硫辛酸抗氧化作用及对小牛主动脉内皮细胞和平滑肌细胞氧化损伤的保护功效研究
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摘要
α-硫辛酸(lipoic acid,LA)广泛存在于各种细胞中。α-硫辛酸在哺乳动物细胞内可以部分转化为还原型的二氢硫辛酸(dihydrolipoic acid,DHLA)。自发现LA以来,对其生物活性已经进行了许多体内、体外和细胞实验研究。研究表明,LA对糖尿病、白内障、局部缺血再灌注和放射性损伤等自由基性疾病具有预防和治疗作用。但LA对保护动脉血管细胞免受氧化损伤的功能研究较为少见。
本研究采用化学发光法和比色法全面检测了LA和DHLA直接清除多种自由基、活性氧和抗脂质过氧化的能力以及对·OH引起的DNA氧化损伤的保护作用,并且体外培养小牛主动脉内皮细胞(endothelial cells,EC)和平滑肌细胞(smooth muscle cells,SMC),建立细胞H_2O_2氧化损伤模型,研究了LA保护EC和SMC免受H_2O_2氧化损伤的功效,以及产生这些功效的自由基生物学机理。
清除自由基和活性氧的实验表明,LA和DHLA不仅可以有效清除相应体系中的羟自由基(·OH)、过氧化氢(H_2O_2)、过氧亚硝基阴离子(ONOO~-)和二苯代苦味肼基自由基(DPPH~·),显著抑制脂质过氧化,而且对·OH引起的DNA损伤也有良好的保护功效。LA不能清除超氧阴离子(O_2~-),而DHLA可以有效清除O_2~-。以上结果提示,DHLA的抗氧化能力明显强于LA。
四甲基偶氮噻唑盐(MTT)比色法实验结果表明,LA可促进EC的增殖而抑制SMC的增殖。LA对EC和SMC由H_2O_2引起的氧化损伤的保护作用的实验结果显示,在H_2O_2胁迫下,LA可剂量依赖性地升高EC和SMC中的超氧化物歧化酶(SOD)活性和过氧化氢酶(CAT)活性及还原型谷胱甘肽(GSH)的含量,抑制细胞内外的ONOO~-和脂质过氧化产物丙二醛(MDA)的产生。
结果表明,LA和DHLA能有效清除多种自由基和活性氧,抑制脂质过氧化,对·OH引起的DNA氧化损伤有保护作用,提示它们是良好的抗氧化剂。LA可促进EC的增殖而抑制SMC的增殖,同时LA可以提高H_2O_2胁迫下EC和SMC的抗氧化水平,改善H_2O_2胁迫下EC和SMC的多种生化指标,对EC和SMC的氧化损伤有保护功效。这些结果提示LA可能具有防止AS的发生和发展,缓解AS病情的作用。本研究为LA和DHLA药用保健价值的进一步开发提供了自由基生物学方面的新的实验依据。
Lipoic acid (LA) exists widely in all kinds of cells .It can be converted into dihydrolipoic acid (DHLA) in mammal cells. Since it was discovered its pharmacological effect has been investigated through a lot of experiment in vivo and in vitro including cells culture technique . It has been shown that LA can be used in the prevention and therapy of diabetes, cataract, ischemia, hypoxia/reoxygenation, irradiation injury, etc. However, the studies about LA activity involved in protecting cardiovascular cells from oxidative damage are limited.In order to further elucidate the free radial biological mechanism ,by which LA and DHLA performs its ant-oxidation activities, the present study analyzed the ability of LA and DHLA to scavenge directly different free radicals and reactive oxygen species (ROS) and prevent DNA oxidative damage caused by ? OH using chemiluminescence and colorimetric systems, established cell oxidative damage model cause by H_2O_2, observed the cardiovascular protection effect of LA using in vitro cultured bovine aortic endothelial cells (EC) and smooth muscle cells (SMC) and established cell oxidative damage model.The free radicals and ROS scavenging experiments indicated that LA and DHLA could effectively scavenge H_2O_2、 ? OH、 1,1-diphenyl-2-picryl-hydrazyl ( DPPH ? ) and ONOO~- , inhibit lipid peroxidation and protect DNA from oxidative damage caused by ? OH. DHLA could effectively scavenge O_2~- but LA could not. It seems that the antioxidation ability of DHLA is stronger than that of LA.The results of MTT showed that LA could promote the proliferation of EC and inhibit the proliferation of SMC. The results dealing with the effects of LA on protecting cultured bovine aortic endothelial cells and smooth muscle cells from oxidative damage caused by H_2O_2, demonstrated that LA could dose-dependently increase the activities of SOD、 CAT and the level of GSH , inhibit malondialdehyde (MDA) and ONOO~- in the EC and SMC , as well as in the culture medium of EC and SMC.The present study indicates both LA and DHLA can effectively scavenge many kinds of free radicals and ROS, inhibit lipid peroxidation and prevent DNA oxidative damage caused by ? OH, they are effective antioxidants. LA can promote the proliferation of EC and inhibit the proliferation of SMC, improve antioxidation level and biochemical index of the EC and SMC injured by H_2O_2, protect EC and SMC from oxidative damage. Our results shows LA can possibly prevent and cure athrosclerosis . It can provide new free radical biological data for the further study on the medical and health-care use of LA and DHLA.
本研究采用化学发光法和比色法全面检测了LA和DHLA直接清除多种自由基、活性氧和抗脂质过氧化的能力以及对·OH引起的DNA氧化损伤的保护作用,并且体外培养小牛主动脉内皮细胞(endothelial cells,EC)和平滑肌细胞(smooth muscle cells,SMC),建立细胞H_2O_2氧化损伤模型,研究了LA保护EC和SMC免受H_2O_2氧化损伤的功效,以及产生这些功效的自由基生物学机理。
清除自由基和活性氧的实验表明,LA和DHLA不仅可以有效清除相应体系中的羟自由基(·OH)、过氧化氢(H_2O_2)、过氧亚硝基阴离子(ONOO~-)和二苯代苦味肼基自由基(DPPH~·),显著抑制脂质过氧化,而且对·OH引起的DNA损伤也有良好的保护功效。LA不能清除超氧阴离子(O_2~-),而DHLA可以有效清除O_2~-。以上结果提示,DHLA的抗氧化能力明显强于LA。
四甲基偶氮噻唑盐(MTT)比色法实验结果表明,LA可促进EC的增殖而抑制SMC的增殖。LA对EC和SMC由H_2O_2引起的氧化损伤的保护作用的实验结果显示,在H_2O_2胁迫下,LA可剂量依赖性地升高EC和SMC中的超氧化物歧化酶(SOD)活性和过氧化氢酶(CAT)活性及还原型谷胱甘肽(GSH)的含量,抑制细胞内外的ONOO~-和脂质过氧化产物丙二醛(MDA)的产生。
结果表明,LA和DHLA能有效清除多种自由基和活性氧,抑制脂质过氧化,对·OH引起的DNA氧化损伤有保护作用,提示它们是良好的抗氧化剂。LA可促进EC的增殖而抑制SMC的增殖,同时LA可以提高H_2O_2胁迫下EC和SMC的抗氧化水平,改善H_2O_2胁迫下EC和SMC的多种生化指标,对EC和SMC的氧化损伤有保护功效。这些结果提示LA可能具有防止AS的发生和发展,缓解AS病情的作用。本研究为LA和DHLA药用保健价值的进一步开发提供了自由基生物学方面的新的实验依据。
Lipoic acid (LA) exists widely in all kinds of cells .It can be converted into dihydrolipoic acid (DHLA) in mammal cells. Since it was discovered its pharmacological effect has been investigated through a lot of experiment in vivo and in vitro including cells culture technique . It has been shown that LA can be used in the prevention and therapy of diabetes, cataract, ischemia, hypoxia/reoxygenation, irradiation injury, etc. However, the studies about LA activity involved in protecting cardiovascular cells from oxidative damage are limited.In order to further elucidate the free radial biological mechanism ,by which LA and DHLA performs its ant-oxidation activities, the present study analyzed the ability of LA and DHLA to scavenge directly different free radicals and reactive oxygen species (ROS) and prevent DNA oxidative damage caused by ? OH using chemiluminescence and colorimetric systems, established cell oxidative damage model cause by H_2O_2, observed the cardiovascular protection effect of LA using in vitro cultured bovine aortic endothelial cells (EC) and smooth muscle cells (SMC) and established cell oxidative damage model.The free radicals and ROS scavenging experiments indicated that LA and DHLA could effectively scavenge H_2O_2、 ? OH、 1,1-diphenyl-2-picryl-hydrazyl ( DPPH ? ) and ONOO~- , inhibit lipid peroxidation and protect DNA from oxidative damage caused by ? OH. DHLA could effectively scavenge O_2~- but LA could not. It seems that the antioxidation ability of DHLA is stronger than that of LA.The results of MTT showed that LA could promote the proliferation of EC and inhibit the proliferation of SMC. The results dealing with the effects of LA on protecting cultured bovine aortic endothelial cells and smooth muscle cells from oxidative damage caused by H_2O_2, demonstrated that LA could dose-dependently increase the activities of SOD、 CAT and the level of GSH , inhibit malondialdehyde (MDA) and ONOO~- in the EC and SMC , as well as in the culture medium of EC and SMC.The present study indicates both LA and DHLA can effectively scavenge many kinds of free radicals and ROS, inhibit lipid peroxidation and prevent DNA oxidative damage caused by ? OH, they are effective antioxidants. LA can promote the proliferation of EC and inhibit the proliferation of SMC, improve antioxidation level and biochemical index of the EC and SMC injured by H_2O_2, protect EC and SMC from oxidative damage. Our results shows LA can possibly prevent and cure athrosclerosis . It can provide new free radical biological data for the further study on the medical and health-care use of LA and DHLA.
引文
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