川芎嗪对缺氧时视网膜血管内皮细胞HIF-1α、VEGF表达及细胞增殖的影响

目的研究川芎嗪对缺氧时体外培养的牛眼视网膜血管内皮细胞（BREC）VEGF表达及细胞增殖活性的影响,探讨川芎嗪治疗视网膜新生血管性疾病的药理机制及可行性。方法 CoCl2模拟缺氧处理培养的BREC细胞,用浓度分别为20、40、120μg/mL的川芎嗪分别加入细胞培养液中24h,采用ELISA法检测细胞培养上清液VEGF含量,免疫组织化学法和生物荧光法分别检测细胞PCNA的表达、细胞内ATP含量以分析细胞增殖能力,采用免疫组织化学法检测HIF-1α的表达。结果川芎嗪可减少缺氧引起的视网膜血管内皮细胞中PCNA的表达,降低细胞内ATP含量,降低培养上清液中的VEGF浓度,且呈剂量依赖性。结论川芎嗪可抑制缺氧引起的视网膜血管内皮细胞的增殖。其机制可能是通过抑制HIF-1α途径来实现的。     

Rhapontigenin inhibited hypoxia inducible factor 1 alpha accumulation and angiogenesis in hypoxic PC-3 prostate cancer cells

Hypoxia inducible factor 1 alpha (HIF-1α) is frequently over-expressed in the numerous types of cancer and plays an important role in angiogenesis. In the present study, the inhibitory mechanism of rhapontigenin isolated from Vitis coignetiae was investigated on HIF-1α stability and angiogenesis in human prostate cancer PC-3 cells. Rhapontigenin significantly suppressed HIF-1α accumulation at protein level but not at mRNA level in PC-3 cells under hypoxia. Also, rhapontigenin suppressed hypoxia-induced HIF-1α activation in various cancer cells, such as colorectal adenocarcinoma (SW620), breast adenocarcinoma (MCF-7), fibrosarcoma (HT-1080) and prostate carcinoma (LNCaP). Interestingly, rhapontigenin had more potency in inhibition of hypoxia-induced HIF-1α expression than that of resveratrol, a known HIF-1α inhibitor. In addition, rhapontigenin promoted hypoxia-induced HIF-1α degradation and cycloheximide (CHX) blocked protein synthesis. A prolyl hydroxylase (PHD) inhibitor dimethyloxalylglycine (DMOG) is usually utilized to examine whether prolyl hydroxylation is involved in inhibition of HIF-1α accumulation. Here, DMOG recovered HIF-1α accumulation inhibited by rhapontigenin. Immunoprecipitation assay also revealed that rhapotigenin enhanced the binding of hydroxylated HIF-1α to von Hippel-Lindau (VHL) tumor suppressor protein. Furthermore, rhapontigenin reduced vascular endothelial growth factor (VEGF) secretion in hypoxic PC-3 cells as well as suppressed tube formation in human umbilical vein endothelial cells (HUVECs) treated by the conditioned media of hypoxic PC-3 cells. However, anti-angiogenic effect of rhapontigenin in hypoxic PC-3 cells was reversed by DMOG. Taken together, these findings suggest that rhapontigenin inhibits HIF-1α accumulation and angiogenesis in PC-3 prostate cancer cells.     

Arsenite induces endothelial cell permeability increase through a reactive oxygen species-vascular endothelial growth factor pathway

As a potent environmental oxidative stressor, arsenic exposure has been reported to exacerbate cardiovascular diseases and increase vascular endothelial cell monolayer permeability. However, the underlying mechanism of this effect is not well understood. In this paper, we test our hypothesis that reactive oxygen species (ROS)-induced vascular endothelial growth factor (VEGF) expression may play an important role in an arsenic-caused increase of endothelial cell monolayer permeability. The mouse brain vascular endothelial cell bEnd3 monolayer was exposed to arsenite for 1, 3, and 6 days. The monolayer permeability, VEGF protein release, and ROS generation were determined. In addition, VE-cadherin and zonula occludens-1 (ZO-1), two membrane structure proteins, were immunostained to elucidate the effects of arsenite on the cell-cell junction. The roles of ROS and VEGF in arsenite-induced permeability was determined by inhibiting ROS with antioxidants and immuno-depleting VEGF with a VEGF antibody. We observed that arsenite increased bEnd3 monolayer permeability, elevated the production of cellular ROS, and increased VEGF release. VE-cadherin and ZO-1 disruptions were also found in cells treated with arsenite. Furthermore, both antioxidant (N-acetyl cysteine and tempol) and the VEGF antibody treatments significantly lowered the arsenite-induced permeability of the bEnd3 monolayer as well as VEGF expression. VE-cadherin and ZO-1 disruptions were also diminished by N-acetyl cysteine and the VEGF antibody. Our data suggest that the increase in VEGF expression caused by ROS may play an important role in the arsenite-induced increase in endothelial cell permeability.     

过氧化氢诱导肺微血管内皮损伤的实验研究

目的　探讨H2 O2 诱导培养肺微血管内皮细胞损伤的机制。方法　观察H2 O2 对大鼠肺微血管内皮细胞 (RP MVEC)的形态、单层通透性、F 肌动蛋白和 β AR的影响。结果　H2 O2 浓度大于 1mmol·L-1作用 48h内观察到细胞脱落和破裂。 10mmol·L-1H2 O2 90min内可使RPMVEC单层通透性增高、F 肌动蛋白发生明显解聚和 β AR显著下调 ;FOR、山莨菪碱和CTX可抑制上述变化。结论　H2 O2引起的RPMVEC脱落与破裂呈浓度和时间依赖性。H2 O2引起RPMVEC单层通透性增高的机制与F 肌动蛋白解聚密切相关 ;β AR参与内皮通透性调节。FOR、山莨菪碱和CTX对H2 O2 引起RPMVEC单层通透性增高有一定的保护作用。     

灯盏花素对血小板活化因子致肺微血管内皮细胞损伤的影响

目的　探讨血小板活化因子 (PAF)诱导肺微血管内皮细胞损伤的机制及灯盏花素的干预作用。方法　观察PAF对体外培养大鼠肺微血管内皮细胞 (RPMVEC)的形态、单层通透性、F 肌动蛋白 (F actin)的影响和灯盏花素干预后的变化 ,F actin用流式细胞仪测定。结果　PAF浓度大于 0 1mg·L-1作用 4 8h内观察到细胞脱落和破裂。 10mg·L-1PAF 12 0min内可使RPMVEC单层通透性增高、F actin解聚 ,灯盏花素可抑制上述变化。结论　①PAF引起RPMVEC脱落和破裂呈时间和剂量依赖性。②PAF引起内皮单层通透性的增高的机制与F actin解聚密切相关。③灯盏花素可抑制PAF引起RPMVEC单层通透性的增高和F actin下降 ,对PAF引起的RPMVEC损伤有一定保护作用。     

Manganese (II) induces chemical hypoxia by inhibiting HIF-prolyl hydroxylase: Implication in manganese-induced pulmonary inflammation

Manganese (II), a transition metal, causes pulmonary inflammation upon environmental or occupational inhalation in excess. We investigated a potential molecular mechanism underlying manganese-induced pulmonary inflammation. Manganese (II) delayed HIF-1α protein disappearance, which occurred by inhibiting HIF-prolyl hydroxylase (HPH), the key enzyme for HIF-1α hydroxylation and subsequent von Hippel-Lindau(VHL)-dependent HIF-1α degradation. HPH inhibition by manganese (II) was neutralized significantly by elevated dose of iron. Consistent with this, the induction of cellular HIF-1α protein by manganese (II) was abolished by pretreatment with iron. Manganese (II) induced the HIF-1 target gene involved in pulmonary inflammation, vascular endothelial growth factor (VEGF), in lung carcinoma cell lines. The induction of VEGF was dependent on HIF-1. Manganese-induced VEGF promoted tube formation of HUVEC. Taken together, these data suggest that HIF-1 may be a potential mediator of manganese-induced pulmonary inflammation.     

Cobalt chloride attenuates hypobaric hypoxia induced vascular leakage in rat brain: molecular mechanisms of action of cobalt chloride

This study reports the efficacy of cobalt preconditioning in preventing hypobaric hypoxia induced vascular leakage (an indicator of cerebral edema) using male Sprague-Dawley rats as model system. Exposure of animals to hypobaric hypoxia led to a significant increase in vascular leakage, reactive oxygen species (ROS), nitric oxide (NO), and vascular endothelial growth factor (VEGF) levels. There was a marked increase in Nuclear Factor κB (NFκB) DNA binding activity and levels of pro-inflammatory cytokines such as Monocyte chemoattractant protein (MCP-1), Interferon-γ (IFN-γ), Interleukin-1 (IL-1), and Tumor Necrosis Factor-α (TNF-α) and cell adhesion molecules such as Vascular Cell Adhesion Molecule-1 (VCAM-1), and P-selectin. Chemical preconditioning by cobalt for 7 days (12.5 mg Co/kg b.w., oral) significantly attenuated cerebral vascular leakage and the expression of inflammatory mediators induced by hypoxia. Administration of NFκB inhibitor, curcumin (50 mg/kg b.w.; i.p.) appreciably inhibited hypoxia induced vascular leakage indicating the involvement of NFκB in causing vascular leakage. Interestingly, cobalt when administered at 12.5 mg Co/kg b.w. (i.p.), 1 h before hypoxia could not prevent the vascular leakage indicating that cobalt per se did not have an effect on NFκB. The lower levels of NFκB observed in the brains of cobalt administered animals might be due to higher levels of antioxidant and anti-inflammatory proteins (hemeoxygenase-1 and metallothionein). To conclude cobalt preconditioning inhibited hypobaric hypoxia induced cerebral vascular leakage by lowering NFκB DNA binding activity and its regulated pro-inflammatory mediators. This is contemplated to be mediated by cobalt induced reduction in ROS/NO and increase in HO-1 and MT.     

常山酮对高原肺动脉高压模型大鼠心肺功能的影响及其机制研究

目的 探讨常山酮（HF）对高原肺动脉高压（HAPH）模型大鼠心肺功能的影响及其机制。方法 根据随机数字表法从50只雄性SD大鼠中抽取40只,分为模型组和低、中、高剂量组,每组10只,于低压低氧舱中模拟海拔6 000 m高原环境构建HAPH大鼠模型;另10只作为对照组于舱外喂养。低、中、高剂量组予HF（给药剂量分别为0.5、1、2 mg/kg）,模型组和对照组予等量蒸馏水,每天灌胃1次,连续4周。测定各组大鼠平均肺动脉压（mPAP）,计算右心室肥厚指数（RVHI）,苏木精-伊红（HE）染色评定肺血管重塑程度,酶联免疫吸附试验（ELISA）检测大鼠血清和肺组织低氧诱导因子-1α(HIF-1α)、血管内皮生长因子（VEGF）、内皮素-1(ET-1)水平。结果 与对照组比较,模型组mPAP和RVHI均明显升高（P<0.05）,肺小动脉管壁明显增厚、管腔狭窄,血清和肺组织HIF-1α、VEGF、ET-1水平均明显升高（P<0.05）;与模型组比较,低、中、高剂量组mPAP和RVHI均明显降低（P<0.05）,中、高剂量组肺血管重塑程度均显著减轻,血清和肺组织HIF-1α、VEG...     

HIF-1α和VEGF在宫颈鳞癌组织中的表达及其意义

目的:探讨缺氧诱导因子-1α(HIF-1α)和血管内皮生长因子(VEGF)在宫颈鳞癌组织中的表达及其意义.方法:采用免疫组化SP法检测HIF-1α仅和VEGF在11例正常宫颈组织,32例宫颈鳞状上皮非典型增生和48例宫颈鳞癌组织的表达特点及相关性.结果:HIF-1α在正常宫颈组织、宫颈上皮内瘤变(CIN)及宫颈癌组织中的表达率依次升高,分别为0%,15.6%(5/32)和47.9%(23/48),3组比较差异有显著性(P<0.05).VEGF在正常宫颈组织、CIN及宫颈癌组织中的表达率依次升高,分别为0%,21.9%(7/32)和54.2%(26/48),3组比较差异有显著性(P<0.05).低分化鳞癌组织中HIF-1α、VEGF的表达率高于高-中分化鳞癌组织(P<0.05).有淋巴结转移鳞癌组织中HIF-1α、VEGF的表达率高于无淋巴结转移鳞癌组织(P<0.05).鳞癌组织中HIF-1α、VEGF的表达之间存在明显关系(P<0.05).结论:HIF-1α、VEGF在宫颈鳞癌的发生发展中起重要作用,HIF-1α可通过调节VEGF的表达促进肿瘤血管形成及其恶性进展.     

Novel vascular endothelial growth factor blocker improves cellular viability and reduces hypobaric hypoxia‐induced vascular leakage and oedema in rat brain

Vascular endothelial growth factor (VEGF) is an important cerebral angiogenic and permeability factor under hypoxia. There is a need to find effective molecules that may ameliorate hypoxia‐induced cerebral oedema. In silico identification of novel candidate molecules that block VEGF‐A site were identified and validated with a Ramachandran plot. The active site residues of VEGF‐A were detected by Pocketfinder, CASTp, and DogSiteScorer. Based on in silico data, three VEGF‐A blocker (VAB) candidate molecules (VAB1, VAB2, and VAB3) were checked for improvement in cellular viability and regulation of VEGF levels in N2a cells under hypoxia (0.5% O₂). Additionally, the best candidate molecule's efficacy was assessed in male Sprague‐Dawley rats for its ameliorative effect on cerebral oedema and vascular leakage under hypobaric hypoxia 7260 m. All experimental results were compared with the commercially available VEGF blocker sunitinib. Vascular endothelial growth factor‐A blocker 1 was found most effective in increasing cellular viability and maintaining normal VEGF levels under hypoxia (0.5% oxygen) in N2a cells. Vascular endothelial growth factor‐A blocker 1 effectively restored VEGF levels, decreased cerebral oedema, and reduced vascular leakage under hypobaric hypoxia when compared to sunitinib‐treated rats. Vascular endothelial growth factor‐A blocker 1 may be a promising candidate molecule for ameliorating hypobaric hypoxia‐induced vasogenic oedema by regulating VEGF levels.     

蛋白激酶Cδ亚型抑制剂对脂多糖诱导肺微血管内皮细胞损伤的影响

目的研究蛋白激酶C(PKC)的亚型PKCδ在大鼠肺微血管内皮细胞(RPMVEC)中的表达及其抑制剂Rottlerin对脂多糖(LPS)导致RPMVEC单层通透性增高的影响。方法取体外培养的RPMVEC进行PKCδ的W estern b lot和免疫组化检测,用针头式滤器检测LPS及LPS+Rottlerin干预后RPMVEC单层滤过系数(K f)的变化。结果PKCδ在RPMVEC中有广泛的表达,其表达部位主要位于胞质。Rot-tlerin能减低LPS导致的RPMVEC单层通透性增高。结论LPS导致RPMVEC损伤的机制与PKC的激活有关,PKCδ亚型抑制剂可减轻LPS诱导的RPMVEC单层通透性增高。