红景天苷对H2O2和高糖诱导损伤的血管内皮细胞保护作用研究

目的:探讨红景天苷对H2O2和高糖诱导人脐静脉内皮细胞(HUVECs)损伤的保护作用。方法:体外培养人脐静脉内皮细胞,分别以H2O2和高糖诱导内皮细胞损伤,分别给予10-6、10-5、10-4mol/L的红景天苷处理,MTT法测定细胞活力,检测LDH、MDA释放量和SOD活性。结果:结果显示红景天苷10-6、10-5、10-4mol/L组对正常人脐静脉内皮细胞皆无损伤作用;红景天苷10-5、10-4mol/L组对H2O2损伤模型的细胞活力有显著改善(P<0.05或P<0.01),红景天苷10-6、10-5、10-4mol/L组对高糖损伤模型的细胞活力均有显著改善(P<0.01),并且红景天苷10-5、10-4mol/L组能显著降低H2O2和高糖损伤组培养液中LDH和MDA释放量,升高细胞内SOD活性(P<0.05或P<0.01)。结论:红景天苷对H2O2和高糖诱导的人脐静脉内皮细胞损伤有明显的保护作用。     

Arnebin-1 promotes the angiogenesis of human umbilical vein endothelial cells and accelerates the wound healing process in diabetic rats

Ethnopharmacological relevance

Zicao is a traditional wound healing herbal medicine that has been used for several hundred years in China. A survey of the published literatures revealed that arnebin-1, one of the naphthoquinone derivatives, played the most important role in wound healing property of this plant. However, whether arnebin-1 affects angiogenesis in vitro and has an effect on wound healing process in diabetic rats remains enigmatic. To investigate the effect of arnebin-1 with or without VEGF on proliferation, migration and tube formation of HUVECs in vitro and the effect of its topical application in the form of ointment on wound healing in a cutaneous punch wound model of alloxan-induced diabetic rats in vivo.

Materials and methods

The pro-angiogenic functions of arnebin-1 on HUVECs including proliferation, migration and angiogenesis were evaluated through MTT assay, wound healing assay, transwell assay and tube formation assay in vitro. Male Sprague-Dawley rats were injected intraperitoneally with alloxan to induce type ? diabetic rats. Three wounds were created in each rat on the dorsal surface, and then divided to be basement treated, arnebin-1 ointment treated and untreated group correspondingly. The indicators including wound closure rate and histological evaluation were investigated on day 4 and 7 post-wounding.

Results

Without VEGF, arnebin-1 did not affect the proliferation of HUVECs significantly, but had a positive effect on cell migration and tube formation. However, in the presence of minimal VEGF, Arnebin-1 could increase the proliferation, enhance the migration and promote the tube formation of HUVECs significantly. The wound closure rate was increased significantly in arnebin-1 treated group compared to that of untreated and basement treated groups in diabetic rats, and the histological evaluation also showed well organized dermal layer, reduced number of macrophages, increased number of fibroblasts, remarkable degree of neovascularization and epithelization in arnebin-1 treated group.

Conclusion

These findings suggest that arnebin-1 has a pro-angiogenic effect, and a synergetic effect with VEGF promotes the wound healing process in diabetic rats.     

姜黄素对肿瘤坏死因子-α所致人脐静脉内皮细胞凋亡的影响

目的观察姜黄素对肿瘤坏死因子-α(TNF-α)诱导的人脐静脉内皮细胞(HUVEC)损伤的保护作用。方法建立TNF-α致HUVEC损伤模型;MTT检测细胞的存活率;光镜观察细胞形态的改变;流式细胞术检测细胞凋亡峰;Hoechst 33258染色荧光显微镜检测凋亡的细胞核;RT-PCR检测caspase-3基因的表达。结果姜黄素处理组与TNF-α组比较其HUVEC贴壁良好;荧光显微镜下可见凋亡小体减少;流式细胞术检测凋亡峰明显降低;caspase-3基因表达下调。结论姜黄素对TNF-α所致的HUVEC凋亡具有保护作用,该作用可能是通过下调caspase-3的基因表达而实现的。     

Sinomenine suppresses TNF-alpha-induced VCAM-1 expression in human umbilical vein endothelial cells

Sinomenine (SN), an alkaloid prepared from the root of Sinomenium acutum Rehd. Et wils, is used to alleviate the symptoms of rheumatism in Chinese medicine. In the present study, the potential inhibition of TNF-alpha-induced VCAM-1 expression on human umbilical vein endothelial cells (HUVECs) was evaluated in vitro. HUVECs were isolated from freshly collected umbilical cords. Positive controls were stimulated with TNF-alpha, omitting SN. Negative controls were cultured omitting TNF-alpha and SN. Experimental groups were co-cultured with TNF-alpha and SN at different concentrations (0.25, 0.5, and 1.0 mol/L), or TNF-alpha and Dexamethasone (Dex) at a concentration of 1.0 x 10(-6) mol/L. Cells were harvested after culturing with the above drugs for 12 h. VCAM-1 mRNA expression was detected by real-time quantitative PCR, and VCAM-1 expression was detected by flow cytometry. The experimental data indicated that VCAM-1 mRNA and VCAM-1 were induced by TNF-alpha. The relative VCAM-1 mRNA expression decreased in the experimental groups (p<0.05). Concentrations of SN at 0.5 and 1.0 mol/L inhibited expression of VCAM-1 (p<0.05). SN at concentration of 0.25 mol/L and Dex at concentration of 1.0 x 10(-6) mol/L did not show an inhibitory effect on VCAM-1 expression in TNF-alpha-induced HUVECs. Our preliminary data indicates that SN has an inhibitory effect in vitro on TNF-alpha-induced VCAM-1 expression at both mRNA level and protein level in HUVECs, and suggests that SN may be a novel method of immunotherapy for rheumatic carditis or rheumatic heart disease.     

三七总皂苷配合双联抗血小板药物对人脐静脉内皮细胞损伤和内皮血小板黏附的影响

目的:探究三七总皂苷配合双联抗血小板药物对人脐静脉内皮细胞损伤和内皮血小板黏附的影响。方法:对人脐静脉细胞进行培养,培养后分为7组,向空白组加入生理盐水,模型组加入80mg/kg的氧化低密度脂蛋白,双联抗血小板药物(简称:双抗组)加入80mg/kg氧化低密度脂蛋白+10mg/kg氯吡格雷+15mg/kg阿司匹林,三七总皂苷组加入80mg/kg氧化低密度脂蛋白+三七总皂苷终浓度100mg/kg,联合组分别加入80mg/kg氧化低密度脂蛋白+10mg/kg氯吡格雷+15mg/kg阿司匹林+三七总皂苷终浓度50、100、200mg/kg。作用48h后采用流式细胞仪检测7组细胞凋亡率以及内皮血小板黏附情况,检测7组细胞乳酸脱氢酶(LDH)、细胞间黏附分子(ICAM)、一氧化氮(NO)水平,并采用Western blot法检测P38MAPK、p-P38MAPK、ERK-1/2、p-ERK-1/2蛋白含量。结果:与空白组比较,模型组细胞凋亡率、LDH、ICAM,CD61平均荧光强度以及p-P38MAPK、p-ERK-1/2蛋白表达均明显为高,NO水平均明显为低;与模型组比较,双抗组、三七总皂苷组、50mg/kg联合组、100mg/kg联合组100mg/kg及200mg/kg联合组细胞凋亡率、LDH、ICAM,CD61平均荧光强度以及p-P38MAPK、p-ERK-1/2蛋白表达均明显降低,NO水平均明显升高;其中200mg/kg联合组细胞凋亡率、LDH、ICAM,CD61平均荧光强度以及p-P38MAPK、p-ERK-1/2蛋白表达明显高于其他各组,NO水平明显低于其他各组。结论:200mg/kg三七总皂苷配合双联抗血小板药物对人脐静脉内皮细胞具有保护作用,且可改善内皮血小板黏附情况,其机制可能与内皮细胞MAPA通路p-P38MAPK、p-ERK-1/2蛋白含量有关。     

阿魏酸对人脐静脉血管内皮细胞ECV304的增殖作用

目的：观察阿魏酸对于培养人脐静脉内皮细胞ECV304增殖的影响,为治疗性血管新生的靶基因选择提供依据。方法：取对数生长期的内皮细胞,分为阿魏酸低、中、高浓度组,同步化于G0期,ELISA法检测BrdU的掺入;RT-PCR法检测血管内皮细胞生长因子（VEGF）mRNA的表达情况。结果：与对照组相比,不同浓度阿魏酸均可显著提高BrdU的掺入量,上调VEGFmRNA的表达,且在10^2ng/ml～10^4ng/ml之间呈一定的量效关系。结论：阿魏酸能显著促进体外培养的ECV304细胞的增殖,可能具有血管新生的作用。     

蛇床子素对高糖诱导人脐静脉血管内皮细胞损伤的保护作用

目的研究蛇床子素对高糖诱导原代培养人脐静脉血管内皮细胞(HUVEC)炎症的保护作用。方法将HUVEC细胞分为对照组、高糖组、对照+蛇床子素组和高糖+蛇床子素组。培养5 d后,采用硝酸还原酶法测定细胞上清液中一氧化氮(NO),荧光酶标仪测定细胞内活性氧(ROS),并通过real-time PCR和Western blot法检测趋化因子CCL5及其受体CCR5 mRNA和蛋白的表达。结果高糖明显降低对照组细胞上清液中NO水平同时升高ROS,蛇床子素能增加高糖组细胞上清液中NO水平同时降低高糖组细胞内ROS;real-time PCR和Western blot结果显示蛇床子素可以降低高糖组细胞CCL5及其受体CCR5 mRNA和蛋白的表达量。结论蛇床子素对高糖诱导的人脐静脉血管内皮细胞的损害具有保护作用是与其抗氧化应激及抗炎作用有关。     

淫羊藿苷对高糖损伤人脐静脉内皮细胞的保护作用及机制研究

目的:探讨淫羊藿苷对抗体外高浓度葡萄糖诱导人脐静脉内皮细胞的损伤及其机制。方法人脐静脉内皮细胞株分别培养在含5.5mmol/L葡萄糖(正常对照组)、5.5mmol/L葡萄糖+24.5mmol/L甘露醇(高渗对照组)、30mmol/L葡萄糖(高糖损伤组)、30mmol/L葡萄糖+10μmol/L淫羊藿苷(低剂量淫羊藿苷组)和30mmol/L葡萄糖+100μmol/L淫羊藿苷(高剂量淫羊藿苷组)的培养基中培养48h,分别进行细胞活力、细胞培养上清液乳酸脱氢酶及一氧化氮含量、细胞丙二醛含量及谷胱甘肽-过氧化物酶活力的测定。结果:高糖损伤组及低、高剂量淫羊藿苷组的细胞活力、乳酸脱氢酶、一氧化氮、丙二醛含量及谷胱甘肽-过氧化物酶活力与正常对照组比差异均有统计学意义(P<0.01);低、高剂量淫羊藿苷组的细胞活力、乳酸脱氢酶、一氧化氮、丙二醛含量及谷胱甘肽-过氧化物酶活力与高糖损伤组比差异均有统计学意义(P<0.01)。高渗对照组各项指标与正常对照组相比无显著性差异。结论淫羊藿苷对体外高糖损伤的内皮细胞具有保护作用,其作用机制可能通过提高内皮细胞抗氧化酶活力,维持NO正常水平;减少细胞膜脂质过氧化损伤,维持膜完整性而实现的。     

天香丹对人脐静脉内皮细胞氧化损伤的保护作用研究

目的:探讨天香丹对tBHP诱导损伤的人脐静脉内皮细胞的保护作用。方法:将HUVECs分为对照组、模型组、阳性对照药(维生素C)组和天香丹剂量组(30、60、90μg/mL)。MTT法筛选tBHP最佳造模浓度,并评价天香丹对内皮细胞氧化损伤的保护作用;微板法和WST法检测各组细胞乳酸脱氢酶(LDH)、还原型谷胱甘肽(GSH)和超氧化物歧化酶(SOD)表达情况;显微镜下观察各组细胞形态学变化,Hoechst 33258荧光染色法检测各组细胞凋亡情况。结果:经tBHP诱导24 h后,模型组细胞存活率、SOD活性和GSH含量较对照组显著降低(P<0.01),LDH释放量和细胞凋亡率明显增加(P<0.01);经维生素C和不同浓度天香丹预处理后,与模型组相比较,各组细胞存活率、SOD活性和GSH含量显著增加,LDH的释放和细胞凋亡率得到减轻(P<0.01)。结论:天香丹具有保护HUVECs免于氧化损伤的作用,其机制可能与抗氧化通路的激活,抗氧化能力的增强及逆转细胞凋亡有关。     

马里苷对高糖损伤人脐静脉内皮细胞的作用

目的观察两色金鸡菊黄酮类化合物马里苷对高糖损伤人脐静脉内皮细胞(HUVEC)的作用。方法取对数生长期的细胞,分为正常对照组(NG)、高糖模型组(HG)及马里苷不同浓度(50、100、200、400μmol/L)给药组。采用CCK-8法检测细胞活力;ELASA法检测MDA含量及SOD、CAT、GSH-PX活力; Western blot法检测炎性因子VEGF、ICAM-1、MCP-1在HUVEC细胞中的蛋白表达。结果与NG组比较,及HG组细胞活力明显降低(P0.05); MDA含量显著升高(P0.05),SOD、CAT、GSH-PX活性降低(P0.05);与HG组(50 mmol/L)比较,马里苷药物干预组(50、100、200、400μmol/L)细胞活性明显增加,差异有统计学意义(P0.05),且随浓度增加细胞活性明显增加。Western blot蛋白印迹结果显示,与NG组比较,HG组VEGF、ICAM-1、MCP-1蛋白表达明显增加,差异有统计学意义(P0.05);与HG组比较,马里苷干预组VEGF、ICAM-1、MCP-1蛋白表达显著降低,差异有统计学意义(P0.05)。结论马里苷对高糖损伤的HUVEC细胞具有一定保护作用。     

Kaempferia parviflora ethanolic extract promoted nitric oxide production in human umbilical vein endothelial cells

The rhizomes of Kaempferia parviflora (KP) (Zingiberaceae) have been used in Thai traditional medicine for health promotion and for the treatment of digestive disorders and gastric ulcer. This study investigated effect of KP on endothelial function. Studies in human umbilical vein endothelial cells (HUVEC) showed that KP dose-dependently increased nitrite concentrations in culture media after 48 h incubation. eNOS mRNA and protein expression were also enhanced. The induction of eNOS mRNA was detected at 4 h and plateau at 48 h while iNOS expression was not observed. These data demonstrate that KP has a great potential for a supplemental use in vascular endothelial health promotion.     

Buddleja officinalis inhibits high glucose-induced matrix metalloproteinase activity in human umbilical vein endothelial cells

The aim of the present investigation was to investigate whether an aqueous extract of Buddleja officinalis (ABO), a traditional Korean herbal medicine, suppresses the endothelial extracellular matrix degradation under high glucose condition. The incubation with high concentration of glucose (25 mM) increased significantly matrix metalloproteinase (MMP)-2/-9 expressions and activities in primary cultured human umbilical vein endothelial cells (HUVEC). Pretreatment with ABO decreased high glucose-induced increase of MMP-2/-9 activities in a dose-dependent manner. Real time qRT-PCR revealed that high glucose-induced MMP-2/-9 mRNA expression levels were attenuated by pretreatment with ABO. High glucose-induced MCP-1 and IL-8 mRNA expression levels also decreased by ABO. ABO decreased high glucose-induced hydrogen peroxide production, oxidative stress marker. These results provide new insights into the pathophysiological mechanisms for anti-inflammatory properties of ABO in vascular diseases associated with diabetes mellitus.     

夏枯草抑制人脐静脉血管内皮细胞增殖的实验研究

目的探讨夏枯草（Spica Prunellae）对体外培养的人脐静脉血管内皮细胞（HUVEC）增殖的抑制作用，并从增殖细胞核抗原（PCNA）方面研究其抑制HUVEC增殖的作用机制。方法采用HUVEC为材料．取生长良好的HUVEC用于实验，利用100umol／L二氯化钴（CoCl2）诱导HUVEC增殖，同时分别将10mg／ml、20mg／ml、40mg／ml的夏枯草作用于增殖状态下的HUVEC24小时，进行以下的研究：①CCK-8法检测不同浓度夏枯草对HUVEC增殖的抑制程度；②流式细胞仪检测夏枯草对HUVEC核内PCNA蛋白表达的影响。结果①夏枯草明显抑制CoCl2诱导的HUVEC增殖，并且有剂量依赖关系。②夏枯草能够显著降低HUVEC核内PCNA蛋白表达，从而抑制细胞增殖，并具有剂量依赖关系。结论夏枯草能够明显抑制HUVEC的增殖。     

康力欣胶囊对人脐静脉血管内皮细胞周期和凋亡的影响

目的研究抗肿瘤药康力欣胶囊对人脐静脉血管内皮HUVEC细胞增殖、细胞周期和细胞凋亡的影响。方法以康力欣胶囊接种于细胞12、24、36、48 h后,采用四甲基偶氮唑盐(MTT)法测定细胞抑制率,采用流式细胞仪检测细胞周期各时相比率的变化,荧光显微镜观察法检测各组细胞凋亡的形态变化。结果 MTT检测结果显示康力欣胶囊能够明显抑制HUVEC的增殖,其抑制率与药物浓度呈正相关;细胞周期检测结果显示,康力欣胶囊将HUVEC细胞周期阻滞在S期;荧光显微镜观察结果显示,康力欣胶囊作用HUVEC后出现大量凋亡细胞。结论康力欣胶囊对HUVEC有明显的抑制作用,该作用可以通过阻滞细胞周期和诱导细胞凋亡实现。     

Z-藁本内酯对人脐静脉内皮细胞保护作用及其机制研究

目的 通过观察Z-藁本内酯对肿瘤坏死因子-α(TNF-α)诱导的人脐静脉内皮细胞(HUVEC)细胞间黏附分子-1(ICAM-1)、血管细胞黏附分子-1(VCAM-1)及核因子-κB (NF-κB)表达的影响,探讨其对炎性因子损伤内皮细胞的保护作用及其机制.方法 向体外培养的HUVEC中加入TNF-α10 ng/mL造成细胞损伤,同时加入不同浓度(5、10、20 μmol/L)的Z-藁本内酯,共同孵育24 h,MTT法检测各组细胞活力,ELISA法测定细胞培养液中 ICAM-1和VCAM-1的量,Westernblotting法检测各组细胞NF-κB蛋白表达.结果 与对照组比较,TNF-α可造成HUVEC明显损伤(P＜0.05、0.01),显著增加HUVEC中ICAM-1和VCAM-1的表达(P＜0.01).与模型组比较,Z-藁本内酯可以剂量相关地降低ICAM-1 (P＜0.01)和VCAM-1 (P＜0.05)的表达,显著减少细胞NF-κB蛋白 的表达(P＜0.05).结论 Z-藁本内酯可减轻TNF-α对HUVEC的损伤,其作用机制可能与通过抑制NF-κB的激活,进而减少ICAM-1和VCAM-1的表达有关.     

Chrysanthemum morifolium Ramat. reduces the oxidized LDL-induced expression of intercellular adhesion molecule-1 and E-selectin in human umbilical vein endothelial cells

Ethnopharmacological relevance

The flower of Chrysanthemum morifolium Ramat. (CM) with antioxidant, cardiovascular protective and anti-inflammatory functions, has been widely used in China for hundreds of years as a healthy beverage and medicine.

Aims of the study

The purpose of the present study is to investigate the effects of HCM (a hot water extract of the flower of Chrysanthemum morifolium Ramat. [CM]), ECM (an ethanol extract of CM), and the abundant flavonoids apigenin and luteolin in CM on the oxidized LDL (oxLDL)-induced expression of ICAM-1 and E-selectin in human umbilical vein endothelial cells (HUVECs). The possible mechanism of these effects was also determined.

Materials and methods

MTT assay was for cell viability. Western blot was used for ICAM-1 and E-selection protein expression, and for activation of protein kinase B (PKB) and cAMP responsive element binding protein (CREB) proteins. Fluorescence flow cytometry was for ICAM-1 and E-selectin expression on cell surface. DCF-DA flow cytometric assay was used for reactive oxygen species (ROS) production.

Results

HCM, ECM, apigenin, and luteolin dose-dependently inhibited ICAM-1 and E-selectin expression and adhesion of HL-60 by oxLDL. HCM, ECM, apigenin, and luteolin reversed the inhibition of phosphorylation of Akt and CREB by oxLDL; however, this reversion was abolished by wortmannin. In addition, wortmannin abrogated the inhibitory effects of CM extracts, apigenin and luteolin on adhesion molecule expression. The ROS scavenging capability of HCM, ECM, apigenin, and luteolin proceeded dose-dependently in the presence of oxLDL.

Conclusion

CM is a plant with cardiovascular-protective potential and the inhibitory effects of CM on ICAM-1 and E-selectin expression are, at least partially, attributed to its antioxidant activity and modulation of the PI3K/Akt signaling pathway.     

蛇床子素通过Akt/eNOS降低氧化低密度脂蛋白诱导血管内皮细胞凋亡

目的:探讨蛇床子素对低密度脂蛋白诱导的人脐静脉血管内皮细胞(HUVEC)凋亡的影响及其机制研究。方法:采用HUVEC细胞,设置正常组、模型组和给药组,细胞培养12 h后,低密度脂蛋白进行细胞损伤,同时给予不同浓度蛇床子素(10、20、40μmol/L)进行干预,应用MTT法检测细胞活力;Annexin V-FITC标记的流式细胞术检测细胞凋亡;检测一氧化氮生成量;免疫印迹法检测p-AKt和p-eN OS蛋白表达水平。结果:与正常组相比,模型组细胞活力和凋亡率有显著差异;与模型组相比,蛇床子素提高细胞活力,抑制细胞细胞凋亡,作用效果呈剂量依赖性,20、40μmol/L蛇床子素均有显著性差异;与模型组相比,20、40μmol/L蛇床子素显著促进细胞一氧化氮合成;蛋白质检测结果显示,与模型组相比,蛇床子素可上调HUVEC细胞中Akt和eN OS蛋白磷酸化水平。结论:蛇床子素能够抑制ox-LDL诱导HUVEC的凋亡而发挥其保护作用,其作用机制可能与其激活Akt/eNOS/NO的信号通路相关。     

活血注射液对ox-LDL诱导人脐静脉内皮细胞活化的影响

目的：观察活血注射液对氧化型低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞(HUVEC) 的细胞间黏附分子-1(ICAM-1)、血管细胞黏附分子-1(VCAM-1)的表达及与人单核细胞黏附作用的影响，并探讨核转录因子kappa B(NF-κB) 在其中的调控作用。方法：HUVEC与单核细胞的黏附率用蛋白定量法检测；ICAM-1，VCAM-1 mRNA和蛋白表达分别用RT-PCR技术和流式细胞仪检测；NF-κB p65的阳性细胞百分率和细胞核/浆染色的灰度比值用免疫细胞化学法测定。结果：ox-LDL作用HUVEC后12,24 h时，人单核细胞与HUVEC的黏附率显著升高, HUVEC中ICAM-1和VCAM-1的mRNA和蛋白表达水平也均明显升高, HUVEC的NF-κB p65的阳性细胞百分率和细胞核/浆染色的灰度比值明显增高，明显高于正常组(P<0.05，P<0.01)，而活血注射液可明显降低人单核细胞与HUVEC的黏附率和HUVEC ICAM-1，VCAM-1 mRNA和蛋白表达水平，以及显著降低HUVEC中NF-κB p65的阳性细胞百分率和细胞核/浆染色的灰度比值，与ox-LDL组相比均有显著性差异(P<0.05，P<0.01)，这种作用随着剂量的增加而增强。结论：活血注射液能通过下调内皮细胞表面ICAM-1，VCAM-1 mRNA和蛋白表达抑制单核-血管内皮细胞黏附,其可能机制是其通过抑制内皮细胞NF-κB p65的活性，从而发挥对血管内皮细胞的保护作用。