Ginsenoside Rg1-induced alterations in gene expression in TNF-α stimulated endothelial cells

Background In China the ginseng root began to be used in medicine over 2000 years ago. Ginsenosides are the most important component isolated from ginseng. The authors investigated the effect of ginsenoside Rg1 on the spectrum of gene expression in the endothelial cells stimulated by TNF-α and further explored the potential molecular mechanism of endothelial protection by ginsenoside Rg1.
Methods Nitric oxide (NO) production in the cultured human umbilical vein endothelial cells(HUVECs) was measured by using an NO assay kit. A home-made oligonucleotide microarray containing approximately 400 cardiovascular disease-related genes was constructed. The alteration of the spectrum of gene expression induced by ginsenoside Rg1 in HUVECs which were activated by TNF-α were detected by oligonucleotide microarray analysis.
Results NO production in HUVECs was decreased significantly after TNF-α treatment, while pretreatment with ginsenoside Rg1 enhanced NO production in TNF-αstimulated HUVECs. Ginsenoside Rg1 affected the expression levels of genes involved in vascular constriction, cell adherence, coagulation, cell growth and signal transduction in TNF-αstimulated HUVECs.
Conclusions Ginsenoside Rg1 could enhance NO production and the expression of eNOS mRNA in TNF-α stimulated HUVECs. Ginsenoside Rg1 regulated sets of genes in endothelial cells and protected endothelial cells from TNF-αactivation. Microarray analysis provided us with valuable insights into the atheroprotective mechanism by gingsenoside Rg1.
     

Effects of TNF-α and curcumin on the expression of VEGF in Raji and U937 cells and on angiogenesis in ECV304 cells

Background To better understand the possibilities of antiangiogenic tumor therapy and to assess possible side effects, we investigated the effect of tumour necrosis factor （TNF）-α and curcumin on the expression of vascular endothelial growth factor （VEGF） in U937 and Raji cell lines and their effect on angiogenesis in a human umbilical vein endothelial cell （HUVECs）-derived cell line （ECV304）, and also the relationship between Notchl and VEGF. The aim of this study was to elucidate potential mechanisms controlling tumor neovascularization. Methods VEGF secreted by U937 and Raji cell lines was determined by ELISA. Angiogenesis was tested by network formation of endothelial cells on Matrigel. Levels of VEGF mRNA in U937 and Raji cells and Notchl mRNA levels in EV304 cells were determined by RT-PCR. Results Secretion of VEGF by U937 and Raji cells was increased by TNF-α treatment and suppressed by curcumin （P 〈 0. 01 ）. The mRNA expression of VEGF165 and VEGF121 （containing 165 and 121 amino acid residues, respectively） were detected in any fractions. TNF-α augmented the expression of VEGF165 and VEGF121 mRNA and curcumin reduced the expression （P 〈0. 01 ）. No networks or cords formed in control and curcumin groups. There was tube formation on matrigel in the supernatants of the Raji culture group and the supernatants groups treated by VEGF group and TNF-α in Raji cell. Notch1 mRNA was detected but there was no significant change in the VEGF group compared with control （P 〉 0. 05）. Conclusions Expressions of VEGF mRNA in U937 and Raji cells were increased by TNF-α and suppressed by curcumin. VEGF and TNF-α can induce angiogenesis, and curcumin can inhibit angiogenesis in ECV304 cells.     

Inhibitory effect of ginsenoside Rg3 on ovarian cancer metastasis

Background Ginsenosides are main components extracted from ginseng, and ginsenoside Rg3 is one of the most important parts. Ginsenoside Rg3 has been found to inhibit several kinds of tumor growth and metastasis. The present study was undertaken to investigate the effect of ginsenoside Rg3 on human ovarian cancer metastasis and the possible mechanism. Methods The experimental lung metastasis models of ovarian cancer SKOV-3 and the assay of tumor-induced angiogenesis were used to observe the inhibitory effects of Rg3 on tumor metastasis and angiogenesis. The effect of Rg3 on invasive ability of SKOV-3 cells in vitro was detected by Boyden chamber, and immunofluorescence staining was used to recognize the expression of matrix metalloproteinase 9 （MMP-9） in SKOV-3 cells. Results In the experimental lung metastasis models of ovarian cancer, the number of tumor colonies in the lung and vessels oriented toward the tumor mass in each ginsenoside Rg3 group, was lower than that of control group. The invasive ability and MMP-9 expression of SKOV-3 cells decreased significantly after treatment with ginsenoside Rg3. Conclusions Ginsenoside Rg3 can significantly inhibit the metastasis of ovarian cancer. The inhibitory effect is partially due to inhibition of tumor-induced an qioqenesis and decrease of invasive ability and MMP-9 expression of SKOV-3 cells.     

Expression of interleukin-15 and its receptor on the surface of stimulated human umbilical vein endothelial cells

Summary： Human interleukin-15 （hIL-15） is an important cytokine to activate endothelial cells and can be regulated by many other cytokines. The aim of this study is to examine the ability of interferon-γ （IFN-γ）, and tumor necrosis factor-or （TNF-α to induce the production of human interleukin-15 （hIL-15） and IL-15 receptor （IL-15Rα by human umbilical vein endothelial cells （HUVECs）. The data are summarized as follows： 1. Northern blot revealed that IL-15 mRNA was up-regulated by IFN-γ and TNF-α 2. Intracellular IL-15 protein was visualized by fluorescence microscopy, whereas the expression of IL-15 on the surface of HUVECs was detected by fluorescence activated cell sorting （FACS）, and no detectable IL-15 in the medium was verified by ELISA. 3. IL-15Rα was detected on the surface of HUVECs by FACS after IFN-α and TNF-α stimulation, whereas Western blotting revealed that the elevated expression on surface IL-15Rα was not due to the increased protein expression. The conclusion demonstrated from our results is that IFN-α and TNF-α play an important role in regulating the expression of IL-15 and IL-15Rα on the surface of HUVECs.     

Activation of peroxisome proliferator-activated receptor α in human endothelial cells increases plasminogen activator inhibitor type-1 expression

Objective To investigate the effect of peroxisome proliferator-activated receptors(PPARs) activators on plasminogen activator inhibitor 1(PAI-1) expression in human umbilical vein endothelial cells and elucidate a possible mechanism.Methods Human umbilical vein endothelial cells(HUVECs) were obtained from normal fetus,and cultured conventionally.Then the HUVEC were exposed to fatty acids and prostaglandin J2 in varying concentrations with fresh media.RT-PCR and ELISA were used to determine the expression of PPAR and PAI-1 in HUVECs.Transient co-transfection of PAI-1 promoter and PPARα gene or PPARγ gene to ECV304 was performed.Results PPARα,PPARγ and PPARγ mNRA in HUVECs were detected by RT-PCR.Treatment of HUVECs with PPARα and PPARγ activators-linolenic acid,linoleic acid,oleic acid and prostaglandin J2,but not with stearic acid could augment PAI-1 mRNA expression and protein secretion in a concentrationdependent manner.Proportional induction of PAI-1 promoter activity was observed through increasing amounts of PPARαDNA in HUVECs throgh a transient gene transfection assay,although the mRNA expression of the 3 subtypes of PPAR with their activators were not changes compared with controls.Conclusions HUVECs express PPARs,PPARs activators may increase PAI-1 expression in endothelial cells(EC).Although PPARs expresslon was not enhanced after being stimulated by their activators in EC,the functionally active PPARαis probably involved in regulating PAI-1 expression in EC.     

Mechanism of ligustrazini against thrombosis

Objective To investigate the mechanism of Chinese medicine ligustrazini against thrombosis, and the effects of ligustrazini on plasminogen activator inhibitor (PAI-1) expression in normal endothelial cells and lipopolysaccharide (LPS) stimulated endothelial cells. Methods Human umbilical vein endothelial cells (HUVECs) were cultured by trypsin digestion method. PAI-1 protein in HUVEC conditioned medium was measured by Sandwich enzyme-linked immunosorbent assay (ELISA), and PAI-1 mRNA expression was determined by Northern blot analysis. Using electrophoretic mobility shift assay (EMSA), we observed HUVEC nuclear factor-kappa B (NF-κB) nuclear translocation. Results LPS treatment of cultured HUVECs resulted in a significant increase in PAI-1 protein and mRNA expression by these cells. However, when HUVECs were incubated with LPS plus ligustrazini, the upregulation of PAI-1 by LPS was abated. Moreover, ligustrazini could decrease the basal level of PAI-1 protein and mRNA in HUVECs as compared with control. Nuclear extracts prepared from HUVECs stimulated by LPS demonstrated that binding to the NF-kB oligo nucleotide increased as compared with the unstimulated cells, but ligustrazini did not change those binding in the absence or presence of LPS. Conclusions Ligustrazini inhibited both basal and LPS-induced PAI-1 protein and mRNA expression in endothelial cells, and the modulation of PAI-1 in HUVECs by ligustrazini might have other mechanisms rather than NF-kB     

RETROVIRUS-MEDIATED TNF-α GENE TRANSFER INTO TCA8113 CELLS

Objective To investigate whether TNF-α gene-modified Tca8113 cells (Tca8113/TNF-α) can be used as vaccine for oral squamous cell carcinoma. Methods TNF-α gene was transduced into Tca8113 cells in vitro with retroviral vector carring genes for both TNF-α and Neo^R. After that, presence and expression of exogenous gene in the transgenic cells, expression of HLA antigen on the cells, expression of TNF-α and survival rate of the cells after irradiation and cryopreservation, and mutagenic activity of the cells were analyzed by PCR technique, ELISA technique, FACS technique, ^60 Co irradiation inactivation test, cryopreservation test, and Ames test, respectively. Results The presence of both TNF-α and Neo^R gene and expression of TNF-α gene were demonstrated in transgenic cells. The levels of the HLA-A, B, C, DR expressed by Tca8113/TNF-α were higher than by the parental cells. Tca8113/TNF-α continued to secrete TNF-α for 14 d, there was a secretion peak time from d4 to d6;and, all the cells died by d14 after irradiation. The Level of TNF-α secreted by Tca8113/TNF-α cryopreserved for 48 h was no different from that cryopreserved for I week after irradiation, the level of TNF-α secreted by the cryopreserved cells was just a little lower than that secreted by the noncryopreserved cells. Both DNA and supernatant of the cells have no mutagenic activity. Conclusion TNF-α gene can be transduced into Tco8113 cells with retroviral vector, and the cells can express TNF-α. Expression of HLA Ⅰ,Ⅱ antigens on Tco8113 cells can be increased by TNF-Ⅱ gene transduction. Irradiation is a reliable inactivation method, and cryopreservation is a feasible conservation method for Tca8113/TNF-α. Ames test result indicate that Tca8113/TNF-α has no mutagenic activity.     

Inhibitory effect of ginsenoside Rg3 combined with cyclophosphamide on growth and angiogenesis of ovarian cancer

Background Ginsenoside Rg3, the main component isolated from ginseng, inhibits some kinds of tumour growth and angiogenesis. The combination of low dose chemotherapy and antiangiogenesis inhibitors suppresses growth of experimental tumours more effectively than conventional therapy. The effect of this combination on ovarian cancer remains to be evaluated. Therefore, we investigated the synergism of ginsenoside Rg3 and cyclophosphamide （CTX） on growth and angiogenesis of human ovarian cancer. Methods Twenty-eight female athymic mice were divided randomly into 4 groups of 7： ginsenoside Rg3, CTX, ginsenoside Rg3 and CTX combination and control, after being transplanted with ovarian cancer cells （SKOV-3）. The mice were given intraperitoneal injection of ginsenoside Rg3 and CTX for the 10 days following inoculation of SKOV-3 cells. The life quality and number of living days of mice were recorded. The size of tumour, tumour inhibitive rate, life elongation rate, proliferating cell nuclear antigen labelling index （PCNALI）, expression of vascular endothelial cell growth factor （VEGF） and microvessel density （MVD） of the tumour tissues were estimated. Results Life quality of mice in ginsenoside Rg3 and combined treatment groups were better and number of living days longer than control. Average tumour weights of each treated group were less than control and there was no significant difference among the treated groups. PCNALI of treated groups was lower than control. The MVD value and VEGF expression in treated groups were significantly lower than control and the MVD values of ginsenoside Rg3 and combined treatment groups were lower than that of CTX group. Conclusions Ginsenoside Rg3 significantly inhibited growth and angiogenesis of ovarian cancer when used alone or combined with CTX. Ginsenoside Rg3 and CTX combination reinforced the antitumour effect each other and improved the living quality and survival time of mice with tumour.     

Effect of N-tosyl-L-phenylalanylchloromethyl Ketone on Tumor Necrosis Factor-alpha -induced NF-κB Activation and Apoptosis in U937 Cell Line

Summary: To investigate the effect of N-tosyl-L-phenylalanylchloromethyl ketone (TPCK) on tumor necrosis factor-alpha-induced NF-κB activation and apoptosis in U937 cell line, changes and subcellular localization of NF-κB/p65 and IκB-α were observed by fluorescencemicroscopy and expression and degradation of IκB-α by flow cytometry. The apoptosis of U937 cells was measured by flow cytometry and electrophoresis of DNA. Immunolfluorescence assay showed that NF-κB/p65,IκB-α only localized in cytoplasm. After TNF-α stimulation, p65 was localized only in nuclei, and IκB-α was only localized in cytoplasm and decreased. The changes of TNF-α stimulation were specifically inhibited by TPCK. Flow cytometry also revealed the downregulation of IκB-α protein during TNF-α-induced apoptosis and the down-regulation was specifically inhibited by TPCK. Flow cytometry also showed the apoptosis of U937 cells after TNF-α induction. DNA ladder can be detected in cells treated by TNF-α. It is concluded that degradation of IκB-α protein and NF-κB/p65 translocation occur during TNF-α-induced apoptosis of U937 cells, suggesting the activation of NF-κB.TPCK-sensitive protease plays an important role in the degradation of IκB-α protein induced by TNF-α in U937 cells. TPCK sensitive protease also plays an important role in the apoptosis of U937 cells induced by TNF-α.     

人参皂苷对波动性高糖致内皮细胞损伤ROS和HO-1表达的影响

目的 :观察人参皂苷对波动性高糖致内皮细胞损伤的活性氧(ROS)和血红素加氧酶-1(HO-1)表达的影响。方法 :体外培养人脐静脉内皮细胞(HUVECs),分为对照组、波动性高糖模型组及人参皂苷低、中、高剂量治疗组。DCFH-DA为荧光探针检测细胞内ROS水平,Western blot及RT-PCR检测各组细胞HO-1的蛋白和m RNA表达。结果:波动性高糖导致内皮细胞ROS水平上升,并上调了HO-1的蛋白和m RNA的表达。人参皂苷能显著降低波动性高糖所致的脐静脉内皮细胞ROS水平,进一步上调HO-1的蛋白和m RNA的表达。结论:人参皂苷可能通过上调HO-1的表达而发挥其抗波动性高糖所致内皮细胞损伤的氧化应激损伤作用。     

人参皂苷Rg1抗同型半胱氨酸诱导人脐静脉内皮细胞凋亡的作用及相关机制研究

目的探讨人参皂苷Rg1抗同型半胱氨酸(Hcy)诱导人脐静脉内皮细胞(HUVECs)凋亡的作用及相关机制。方法体外培养HUVECs细胞,采用琼脂糖凝胶电泳和末端脱氧核糖核苷酸转移酶介导的缺口末端标记法(TUNEL)染色检测细胞凋亡情况,逆转录-聚合酶链反应(RT-PCR)和Western blotting分析各组内皮型一氧化氮合酶(eNOS)mRNA和蛋白质的表达。结果琼脂糖凝胶电泳显示:与对照组和Rg1组比较,Hcy组可见特征性DNA的"梯状"条带。TUNEL染色显示:与对照组和Rg1组比较Hcy组细胞凋亡率增加差异有统计学意义(P<0.01);RT-PCR显示:与对照组和Rg1组相比,Hcy组eNOS mRNA水平表达降低差异有统计学意义(P<0.01);与Hcy组相比,Hcy+Rg1组eNOS mRNA水平表达增加差异有统计学意义(P<0.01),但仍低于对照组和Rg1组,差异有统计学意义(P<0.01)。Western blotting显示:与对照组和Rg1组相比,Hcy组eNOS蛋白水平表达降低差异有统计学意义(P<0.01);与Hcy组相比,Hcy+Rg1组eNOS蛋白水平表达增加差异有统计学意义(P<0.01),但仍低于对照组和Rg1组,差异有统计学意义(P<0.01)。结论人参皂苷Rg1能够抑制Hcy诱导的人脐静脉内皮细胞凋亡,此作用可能与上调血管内皮细胞eNOS水平有关。     

奥美拉唑对人脐静脉内皮细胞全基因表达谱的影响及机制分析

目的研究奥美拉唑对体外培养人脐静脉内皮细胞(EA.hy926)基因表达谱的影响,探讨奥美拉唑对内皮细胞作用的分子机制。方法 10-5mol/L奥美拉唑与人脐静脉内皮细胞株共培养48 h,运用Affymetrix U133 plus 2.0全基因组表达芯片,检测奥美拉唑对人脐静脉内皮细胞基因表达谱的影响,分子注释系统MAS3.0软件进行聚类分析。RT-PCR验证基因表达谱结果,Western blotting验证相关蛋白水平的变化。结果奥美拉唑作用内皮细胞48 h后,基因芯片分析筛选出差异表达大于1.5倍的基因282个,其中上调基因236个,下调基因46个,包括转录调节、炎症反应、免疫反应、细胞粘附、抗凋亡、信号转导等相关基因,RT-PCR和Western blotting结果与基因芯片结果一致。结论奥美拉唑在基因水平通过多条通路调节内皮功能。     

人参皂甙Rg1对同型半胱氨酸诱导人脐静脉内皮细胞凋亡的逆转作用

目的人参皂甙Rg1对同型半胱氨酸(homocy steine,Hcy)诱导人脐静脉内皮细胞(HUVECs)凋亡的逆转作用。方法体外培养HUVECs,采用台盼蓝染色方法筛选人参皂甙Rg1最适浓度。将细胞分为对照组(无任何诱导物)、Hcy组(10μmol·L-1Hcy)、Hcy+Rg1组(10μmol·L-1Hcy和40mg·L-1Rg1)和Rg1组(单独用40mg·L-1Rg1处理的细胞),作用时间为24h。琼脂糖凝胶电泳和末端脱氧核糖核苷酸转移酶介导的缺口末端标记法(TUNEL)染色检测细胞凋亡情况。结果台盼蓝染色结果显示:人参皂甙Rg1的最适浓度40mg·L-1。琼脂糖凝胶电泳结果显示:对照组和Rg1组未见凋亡条带,Hcy组可见清楚的细胞凋亡特征性的"梯状"条带,而Hcy+Rg1组可见不明显的细胞凋亡特征性的"梯状"DNA断裂条带。TUNEL染色结果显示:与对照组和Rg1组相比,Hcy组细胞凋亡数量显著增加(P<0.01);与Hcy组相比,Hcy+Rg1组细胞凋亡数量显著减少(P<0.01),凋亡百分比由33.733%下降至9.200%,但仍高于对照组和Rg1组(P<0.01)。结论 40mg·L-1人参皂甙Rg1可一定程度逆转Hcy诱导的HUVECs凋亡。     

人参皂苷Rg1对急性心肌梗死大鼠血管新生的作用

目的:研究人参皂苷Rg1对急性心肌梗死(AMI)大鼠血管新生的影响及其机制。方法:建立Wistar大鼠急性心肌梗死模型,分假手术组、急性心肌梗死对照组、人参皂苷Rg1低剂量治疗组(1mg/kg)和高剂量治疗组(5mg/kg),于术后3,7,10,14d测定血清心肌酶、心肌梗死面积、梗死区微血管密度,RT-PCR法检测梗死区心肌组织血管内皮生长因子(VEGF)mRNA的表达。结果:心肌梗死组织中各时段假手术组的微血管密度、VEGFmRNA表达均低于手术各组(P<0.01);治疗组心肌酶及心肌梗死面积明显降低(P<0.05),梗死区血管生成数量稳定持续增加,与对照组有显著差异(P<0.01);心肌梗死后VEGFmRNA表达随缺血时间的延长(3,7,10d组)有增高趋势,治疗组明显升高(P<0.01),14d时VEGFmRNA的增长出现停止或下降。结论:严重缺血急性期可刺激心肌组织产生大量的VEGF起自我保护作用,人参皂苷Rg1增加其表达,并可刺激心肌梗死区的血管生成。     

聚乙二醇修饰人参皂苷Rg1与原人参三醇的制备及其在胃中稳定性

目的:对人参皂苷Rg1与原人参三醇进行聚乙二醇修饰和表征,并考察PEG修饰前后人参皂苷Rg1在胃中的稳定性,比较修饰前后的变化。方法:选择单甲氧基聚乙二醇2000为原料,经过与丁二酸酐的催化酯化反应活化为单甲氧基聚乙二醇2000-琥珀酸单酯后,再分别与人参皂苷Rg1和原人参三醇分子耦联,并采用核磁共振等方法来表征合成产物;以大鼠为实验动物,采用UPLC方法分析样品,分别考察人参皂苷Rg1与PEG-Rg1在离体胃中的稳定性情况,并对修饰前后的变化进行比较。结果:成功合成了原人参三醇与人参皂苷Rg1的聚乙二醇修饰产物,产率分别为16.2%和17.5%;人参皂苷Rg1在胃中容易降解,2 h时降解73.2%,PEG-Rg1在2 h时仅降解18.2%,稳定性提高。结论:成功合成了人参皂苷Rg1与原人参三醇的聚乙二醇修饰产物。修饰之后可以大大提高人参皂苷Rg1在胃中的稳定性。     

高效液相色谱法测定调经养颜片中人参皂苷Rg_1的含量

目的:研究调经养颜片(黄芪、女贞子等)中人参皂苷Rg1的含量测定方法。方法:采用高效液相色谱法测定人参皂苷Rg1的含量。结果:人参皂苷Rg1的平均加样回收率为97.89%,RSD为1.1(n=6),在0.4μg~10μg范围内,有良好线性关系(r=0.9991)。结论:该方法简便、灵敏、重复性好,可作为调经养颜片中人参皂苷Rg1的含量测定方法。