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目的:观察氧化低密度脂蛋白(ox-LDL)对培养的人脐静脉内皮细胞(HUVECs)二甲精氨酸-二甲赖氨酸水解酶(DDAH)活性及表达的影响,以探讨不对称二甲精氨酸(ADMA)代谢机制及卡托普利作用。方法:采用改良的Jaffe法培养原代HUVECs,取生长良好的3～6代HUVECs用于实验,分为①空白对照组:加DMEM培养液;②ox-LDL组:加入ox-LDL(100,150mg/L);③ox-LDL加卡托普利组:同时加入150mg/Lox-LDL及卡托普利100mg/L,共孵24h后,检测上清液中一氧化氮(NO)、内皮素(ET)、一氧化氮合酶(NOS)活性、ADMA含量、反应DDAH酶活性的L-胍氨酸(L-cit)浓度,采用Western blot测定细胞裂解液中DDAH的蛋白表达。结果:ox-LDL条件培养下,内皮细胞的代谢产物ADMA、ET的量均较空白对照组高,而NO的量及NOS的活性减少;L-cit浓度显著降低,且有浓度依赖性,而DDAH的表达无明显变化。卡托普利干预后,AD-MA、ET的量较ox-LDL组降低,NOS活性及NO增加,L-cit浓度明显升高。结论:ox-LDL诱导下,内皮损伤ADMA的增加与DDAH的活性减弱有关,而与DDAH的表达无关。卡托普利通过增加DDAH活性促进AD-MA代谢,使NOS活性增高,抑制ox-LDL对内皮功能的损伤。
Objective: To investigate the effect of ox-LDL on the activity and expression of dimethylarginine-dimethyllysine hydrolase (DDAH) in cultured human umbilical vein endothelial cells (HUVECs) Metabolic mechanism of dimethylarginine (ADMA) and captopril effect. Methods: Primary HUVECs were cultured with modified Jaffe method. HUVECs from 3rd to 6th generation with good growth were used in the experiment and divided into ① blank control group and DMEM culture medium; ② ox-LDL group: ox-LDL (100,150mg / L); ③ox-LDL plus captopril group: adding 150mg / Lox-LDL and captopril 100mg / L at the same time, incubated 24h, the supernatant was detected nitric oxide (NO), endothelin ), Nitric oxide synthase (NOS) activity, ADMA content, and L-cit (D-Lysine) activity in response to DDAH. Western blot was used to detect the protein expression of DDAH in cell lysate. Results: Under ox-LDL condition, the amount of ADMA and ET in endothelial cells were higher than that in blank control group, while the amount of NO and the activity of NOS decreased. The concentration of L-cit was significantly decreased and concentration-dependent DDAH expression did not change significantly. After captopril intervention, the levels of AD-MA and ET were lower than that of ox-LDL group, NOS activity and NO were increased, and the concentration of L-cit was significantly increased. CONCLUSION: The increase of ADMA induced by ox-LDL is related to the weakened activity of DDAH, but not to the expression of DDAH. Captopril promotes AD-MA metabolism by increasing DDAH activity, resulting in increased NOS activity and inhibition of endothelial dysfunction by ox-LDL.