Correlating endothelin releasing function of vascular endothelial cells to tea polyphenols and
angiotensin Ⅱ*
Liu Yu1, Sun Yun-feng2, Ma Gui-xi1, Li Jing1, Meng Guo1, Han Lei1, Liu Xin1, Li Ming-gao1
Abstract
BACKGROUND: Angiotensin Ⅱ (Ang-Ⅱ) can stimulate vascular endothelial cells to excrete endothelin, a kind of potent vasoconstrictor. The content of endothelin in blood or cell culture media directly reflects the function and injured status of vascular endothelial cells. Therefore, it is significant for strengthening vascular endothelial cells to resist the injured factors. Tea polyphenols is a mainly active component of tea, and it is considered as a reagent for anti-atherosclerosis, protecting injuries of vascular endothelial cells and preventing cardiovascular diseases.
OBJECTIVE: To observe the effects of tea polyphenols in different concentrations on endothelin content in vascular endothelial cells induced by Ang-Ⅱ at various time points through establishing Ang-Ⅱ-induced vascular endothelial cell injury models and further to investigate the protective effect of tea polyphenols on vascular endothelial cells.
DESIGN: Observational study.
SETTING: Aerospace and Diving Medical Center, Navy General Hospital of Chinese PLA.
MATERIALS: The experiment was carried out in Laboratory of Aerospace and Diving Medical Center, Navy General Hospital of Chinese PLA from March to September 2005. Main materials were detailed as follows: Ang-Ⅱ (Sigma Company), tea polyphenols (Department of Tea Science, Zhejiang University) and vascular endothelial cells (human large artery vascular endothelial cell system, CBI Company, USA).
METHODS: Cultured vascular endothelial cells were divided into 4 groups: ① Control group: The normal culture media was added in the isopyknic vascular endothelial cells, and 100 μL supernatant was extracted before and at 0.5, 6 and 24 hours after filling moisturized liquid. ② Ang-Ⅱ group: Cell culture media containing 10-7 mol/L Ang-Ⅱ was added in the vascular endothelial cells, and other operations were as the same as those in the control group. ③ High-concentration tea polyphenols + Ang-Ⅱ group: Cell culture media containing 50 mg/L tea polyphenols was added in the vascular endothelial cells, and other operations were as the same as those in the Ang-Ⅱ group. ④ Low-concentration tea polyphenols + Ang-Ⅱ group: Cell culture media containing 25 mg/L tea polyphenols. 100 μL supernatant was extracted before and after 0.5, 6 and 24 hours treatment in each group. Thereafter, radioimmunoassay was used to measure the content of endothelin.
MAIN OUTCOME MEASURES: Content of endothelin.
RESULTS: ① Content of endothelin in Ang-Ⅱ group was higher than that in the control group (P < 0.01). ② At 6 and 24 hours after high-concentration tea polyphenols incubation, content of endothelin in high-concentration tea polyphenols + Ang-Ⅱ group was lower than that in Ang-Ⅱ group (P < 0.01). Moreover, the content of endothelin in low-concentration tea polyphenols + Ang-Ⅱ group was lower than that in both high-concentration tea polyphenols + Ang-Ⅱ group and angiotensin Ⅱ group (P < 0.01).
CONCLUSION: Tea polyphenols has inhibitory effects on endothelin releasing function of vascular endothelial cells induced by Ang-Ⅱ, suggesting that tea polyphenols has protective effect on vascular endothelial cells, and the effect of low-concentration tea polyphenols is superior to that of the high-concentration one.
INTRODUCTION
Vascular endothelial cell is not only a pute vascular luminal liner, but also it can excrete endothelin and angiotensin Ⅱ (Ang-Ⅱ). Both of them are potent vasoconstrictors in organism[1] and play important role in maintaining normal function of vessels and arteriosclerosis[2]. Endothelin has positive feedback regulatory mechanism with Ang-Ⅱ for enhancing activities each other[3]. Ang-Ⅱ can stimulate vascular endothelial cells to excrete endothelin, the content of endothelin in blood or cell culture media directly reflects the function and injured status of vascular endothelial cells. Therefore, it is significant for strengthening vascular endothelial cells to resist the injured factors. Tea polyphenols is a mainly active component of tea, and it is considered as anti-oxidation, anti-mutagenesis, neuroprotective effect, inhibitory effect on neoplasm onset, reducing lipid and protective effect on cardiovascular system. This study was designed to observe effects of tea polyphenols in different concentrations on endothelin release in vascular endothelial cells induced by angiotensin Ⅱ at various time points through establishing angiotensin Ⅱ-induced vascular endothelial cell injury models and investigate the protective effect of tea polyphenols on vascular endothelial cells.
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MATERIALS AND METHODS
Main reagents and equpiments
Main reagents and equipments were detailed as follows: Ang-Ⅱ (Sigma Company); tea polyphenols (Department of Tea Science, Zhejiang University); vascular endothelial cells (human large artery vascular endothelial cell system, CBI Company, USA); culture media and growth factors of vascular endothelial cells (CBI Company); endothelin kit (Huaying Immune Technology Institute); cell incubator (Asheville, N.C Company, USA); 24-well cell culture plate (NUNC Company, Denmark); depurative operating table (Beijing Guanpeng Depurative Instrument Limited-liability Company).
Culture and generation of vascular endothelial cells
Cells were given routine resuscitation, inoculated in a 75 cm2 culture bottle after regulating an appropriate concentration and cultured in an incubator with 0.05 volume fraction of CO2 and saturation humidity at 37 ℃. And then, the culture media was changed every two days. When cells fully covered the bottom of bottle, they were digested and generated with pancreatin. In the third passages, cells were cultured in 24-well plate with the inoculating density of 5 ×103/cm2. After the growth confluence, the cells were applied in the interventional experiment.
Grouping and processing
Cultured vascular endothelial cells were divided into 4 groups, including control group, Ang-Ⅱ group, high-concentration tea polyphenols + Ang-Ⅱ group and low-concentration tea polyphenols + Ang-Ⅱ group, with 8 exponents for each. ① Control group: The normal culture media was added in the isopyknic vascular endothelial cells, and 100 μL supernatant was extracted before and at 0.5, 6 and 24 hours after filling moisturized liquid. ② Ang-Ⅱ group: Cell culture media containing 10-7 mol/L Ang-Ⅱ was added in the vascular endothelial cells, and other operations were as the same as those in the control group. ③ High-concentration tea polyphenols + Ang-Ⅱ group: Cell culture media containing 50 mg/L tea polyphenols was added in the vascular endothelial cells, and other operations were as the same as those in the Ang-Ⅱ group. ④ Low-concentration tea polyphenols + Ang-Ⅱ group: Cell culture media containing 25 mg/L tea polyphenols. 100 μL supernatant was extracted before and after 0.5, 6 and 24 hours treatment in each group. Thereafter, radioimmunoassay was used to measure the content of endothelin. All above-mentioned exponents were maintained in aprotinin-treated centrifuge tube and frozen at -20℃. (Note: On the experimental day, the general concentration of tea polyphenols was 25-40 mg/L. In this study, 25 mg/L was regarded as the low concentration, while 50 mg/L as the higher one.)
Measurement of endothelin
Radioimmunoassay (Huaying Technology Institute) was used to measure the concentration of endothelin in supernatant before and after 0.5, 6 and 24 hours Ang-Ⅱ or tea polyphenols incubation.
Statistical analysis
SPSS software was used by the sixth author in this study. Data were expressed as Mean ± SD. Mean at each time point was compared with one-way analysis of variance, and differences among groups were compared with LSD t test.
RESULTS
Effect of Ang-Ⅱ on endothelin release in vascular endothelial cells
There were no significant differences in content of endothelin in supernatant at various time points before and after filling moisturized liquid in the control group (P > 0.05). The concentration of endothelin in Ang-Ⅱ group was higher than that of control group (P < 0.01, Table 1).
Interventional effect of tea polyphenols on Ang-Ⅱ-induced endothelin release in vascular endothelial cells
At 0.5 hour after culture, there was no significant difference in the content of endothelin between high-concentration tea polyphenols + Ang-Ⅱ group and Ang-Ⅱ group (P > 0.05). However, the content of endothelin was decreased at 6 and 24 hours after high-concentration tea polyphenols incubation, and there was significant difference (P < 0.01). Moreover, the content of endothelin in low-concentration tea polyphenols + Ang-Ⅱ group was significantly different from that in the Ang-Ⅱ group (P < 0.01). This result suggested that tea polyphenols could notably decrease Ang-Ⅱ-induced endothelin release and protect Ang-Ⅱ-induced vascular endothelial cell injury (Table 1).
Comparison of interventional effect of tea polyphenols in various concentrations
After Ang-Ⅱ incubatiuon 0.5 hour, tea polyphenols in a high concentration did not notably decrease the release of endothelin (P > 0.05). However, at 6 and 24 hours, tea polyphenols in a high concentration could protect vascular endothelial cells (P < 0.01). At 0.5, 6 and 24 hours after the incubation, the content of endothelin in low-concentration tea polyphenols + Ang-Ⅱ group was lower than that of in high-concentration tea polyphenols + Ang-Ⅱ group (P < 0.01). These results suggested that the protective effect of low-concentration tea polyphenols was superior to that of the high-concentration one (Table 1).
DISCUSSION
Ang-Ⅱ is an important vascular bioactive peptide to constrict vessels by the direct effect of rennin-angiotensin system on vascular bed[4]. It can promote the proliferation and migration of smooth muscle cells and has pathological and physiological significance in hypertension, arteriosclerosis, cardiac insufficiency and other diseases[5]. Vascular endothelial cells not only play a natural barrier role, but also have metabolic and endocrine functions. Endothelin is a kind of potent vasoconstrictor[6]. Contents of Ang-Ⅱ and endothelin in the plasma are increased remarkably in patients with hypertension, nephrotic syndrome, cardiac insufficiency, angina pectoris and long-term hypoxic diseases[7-9]. Previous researches[10] have demonstrated that, among various indexes of coronary arterial endothelial injury, endothelin is remarkably positive correlation with Ang-Ⅱ of local heart, which suggests that coronary arterial endothelial injury has the correlation with Ang-Ⅱ surrounding vascular endothelial cells. A lot of scholars think that increase of Ang-Ⅱ is related to increase of endothelin each other.
Our results indicated that Ang-Ⅱ could remarkably increase the concentration of endothelin in vascular endothelial cells, especially at 6 hours after induction. In addition, the concentration was still higher than that in the control group at 24 hours after induction. This suggested that Ang-Ⅱ could enhance the endothelin release function of vascular endothelial cells for a long period. Ang-Ⅱ could enhance endothelin release function of vascular endothelial cells by: ① activating NF-κB, promoting vascular endothelial cells to express MCP-1, VCAM, ICAM and IL-6 and aggravating inflammatory reaction[11]; ② activating AT1 receptor, increasing the content of calcium ions and causing calcium overload[12]; ③ activating NADH/NADPH oxidase at the surface of cell membrane, generating puperoxide anion[13], changing oxidation-reduction status of vascular endothelial cells[14], inducing lipid peroxidation injury of cell membrane, enhancing and damaging membrane permeability so as to cause plentiful releasing of endothelin in blood or culture media. Under a normal condition, the concentration of Ang-Ⅱ in organism was higher than that in this study. Therefore, Ang-Ⅱ did not cause any damages on vascular endothelial cells due to the balance between vasoconstrictor factors (Ang-Ⅱ) and vasodilative factors (nitrogen monoxide). When the above-mentioned balance was damaged or in tendency to vasoconstrictor factors, Ang-Ⅱ might damage vascular endothelial cells so as to cause other related diseases.
Tea polyphenols is mainly active component of tea leaves and counts for 20%-35% of dry weight. Its main components contain epicatechin, epigallocatechin and epigallocatechin gallate. All the three materials have a strong anti-oxidative activity and also have great effects on anti-bacterium, anti-virus and anti-neoplasm[15, 16]. Some epidemiological studies indicate that tea consumption is negative correlation with risk factors of cardiovascular diseases[17]. Tea polyphenols has the effects on anti- atherosclerosis, anti-vascular endothelial cell injury and prevention of cardiovascular diseases[18-20].
Results in this study demonstrated that Ang-Ⅱ could remarkably increase the concentration of endothelin in vascular endothelial cells, but tea polyphenols could decrease the concentration in the Ang-Ⅱ group, especially the concentration of endothelin was the lowest in the low-concentration tea polyphenols + Ang-Ⅱ group. This suggested that tea polyphenols could obviously decrease the concentration of endothelin increased by Ang-Ⅱ; meanwhile, tea polyphenols had the protective effects on cell injury induced by Ang-Ⅱ. While the effect of low-concentration tea polyphenols was superior to that of the high-concentration one. The reasons were detailed as follows: ① Tea polyphenols in the high concentration could generate oxygen-derived free radials and polyphenolic semiquinone negative free radials so as to relieve the toxic reaction and clearance of free radials[21]. ② Tea polyphenols in a certain concentration could completely block the damaging effect of Ang-Ⅱ on vascular endothelial cells and reduce the endothelin releasing content of vascular endothelial cells caused by Ang-Ⅱ. However, tea polyphenols in a high concentration did not further reduce excretion of endothelin; therefore, tea polyphenols in a high concentration did not have the further protective effect. The protective effects of tea polyphenols were detailed as follows: ① Tea polyphenols could reduce calcium overload which was one of important factors to cause cell injury; ② Almost cell injuries were caused by lipid peroxidation injury induced by increase of free radials, while tea polyphenols could properly reduce the generation of free radials; ③ Tea polyphenols could increase the content of nitrogen monoxide in serum or culture media so as to activate the protective effects through improving metabolism of vascular endothelial cells.
Recently, protective effects of tea polyphenols involve in wide regions, especially in cardio-cerebrovascular diseases and neoplasm; however, detailed mechanisms need to be further studied in the future.
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血管内皮细胞内皮素分泌功能与茶多酚及血管紧张素Ⅱ的相关性*
刘 玉1，孙运峰2，马贵喜1，李 靖1，蒙 果1，韩 磊1，刘 昕1，李鸣皋1
1解放军海军总医院航空潜水医学中心，北京市 100037；2解放军济南军区青岛第二疗养院，山东省青岛市 266071
刘 玉，男，1966年生，山东省高密市人，汉族，1991年解放军第二军医大学毕业，副主任医师，主要从事航空医学研究。
海军后勤科研基金（00-3321）*
通讯作者：李鸣皋，解放军海军总医院航空潜水医学中心，北京市 100037
摘要
背景：内皮素是一种强效血管收缩物质，血管紧张素Ⅱ可以刺激血管内皮细胞分泌内皮素，通过检测血液或细胞培养液中的内皮素含量，可以间接反映血管内皮细胞的功能及损伤情况，因此，增强血管内皮细胞对损伤因素的抵抗作用意义重大。茶多酚是茶叶药效的主要活性成分，具有抗动脉粥样硬化、对抗血管内皮细胞损伤、预防心血管疾病等作用。
目的：通过建立血管紧张素Ⅱ诱导血管内皮细胞损伤模型，观察了血管紧张素Ⅱ作用不同时间后血管内皮细胞分泌内皮素含量的变化以及不同浓度的茶多酚对这种变化的影响，以探讨茶多酚对血管内皮细胞的保护作用。
设计：观察性实验。
单位：解放军海军总医院航空潜水医学中心。
材料：实验于2005-03/09在解放军海军总医院航空潜水医学中心实验室完成。主要材料：血管紧张素Ⅱ（Sigma公司）；茶多酚（浙江大学茶学系）；血管内皮细胞（美国CBI公司的人大动脉血管内皮细胞体系）。
方法：将培养的血管内皮细胞分为4个组：①对照组：不加特殊试剂，加入等体积的细胞培养液，分别在加液前和加液后0.5，6，24 h提取上清液100μL。②血管紧张素Ⅱ组：在细胞培养液中加入血管紧张素Ⅱ，使培养液中血管紧张素Ⅱ的终浓度为10-7 mol/L，余同对照组。③高浓度茶多酚+血管紧张素Ⅱ组：将茶多酚加入细胞培养液中，使培养液中茶多酚终浓度为50 mg/L，余同血管紧张素Ⅱ组。④低浓度茶多酚+血管紧张素Ⅱ组：加入茶多酚，使培养液中茶多酚终浓度为25 mg/L，余同血管紧张素Ⅱ组。采用放射免疫法测定血管紧张素Ⅱ及茶多酚作用前及作用后0.5，6，24 h各组细胞培养上清中的内皮素含量。
主要观察指标：内皮素含量。
结果：①血管紧张素Ⅱ组培养上清的内皮素含量显著高于对照组（P < 0.01）。②高浓度茶多酚在作用6 h和24 h 后，内皮素含量较血管紧张素Ⅱ组显著下降（P < 0.01）；而低浓度茶多酚在各作用时间点均较高浓度茶多酚组及血管紧张素Ⅱ组显著下降（P < 0.01）。
结论：茶多酚对血管紧张素Ⅱ所致血管内皮细胞分泌内皮素的功能具有抑制作用，提示茶多酚对血管内皮细胞具有保护作用；且低浓度茶多酚的保护作用要强于高浓度茶多酚。
关键词：茶多酚；血管紧张素Ⅱ；血管内皮细胞；内皮素
中图分类号: R329.412 文献标识码: A 文章编号: 1673-8225(2008)02-00381-04
刘玉，孙运峰，马贵喜，李靖，蒙果，韩磊，刘昕，李鸣皋. 血管内皮细胞内皮素分泌功能与茶多酚及血管紧张素Ⅱ的相关性 [J].中国组织工程研究与临床康复,2008,12(2):381-384
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(Edited by Yu XP/Ji H/Wang L)