乙醇诱导人脐静脉内皮细胞损伤及ADMA/NOS/NO信号机制

Damage of Human Umbilical Vein Endothelial Cells Induced by Ethanol and Whether Its Injuring Mechanism is Related to the Signal Pathway of ADMA/NOS/NO

目的探讨乙醇诱导人脐静脉内皮细胞损伤效应及ADMA/NOS/NO信号机制。方法用不同浓度的乙醇与人脐静脉内皮细胞共培养,观察细胞形态和活性,通过检测细胞上清液丙二醛（MDA）、非对称性二甲基精氨酸（AD-MA）、一氧化氮（NO）的含量和一氧化氮合酶（NOS）的活性等指标,观察乙醇对人脐静脉内皮细胞损伤的量-效关系（乙醇处理细胞24 h）和时-效关系（终浓度为100 mmol/L的乙醇分别于处理0、3、6、12、24、48 h后观察检测指标）。实验分为正常对照组（加入与处理组等体积的D-Hank’s液,乙醇浓度为0 mmol/L）、乙醇处理组（终浓度分别为25、50、100、200mmol/L的乙醇）、乙醇＆VitC组（乙醇终浓度为100 mmol/L,VitC终浓度为100 mg/L）和阳性对照组（终浓度为500μmol/L的过氧化氢）。结果50-200 mmol/L乙醇能显著改变细胞形态、降低细胞活性（OD值：0.91±0.10-0.58±0.04,细胞生长抑制率：0.00±0.00-35.57±3.13,P〈0.01）;能显著性诱导内皮细胞MDA升高（258.72±7.36-524.07±8.25,P〈0.01）。50-200 mmol/L的乙醇还能显著性诱导：内皮型一氧化氮合酶（eNOS）活性下降（6.27±0.10-0.47±0.23,P〈0.05）及总NOS活性下降（10.69±0.54-3.77±0.32,P〈0.05）;NO含量降低（191.61±7.96-88.38±5.07,P〈0.05）。而且上述生物学效应均具有显著的时间依赖性,各处理组间两两比较也具有显著性（P〈0.05或P〈0.01）。100 mmol/L乙醇处理时细胞诱导型一氧化氮合酶（iNOS）活性及ADMA含量最高,且分别在处理后24 h到达最高峰。VitC均能扭转上述生物学效应,且具有显著性（P〈0.01）。结论50-200 mmol/L的乙醇能显著诱导人脐静脉内皮细胞损伤;ADMA在乙醇诱导人脐静脉内皮细胞损伤中有着重要作用。

Objective To study the damage of human umbilical vein endothelial cells induced by ethanol and whether its injuring mechanism is related to the signal pathway of ADMA/NOS/NO. Methods HUVEOs - 12 was cultured in DMEM containing 10 % FOS. At 80% confluence, cells were washed with D Hank＇s fluid and cultured in DMEM containing 1% FOS for 24h. Cells were incubated in the presence or absence （control） of ethanol. To test the dose effect of ethanol- induced endothelial cells damage, the cells were treated with ethanol at the doses of 2.5, .50, 100, and 200mmol/L for 24h. And the time - effect was also tested by culturing cells with ethanol at the dose of 100mmol/L for 0, 3, 6, 12, 24, and 48 h respectively. To explore the injuring feature of ethanol on endothelial cells, hydrogen peroxide （500μmol/L）, a potent free radical, was selected as a positive control; vitamin O （ 100mg/L）, an anti - oxidant medicine, was selected as a protective group. The cell morphology and viability of HUVEOs- 12 （MTT assay） were observed to evaluate the injuring feature of ethanol. The levels of malonaldehyde （MDA）, asymmetric dimethylarginine （ADMA）, nitric oxide （NO）, and nitric oxide synthase （NOS） activity were detected to elucidate the injuring mechanism of ethanol. Results The cell morphology and viability were changed conspicuously by ethanol at the final concentration 50-200mmol/L （OD： 0.9] ± 0.10 - 0..58 ± 0.04, inhibition ratio of cells＇ growth ： 0.00 ± 0.00 - 35.57 ± 3. ] 3, P〈 0.0 1 ）. And with concentration up （50 - 200mmol/L）, the levels of MDA stepped up significantly versus vitamin C protective group （258.72 ± 7.36 - 524.07 ± 8.25, P〈 0.01 ）. The activity of eNOS and the ac- tivity of total NOS were descended （6.27 ± 0. ] 0 - 0.47 ± 0.23, P 〈 0.05 ;1 0.69 ± 0.54 - 3.77 ± 0.32, P 〈 0.05）. The synthesis of NO was also inhibited significantly （ 191.61 ± 7.96 -88.38 ± 5.07, P〈 0.05）. All the biological effect we have tested were time - related and the statistical significance was also conspicuous between every two ethanol treated groups （P 〈 0.05 or P〈 0.01 ）. The level of ADMA was the highest in 100mmol/L ethanol treated group and the peak value appeared after 24 h. Conclusions Ethanol at the concentration of 50 - 200mmol/L can induce significant damage on human umbilical vein endothelial cells, and ADMA plays an important role in this progress.