significantly in the presence of DATS in a concentration-dependent manner, with an IC50 of approximately 4 microM. The DATS-mediated suppression of HUVEC survival was associated with apoptosis induction characterized by accumulation of subdiploid cells, cytoplasmic histone-associated DNA fragmentation, and cleavage of caspase-3 and poly-(ADP-ribose)-polymerase. The DATS-induced DNA fragmentation was significantly attenuated in the presence of pan-caspase inhibitor zVAD-fmk and specific inhibitors of caspase-9 (zLEHD-fmk) and caspase-8 (zIETD-fmk). DATS treatment inhibited the formation of capillary-like tube structure and migration by HUVECs in association with suppression of vascular endothelial growth factor (VEGF) secretion and VEGF receptor-2 protein level and inactivation of Akt kinase. DATS treatment also caused activation of extracellular signal-regulated kinase 1/2 (ERK1/2) but not c-Jun NH2-terminal kinase (JNK) or p38 mitogen-activated protein kinase (p38MAPK).DATS-mediatedapoptosis induction and inhibition of HUVEC tube formation was partially but statistically significantly attenuated by pharmacologic inhibition of ERK1/2 but not JNK or p38MAPK. The present study demonstrates, for the first time, that DATS has the ability to inhibit angiogenic features of human endothelial cells.     

乙醇诱导人脐静脉内皮细胞损伤及ADMA/NOS/NO信号机制

目的探讨乙醇诱导人脐静脉内皮细胞损伤效应及ADMA/NOS/NO信号机制。方法用不同浓度的乙醇与人脐静脉内皮细胞共培养,观察细胞形态和活性,通过检测细胞上清液丙二醛（MDA）、非对称性二甲基精氨酸（AD-MA）、一氧化氮（NO）的含量和一氧化氮合酶（NOS）的活性等指标,观察乙醇对人脐静脉内皮细胞损伤的量-效关系（乙醇处理细胞24 h）和时-效关系（终浓度为100 mmol/L的乙醇分别于处理0、3、6、12、24、48 h后观察检测指标）。实验分为正常对照组（加入与处理组等体积的D-Hank’s液,乙醇浓度为0 mmol/L）、乙醇处理组（终浓度分别为25、50、100、200mmol/L的乙醇）、乙醇＆VitC组（乙醇终浓度为100 mmol/L,VitC终浓度为100 mg/L）和阳性对照组（终浓度为500μmol/L的过氧化氢）。结果50-200 mmol/L乙醇能显著改变细胞形态、降低细胞活性（OD值：0.91±0.10-0.58±0.04,细胞生长抑制率：0.00±0.00-35.57±3.13,P〈0.01）;能显著性诱导内皮细胞MDA升高（258.72±7.36-524.07±8.25,P〈0.01）。50-200 mmol/L的乙醇还能显著性诱导：内皮型一氧化氮合酶（eNOS）活性下降（6.27±0.10-0.47±0.23,P〈0.05）及总NOS活性下降（10.69±0.54-3.77±0.32,P〈0.05）;NO含量降低（191.61±7.96-88.38±5.07,P〈0.05）。而且上述生物学效应均具有显著的时间依赖性,各处理组间两两比较也具有显著性（P〈0.05或P〈0.01）。100 mmol/L乙醇处理时细胞诱导型一氧化氮合酶（iNOS）活性及ADMA含量最高,且分别在处理后24 h到达最高峰。VitC均能扭转上述生物学效应,且具有显著性（P〈0.01）。结论50-200 mmol/L的乙醇能显著诱导人脐静脉内皮细胞损伤;ADMA在乙醇诱导人脐静脉内皮细胞损伤中有着重要作用。     

The flavonoid luteolin induces nitric oxide production and arterial relaxation

Purpose

Luteolin, a flavone present in many foods and medicinal plants, may have beneficial effects on various human chronic diseases. In the present study, we investigated the hypothesis that luteolin can directly act on vascular endothelial cells (ECs), leading to nitric oxide (NO) production and subsequent vascular relaxation.

Methods

Rat aortic rings were mounted in organ bath. Luteolin was added cumulatively, and vessel relaxation of rat aortic rings precontracted with phenylephrine (PE) or potassium was recorded. Endothelial nitric oxide synthase (eNOS) phosphorylation at Ser1177 and NO production from aortic rings and primary human aortic endothelial cells (HAECs) exposed to luteolin were measured by using Western blot and fluorometric assay, respectively.

Results

Luteolin dose-dependently (10–100 μmol/L) elicited relaxation of PE- or potassium-contracted aortic rings. The vasorelaxation effect of luteolin was attenuated by the eNOS inhibitor, N-nitro-l-arginine methyl ester, suggesting that this luteolin action is at least partially mediated by activating eNOS activity. We further found that luteolin dose-dependently (10–100 μmol/L) increased eNOS phosphorylation at Ser1177 (up to 1.9-fold) in isolated rat rings. Consistently, exposure of HAECs to luteolin also increased eNOS phosphorylation and NO production.

Conclusions

Luteolin may be a vascular protective agent by directly acting on vascular ECs to stimulate NO-dependent vascular dilatation.     

维生素E抑制子痫前期脐血清诱导脐静脉内皮细胞中NOSTRIN表达上调的体外研究

目的:探讨子痫前期患者脐血清诱导脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)中人内皮型一氧化氮合酶运输介导物(endothelial nitric oxide synthase traffic inducer,NOSTRIN)的表达及维生素E对其表达的调节作用。方法:采用消化培养法培养正常晚孕妇女的HUVEC,传代后待细胞长满至70%~80%后,加或不加维生素E作用30min后分别加入正常妊娠(正常组)及子痫前期(子痫前期组)脐静脉血清,培养48 h后,MTT测定细胞活力,RT-PCR测定NOSTRINmRNA的表达,分光光度法测定细胞上清中eNOS的活性。结果:子痫前期患者脐静脉血清培养的HUVEC,其细胞的活力明显低于正常组(P<0.05),细胞中NOSTRINmRNA的表达量明显高于正常妊娠组(P<0.01),细胞上清中eNOS的活性明显低于正常组(P<0.01);加维生素E预处理后,子痈前期组细胞的活力明显升高(P<0.05),其细胞中NOSTRIN的表达水平明显下降(P<0.01),eNOS活性明显升高(P<0.01)。结论:子痫前期患者脐静脉血清可使HUVEC细胞活力降低、NOSTRIN表达增加以及eNOS活性降低,维生素E可抑制子痫前期患者脐静脉血清诱导的NOSTRIN表达的增加以及eNOS活性降低。     

Monomethylarsonous Acid Induced Cytotoxicity and Endothelial Nitric Oxide Synthase Phosphorylation in Endothelial Cells

Chronic arsenic poisoning is reported to be associated with peripheral and cardiovascular disease, arteriosclerosis, Raynaud’s syndrome, hypertension, and Blackfoot disease. Monomethylarsonous acid (MMA^III) is a reactive metabolite of inorganic arsenic and a potent inhibitor of endothelial nitric oxide synthase (eNOS). Arsenic is also reported to phosphorylate eNOS in cultured keratinocyte and Human T cell leukemia Jurkat cells, respectively. In the present study, we examined the cytotoxicity and eNOS phosphorylation by MMA^III exposure in cultured bovine aortic endothelial cells (BAEC). Results showed that MMA^III is more toxic than arsenite in BAEC cells. The IC₅₀ values for MMA^III and arsenite were determined to be approximately 1.7 and 24.1 μmol/L, respectively. Exposure of BAEC to MMA^III (0.75 μmol/L) caused a significant eNOS phosphorylation 15 min after MMA^III exposure. However, a complex of MMA^III with dithiothreitol (DTT) that lacks the reactivity with vicinal thiols unaffected eNOS phosphorylation. The present study shows that MMA^III generated during biomethylation of arsenic is highly toxic in BAEC. Our study also suggests that MMA^III could induce the eNOS phosphorylation through modification to cellular thiols of the eNOS enzyme. And the initial up-regulation of eNOS phosphorylation by MMA^III seems to be an adaptive response against disruption of eNOS bioactivity during arsenic exposure.     

茶多酚影响同型半胱氨酸对内皮细胞的作用

目的:探讨茶多酚能否减弱同型半胱氨酸(HCY)对内皮细胞(EC)的损伤作用。方法:体外培养牛主动脉内皮细胞,随机分为5组,正常对照组,用含20%小牛血清DMEM培养液培养;0.25 mmol/L HCY组,培养液中加入HCY使其终浓度为0.25 mmol/L;1 mg/L茶多酚 +0.25 mmol/L HCY组;2 mg/L茶多酚 +0.25 mmol/L HCY组;4 mg/L茶多酚 +0.25 mmol/L HCY组。培养72h后,用流式细胞仪检测细胞周期,观察细胞生长情况,同时检测培养液中的一氧化氮(NO)、 一氧化氮合酶(NOS)、内皮素(ET)、谷胱甘肽过氧化酶(GSH-Px)、丙二醛(MDA)浓度或活性。结果:浓度为1、2、4、8 mg/L茶多酚组与HCY组相比,(1)细胞形态规则性增强,立体感增加,细胞内颗粒减少;(2)细胞周期检测提示茶多酚组细胞S期比例增多;(3)功能实验提示,茶多酚组EC释放的血管舒张因子NO浓度较HCY组高;而血管收缩因子ET低;(4)MDA含量和GS H-Px活性无明显改变。结论:茶多酚有抑制HCY对EC的损伤作用。     

高甘油三酯血清对牛主动脉内皮细胞一氧化氮合酶基因表达的影响及其机制的研究

目的研究高甘油三酯血清（HTS）对原代牛主动脉内皮细胞（BVEC）一氧化氮合酶（eNOS）基因表达的影响及其可能机制。方法采用Western印迹法从蛋白质水平检测高甘油三酯血清对牛主动脉内皮细胞eNOS和一氧化氮（NO）活性的影响。在证实其效应的基础上，采用Western印迹法检测经HTS作用后，PKC—MAPK—MAPKK信号转导通路的改变。进一步从Ⅳ型高脂蛋白血症血浆提取极低密度脂蛋白（VLDL）（HTG—VLDL）并采用Western印迹法研究HTG—VLDL对eNOS的蛋白质表达的影响、结果HTS以浓度依赖的方式抑制eNOS的蛋白质表达，减少一氧化氮的产生．并以剂量和时间依赖的方式促进MAPK的磷酸化，而经PKC、MAPKK和MAPK抑制剂预处理后，HTS对eNOS的蛋白质表达的抑制作用更明显：另外发现HTG-VLDL可抑制eNOS的蛋白质表达水平。结论HTS抑制eNOS的蛋白质表达水平，可能部分通过HTG—VLDL对eNOS的影响，但未经PKC—MAPKK-MAPK通路，而且PKC—MAPKK—MAPK可能参与了eNOS正常的蛋白质表达，HTS增加MAPK的磷酸化水平有可能是一种细胞自身保护机制．     

高甘油三酯血清对牛主动脉内皮细胞一氧化氮合酶基因表达的影响及其机制的研究

目的研究高甘油三酯血清(HTS)对原代牛主动脉内皮细胞(BVEC)一氧化氮合酶(eNOS)基因表达的影响及其可能机制。方法采用Western印迹法从蛋白质水平检测高甘油三酯血清对牛主动脉内皮细胞eNOS和一氧化氮(NO)活性的影响。在证实其效应的基础上,采用Western印迹法检测经HTS作用后,PKC-MAPK-MAPKK信号转导通路的改变。进一步从Ⅳ型高脂蛋白血症血浆提取极低密度脂蛋白(VLDL)(HTG-VLDL)并采用Western印迹法研究HTG-VLDL对eNOS的蛋白质表达的影响。结果HTS以浓度依赖的方式抑制eNOS的蛋白质表达,减少一氧化氮的产生,并以剂量和时间依赖的方式促进MAPK的磷酸化,而经PKC、MAPKK和MAPK抑制剂预处理后,HTS对eNOS的蛋白质表达的抑制作用更明显;另外发现HTG-VLDL可抑制eNOS的蛋白质表达水平。结论HTS抑制eNOS的蛋白质表达水平,可能部分通过HTG-VLDL对eNOS的影响,但未经PKC-MAPKK-MAPK通路,而且PKC-MAPKK-MAPK可能参与了eNOS正常的蛋白质表达,HTS增加MAPK的磷酸化水平有可能是一种细胞自身保护机制。