C-reactive protein activates the nuclear factor-kappaB pathway and induces vascular cell adhesion molecule-1 expression through CD32 in human umbilical vein endothelial cells and aortic endothelial cells

C-reactive protein (CRP) contributes to the process of atherosclerosis by inducing pro-inflammatory changes in endothelial cells. However, the exact receptor involved in CRP-induced endothelial changes remains unclear. Human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) were used for the experiments. After incubation with CRP, immunoblotting showed a significant decrease of IkappaB protein and electrophoretic mobility shift assay showed a significant increase of nuclear NF-kappaB binding capacity. These changes were associated with a significant increase of vascular cell adhesion molecule-1 (VCAM-1) expression. The mRNA level of CD32, the major binding protein for CRP in endothelial cells, increased significantly as measured by Northern blot and Western blot. When these cells were transfected with siRNA directed against CD32, the mRNA of CD32 decreased significantly. The IkappaB degradation, NF-kappaB nuclear translocation and VCAM-1 up-regulation induced by CRP were all inhibited by treatment with siRNA against CD32. SB203580, a P38 inhibitor, significantly attenuated the CRP-induced responses while SP600125 (c-jun kinase inhibitor) did not. In conclusion, CRP-induced IkappaB degradation, NF-kappaB nuclear translocation and VCAM-1 protein expression in HUVECs and HAECs. CRP also increased the expression of CD32, which might serve as the receptor for CRP in endothelial cells and mediated the effects of CRP.     

血管紧张素Ⅱ诱导人脐静脉内皮细胞衰老及苦碟子注射液的干预研究

目的:观察血管紧张素Ⅱ(AngⅡ)对人脐静脉内皮细胞(HUVECs)衰老的诱导作用,及苦碟子注射液的干预作用。方法:体外培养HUVECs,用AngⅡ(在10-6mol/LAngⅡ的DMEM培养液中48h)干预,分为实验对照组、AngⅡ诱导组和苦碟子注射液组(给予AngⅡ刺激前1h先加用10%苦碟子注射液)。采用CCK-8法检测细胞存活率,以衰老相关β-半乳糖苷酶活性和细胞的增殖能力两种衰老标志物为主要观察指标。其中衰老相关β-半乳糖苷酶活性采用免疫化学染色方法,流式细胞术检测细胞周期来反应细胞的增殖能力;利用Western印迹法检测小凹蛋白-1(Caveolin-1,Cav-1)的表达。结果:与对照组比较,AngⅡ诱导组细胞存活率明显下降,β-gal阳性染色率明显增多,细胞周期多停滞于G0/G1期,S期细胞逐渐减少,Cav-1蛋白表达水平明显上调,表明AngⅡ成功诱导了HUVECs衰老,而给予苦碟子注射液干预后上述变化改善。结论:苦碟子注射液可以延缓AngⅡ诱导HUVECs衰老,其作用机制可能与下调Cav-1蛋白表达有关。     

丙酮酸乙酯对人脐静脉内皮细胞HMGB1表达的影响

目的：本研究意在探讨丙酮酸乙酯（EP）对炎症因子HMGB1表达的影响,从而证实EP在急性脓毒症中的作用及机制。方法：采用原代培养的人脐静脉内皮细胞（HUVECs）,免疫组化法鉴定其纯度,台盼蓝拒染实验鉴定细胞活力。观察不同计量的EP对人脐静脉内皮细胞分泌HMGB1的作用。细胞分三组：LPS 100 ng/mL＋EP 0μM组、LPS 100 ng/mL＋EP 5μM组、LPS 100 ng/mL＋EP 10μM组。采用Western blot方法检测培养上清液HMGB1的含量。结果：各细胞组于LPS 100 ng/mL刺激16 h后,EP 5μM剂量组对HMGB1分泌具有一定的抑制作用,与EP 0μM组比,HMGB1含量明显降低（t=21.27,P〈0.05）;10μM剂量组与0μM组比,降低HMGB1作用更为明显（t=26.38,P〈0.05）。结论：丙酮酸乙酯可以减少血管内皮细胞释放HMGB1,对急性脓毒症有改善作用。     

甘草素对人脐静脉内皮细胞血管生成的作用及其机制探讨

目的研究甘草素(liquirigenin,LQ)对人脐静脉内皮细胞(HUVECs)血管生成的作用及其分子机制。方法采用四甲基氮唑兰MTT比色法检测甘草素对HUVEC细胞增殖的影响;划痕试验观察甘草素对细胞迁移的影响;蛋白质印迹法分析甘草素对血管生成相关蛋白和通路的影响。结果甘草素可抑制HUVEC细胞增殖,75μmol/L时,存活率为76.8%;甘草素可降低HUVEC细胞的迁移能力,随剂量浓度增加,细胞迁移能力减弱;甘草素可阻断磷脂酰肌醇3-激酶/蛋白激酶B/p70S6激酶(PI3K/AKT/p70S6K)信号通路,并下调血管内皮生长因子(vascular endothelial growth factor,VEGF)和缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)蛋白的表达。结论甘草素抑制人脐静脉内皮细胞增殖和迁移,并可经PI3K/AKT/p70S6K信号通路介导抑制血管生成靶向因子VEGF和HIF-1α的表达。     

Connexin43 Modulates X-Ray-Induced Pyroptosis in Human Umbilical Vein Endothelial Cells

Objective Pyroptosis is an inflammatory form of programmed cell death. This phenomenon has been recently reported to play an important role in radiation-induced normal tissue injury. Connexin43(Cx43) is a gap junction protein that regulates cell growth and apoptosis. In this study, we investigated the effect of Cx43 on X-ray-induced pyroptosis in the human umbilical vein endothelial cells(HUVECs). Methods HUVECs, Cx43 overexpression, and Cx43 knockdown strains were irradiated with 10 Gy. Proteins were detected using western blot analysis. Cell pyroptosis was evaluated using the fluorescence-labeled inhibitor of caspase assay(FLICA) and propidium iodide staining through flow cytometry and confocal microscopy. Cell morphology and cytotoxicity were detected by scanning electron microscopy and lactate dehydrogenase release assay, respectively. Results Irradiation with 10 Gy X-ray induced pyroptosis in the HUVECs and reduced Cx43 expression. The pyroptosis in the HUVECs was significantly attenuated by overexpression of Cx43 as it decreased the level of active caspase-1. However, interference of Cx43 expression with si RNA significantly promoted pyroptosis by increasing the active caspase-1 level. Pannexin1(Panx1), a gap junction protein regulates pyroptosis, and its cleaved form is used to evaluate channel opening and active state. The level of cleaved Panx1 in the HUVECs and Cx43 knockdown strains increased in the presence of X-ray, but decreased in the Cx43 overexpression strains. Furthermore, interference of Panx1 with si RNA alleviated the upregulation of pyroptosis caused by Cx43 knockdown. Conclusion Results suggest that single high-dose X-ray irradiation induces pyroptosis in the HUVECs. In addition, Cx43 regulates pyroptosis directly by activating caspase-1 or indirectly by cleaving Panx1.     

Non-functionalized multi-walled carbon nanotubes alter the paracellular permeability of human airway epithelial cells

Little information is available upon the effects of carbon nanotubes (CNT) on the airway barrier. Here we study the barrier function of Calu-3 human airway epithelial cells, grown on permeable filters, after the exposure to commercial single-walled or multi-walled CNT, produced through chemical vapour deposition. To assess changes in the paracellular permeability of CNT-treated Calu-3 monolayers, we have measured the trans-epithelial electrical resistance (TEER) and the permeability to mannitol. Multi-walled CNT caused a large decrease in TEER and an increase in mannitol permeability but no substantial alteration in monolayer viability. Single-walled CNT produced much smaller changes of TEER while CNT, synthesized through the arc discharge method, and Carbon Black nanoparticles had no effect. If commercial multi-walled CNT were added during the formation of the tight monolayer, no further increase in trans-epithelial resistance was observed. Moreover, the same nanomaterials, but neither single-walled counterparts nor Carbon Black, prevented the TEER recovery observed after the discontinuation of interleukin-4, a Th2 cytokine that causes a reversible barrier dysfunction in airway epithelia. These findings suggest that commercial multi-walled CNT interfere with the formation and the maintenance of tight junctional complexes in airway epithelial cells.     

糖化LDL对人脐静脉血管内皮细胞SOD活性及基因表达的影响

目的 观察糖化低密度脂蛋白(gly-LDL)对人血管内皮细胞生长及抗氧化相关因子的影响,探讨糖尿病患者血管内皮损伤与gly-LDL的相关性及可能机制。方法 用完全培养基及不同浓度(G1组 0.06mg/L, G2组0.12mg/L, G3组0.24mg/L)的gly-LDL处理人脐静脉血管内皮细胞(HUVECs)24h、48h及72h,设对照组(Ctr),取细胞培养上清液检测SOD和MDA水平,同时用细胞划痕实验观察gly-LDL对内皮细胞迁移功能的影响,用实时半定量PCR(realtime quantitative PCR,RT-qPCR)方法测定Mn-SOD和eNOS的mRNA的表达情况。结果 与对照组相比不同浓度的gly-LDL均可抑制血管内皮细胞的迁移功能。较低或较高浓度的gly-LDL作用于人脐静脉内皮细胞时,随时间的增加SOD的活性及MDA的含量是升高的;中间浓度的gly-LDL作用于人脐静脉内皮细胞时,随时间的增加SOD活性是逐渐下降的,MDA含量是先增高后降低的。各组Mn-SOD的mRNA表达水平48h和72h均下调,eNOS的表达水平于24h和48h均下调,72h时上调。结论 Gly-LDL与糖尿病患者血管并发症可能存在一定的相关性,但不能肯定其是恶化因素之一。     

The roles of CYP450 epoxygenases and metabolites, epoxyeicosatrienoic acids, in cardiovascular and malignant diseases

Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid to biologically active eicosanoids. The primary epoxidation products are four regioisomers of cis-epoxyeicosatrienoic acid (EET): 5,6-, 8,9-, 11,12-, and 14,15-EET. CYP2J2, CYP2C8, and CYP2C9 are the predominant epoxygenase isoforms involved in EET formation. CYP2J and CYP2C gene families in humans are abundantly expressed in the endothelium, myocardium, and kidney. The cardiovascular effects of CYP epoxygenases and EETs range from vasodilation, anti-hypertension, pro-angiogenesis, anti-atherosclerosis, and anti-inflammation to anti-injury caused by ischemia-reperfusion. Using transgenic animals for in vivo analyses of CYP epoxygenases revealed comprehensive and marked cardiovascular protective effects. In contrast, CYP epoxygenases and their metabolites, EETs, are upregulated in human tumors and promote tumor progression and metastasis. These biological effects result from the anti-apoptosis, pro-mitogenesis, and anti-migration roles of CYP epoxygenases and EETs at the cellular level. Importantly, soluble epoxide hydrolase (sEH) inhibitors are anti-hypertensive and anti-inflammatory and, therefore, protect the heart from damage, whereas the terfenadine-related, specific inhibitors of CYP2J2 exhibit strong anti-tumor activity in vitro and in vivo. Thus, CYP2J2 and arachidonic acid-derived metabolites likely play important roles in regulating cardiovascular functions and malignancy under physiological and/or pathological conditions. Moreover, although challenges remain to improving the drug-like properties of sEH inhibitors and identifying efficient ways to deliver sEH inhibitors, sEH will likely become an important therapeutic target for cardiovascular diseases. In addition, CYP2J2 may be a therapeutic target for treating human cancers and leukemia.     

剪应力联合参莲提取物对血管内皮细胞炎症蛋白表达的影响

目的:观察流动剪应力联合参莲提取物对血管内皮细胞炎症通路E选择素(E-selectin)及核因子NF-κB的影响。方法:本研究采用生物力药理学的研究方法,2×2析因设计分组,利用BioFlux 1000控剪应力微流培养系统,以2水平剪应力(1,10 dyn·cm-2)联合2剂量参莲提取物对人脐静脉血管内皮细胞(HUVECs)进行预处理10 h,以免疫荧光法分析流动剪应力与药物对肿瘤坏死因子-α(TNF-α)引起的内皮炎症蛋白表达的影响。结果:剪应力与药物在抑制内皮细胞膜表达E-selectin方面,具有交互作用,10 dyn·cm-2剪应力可以显著提高药效。作为单因素,流动剪应力及参莲提取物均可抑制TNF-α诱导的血管内皮细胞NF-κB表达,二者未见交互作用。结论:血流剪应力可影响药效,参莲提取物与正常血流剪应力联合作用可通过NF-κB路径抑制E-selectin表达。