牛磺酸对低密度脂蛋白所致血管内皮功能障碍的保护作用与DDAH/ADMA途径的关系
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摘要
氧化型低密度脂蛋白(ox-LDL)是诱发血管内皮功能障碍的重要因素。L-精氨酸的同类物非对称二甲基精氨酸(ADMA)能够竞争性抑制一氧化氮合酶(NOS),阻止一氧化氮(NO)生成和诱导血管内皮功能障碍。本实验室前期研究发现,在体大鼠静脉单次给予低密度脂蛋白(LDL)能显著升高血浆ADMA水平,同时伴血管内皮依赖性舒张反应功能降低。在培养的内皮细胞,ox-LDL能诱导ADMA特异性水解酶二甲精氨酸二甲胺水解酶(DDAH)氧化失活,增加ADMA的累积。牛磺酸是含磺酸基的必需氨基酸,具有抗炎、抗氧化作用。最近研究证明,牛磺酸能保护血管内皮功能和抑制动脉粥样硬化形成,其作用机制尚未完全清楚。因此,本实验探究牛磺酸对LDL诱导的血管内皮损伤的保护作用与DDAH/ADMA途径的关系。
方法:实验分两部分,(1)动物实验:雄性SD大鼠在乙醚麻醉下,舌下静脉注射人血清LDL(4 mg/kg)诱发血管内皮功能损伤。大鼠在3%戊巴比妥钠麻醉下,颈动脉插管取血,观察离体胸主动脉环的内皮依赖性舒张反应,检测血中ADMA、NO含量,以及丙二醛(MDA)、肿瘤坏死因子α(TNF-α)的含量。(2)细胞实验:培养人脐带静脉血管内皮细胞(HUVECs),用ox-LDL孵育内皮细胞24 h.检测血管内皮细胞培养液中乳酸脱氢酶(LDH)、ADMA、TNF-α、MDA、NO含量和DDAH活性。
结果:(1)单次静注LDL(4 mg/kg)显著抑制乙酰胆碱(ACh)诱导的内皮依赖性舒张反应,增高血浆ADMA、MDA和TNF-α水平,降低NO水平。两个剂量牛磺酸(60或180 mg/kg)均能显著减轻LDL所致ACh诱导内皮依赖性舒张的损伤,而较大剂量牛磺酸作用较小剂量牛磺酸组作用为强;两个剂量牛磺酸均能抑制LDL所致ADMA、MDA和TNF-α浓度升高;(2)用ox-LDL(100μg/ml)孵育人脐带静脉内皮细胞(HUVECs)24 h,牛磺酸(1或5μg/ml)明显地抑制细胞培养液中LDH、ADMA、MDA和TNF-α水平的提高,而减少NO水平下降和DDAH活性降低。
结论:牛磺酸能显著改善LDL诱导的血管内皮功能障碍,其保护作用与抑制氧化应激和减少炎症因子释放,改善DDAH活性和降低ADMA水平有关。
Oxide low-density lipoprotein(ox-LDL)is an important factor contributing the development of atherogenesis.L-arginine congener compounds asymmetric dimethylarginine(ADMA),an endogenous inhibitor of nitric oxide synthase(NOS),inhibit nitric oxide(NO) production.Our previous work has showed that a single injection of low-density lipoprotein(LDL)significantly increases the serum level of endogenous ADMA concomitantly with endothelial dysfunction: Incubation human umbilical vein endothelial cells(HUVECs)with ox-LDL,dimethylarginine dimethylaminohydrolase(DDAH),a major hydrolase of ADMA were devitalization,and cause accumulation of ADMA.Taurine is a sulfur-containing semiessential amino acid,an antioxidant and anti-inflammatory drug.Recently it has been reported that taurine preserves endothelial function and inhibit atherogenesis. However,the mechanisms responsible for the beneficial effect of taurine on endothelial cells remain unclear.Based on the elevated level of ADMA by lipid peroxides and antioxidant properties of taurine,in the present study we tested the protective effect of taurine on damages of the endothelium induced by Low-density lipoprotein(LDL),and whether the protective effect of taurine is related to the DDAH/ADMA pathway.
METHODS:study were divided to two part.(1)Animal treatment: Blood samples were collected from carotid artery.Vasodilator responses to acetylcholine(Ach)in the isolated aortic rings were determined,and serum concentrations of asymmetric dimethylarginine(ADMA), malondialdehyde(MDA),tumour necrosis factor-α(TNF-α),Nitric oxide (NO).(2)Cell culture and treatment:Incubation human umbilical vein endothelial cells(HUVECs)with ox-LDL(100μg/ml)for 24h,the medium levels of lactate dehydrogenase(LDH),ADMA,TNF-α,NO, MDA and the intracellular activity of DDAH were measured.
RESULTS:(1)A single injection of LDL(4mg/kg)significantly decreased vasodilator responses to Ach,increased the serum level of ADMA,MDA and TNF-α,and decreased the serum level of NO.Taurine (60 or 180mg/kg)markedly reduced the inhibition of vasodilator responses to Ach by LDL,and the protective effect of taurine at the high dose was greater compared with the lower group.Taurine inhibited the increased level of ADMA,MDA and TNF-αinduced by LDL,and increased the serum level of NO.(2)Incubation human umbilical vein endothelial cells(HUVECs)with ox-LDL(100μg/ml)for 24h,Taurine (1 or 5μg/ml)markedly decreased the medium levels of LDH,ADMA, MDA and TNF-α,and increased the level of NO in the medium and the intracellular activity of DDAH.
CONCLUSIONS:taurine protects the endothelium against damages elicited by LDL in vivo and in culture,and the protective effect of taurine on endothelium is related to the DDAH/ADMA pathway.
方法:实验分两部分,(1)动物实验:雄性SD大鼠在乙醚麻醉下,舌下静脉注射人血清LDL(4 mg/kg)诱发血管内皮功能损伤。大鼠在3%戊巴比妥钠麻醉下,颈动脉插管取血,观察离体胸主动脉环的内皮依赖性舒张反应,检测血中ADMA、NO含量,以及丙二醛(MDA)、肿瘤坏死因子α(TNF-α)的含量。(2)细胞实验:培养人脐带静脉血管内皮细胞(HUVECs),用ox-LDL孵育内皮细胞24 h.检测血管内皮细胞培养液中乳酸脱氢酶(LDH)、ADMA、TNF-α、MDA、NO含量和DDAH活性。
结果:(1)单次静注LDL(4 mg/kg)显著抑制乙酰胆碱(ACh)诱导的内皮依赖性舒张反应,增高血浆ADMA、MDA和TNF-α水平,降低NO水平。两个剂量牛磺酸(60或180 mg/kg)均能显著减轻LDL所致ACh诱导内皮依赖性舒张的损伤,而较大剂量牛磺酸作用较小剂量牛磺酸组作用为强;两个剂量牛磺酸均能抑制LDL所致ADMA、MDA和TNF-α浓度升高;(2)用ox-LDL(100μg/ml)孵育人脐带静脉内皮细胞(HUVECs)24 h,牛磺酸(1或5μg/ml)明显地抑制细胞培养液中LDH、ADMA、MDA和TNF-α水平的提高,而减少NO水平下降和DDAH活性降低。
结论:牛磺酸能显著改善LDL诱导的血管内皮功能障碍,其保护作用与抑制氧化应激和减少炎症因子释放,改善DDAH活性和降低ADMA水平有关。
Oxide low-density lipoprotein(ox-LDL)is an important factor contributing the development of atherogenesis.L-arginine congener compounds asymmetric dimethylarginine(ADMA),an endogenous inhibitor of nitric oxide synthase(NOS),inhibit nitric oxide(NO) production.Our previous work has showed that a single injection of low-density lipoprotein(LDL)significantly increases the serum level of endogenous ADMA concomitantly with endothelial dysfunction: Incubation human umbilical vein endothelial cells(HUVECs)with ox-LDL,dimethylarginine dimethylaminohydrolase(DDAH),a major hydrolase of ADMA were devitalization,and cause accumulation of ADMA.Taurine is a sulfur-containing semiessential amino acid,an antioxidant and anti-inflammatory drug.Recently it has been reported that taurine preserves endothelial function and inhibit atherogenesis. However,the mechanisms responsible for the beneficial effect of taurine on endothelial cells remain unclear.Based on the elevated level of ADMA by lipid peroxides and antioxidant properties of taurine,in the present study we tested the protective effect of taurine on damages of the endothelium induced by Low-density lipoprotein(LDL),and whether the protective effect of taurine is related to the DDAH/ADMA pathway.
METHODS:study were divided to two part.(1)Animal treatment: Blood samples were collected from carotid artery.Vasodilator responses to acetylcholine(Ach)in the isolated aortic rings were determined,and serum concentrations of asymmetric dimethylarginine(ADMA), malondialdehyde(MDA),tumour necrosis factor-α(TNF-α),Nitric oxide (NO).(2)Cell culture and treatment:Incubation human umbilical vein endothelial cells(HUVECs)with ox-LDL(100μg/ml)for 24h,the medium levels of lactate dehydrogenase(LDH),ADMA,TNF-α,NO, MDA and the intracellular activity of DDAH were measured.
RESULTS:(1)A single injection of LDL(4mg/kg)significantly decreased vasodilator responses to Ach,increased the serum level of ADMA,MDA and TNF-α,and decreased the serum level of NO.Taurine (60 or 180mg/kg)markedly reduced the inhibition of vasodilator responses to Ach by LDL,and the protective effect of taurine at the high dose was greater compared with the lower group.Taurine inhibited the increased level of ADMA,MDA and TNF-αinduced by LDL,and increased the serum level of NO.(2)Incubation human umbilical vein endothelial cells(HUVECs)with ox-LDL(100μg/ml)for 24h,Taurine (1 or 5μg/ml)markedly decreased the medium levels of LDH,ADMA, MDA and TNF-α,and increased the level of NO in the medium and the intracellular activity of DDAH.
CONCLUSIONS:taurine protects the endothelium against damages elicited by LDL in vivo and in culture,and the protective effect of taurine on endothelium is related to the DDAH/ADMA pathway.
引文
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