摘要
目的观察葛根素对氧自由基(oxidized free radical,OFR)培养的人脐静脉内皮细胞(human um-bilical vein endothelial cells,HUVECs)二甲基精氨酸-二甲基赖氨酸水解酶(di methylarginine di methy-laminohydrolase,DDAH)活性及表达的影响,以探讨葛根素对不对称二甲精氨酸(ADMA)代谢机制的影响。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;(2)OFR组:加入OFR0.1mmol/L;(3)葛根素Ⅰ组:加0.1mmol/L OFR及0.5mg/ml葛根素;(4)葛根素Ⅱ组:加0.1mmol/L OFR和1.0mg/ml葛根素。共孵24h后,检测上清液中NO、NOS活性、ET、ADMA含量、L-胍氨酸(L-cit)浓度,采用Western blotting测定细胞裂解液中DDAH的蛋白表达。结果OFR条件培养下,内皮细胞的代谢产物ADMA、ET含量均较空白对照组高,而NO及NOS的活性减少;反应DDAH酶活性的L-cit浓度显著降低,而DDAH的表达无明显变化。葛根素干预后,ADMA、ET含量较OFR组降低,NOS活性及NO增加,L-cit浓度明显升高。结论OFR培养下,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。葛根素通过增加DDAH活性促进ADMA代谢,使NOS活性增加,抑制OFR对内皮功能的损伤。
Objective To observe the effects of puerarin on activity of dimethylarginine dimethylaminohydrolase （DDAH） in human umbilical vein endothelial cells （HUVECs） cultured with oxidized free radical （OFR）, to explore the effect of puerarin on metabolic mechanism of asymmetric dimethylarginine （ADMA）. Methods HUVECs of the 3rd- 6th passage cultured with modified Jaffe＇s method were divided into 4 groups, the blank control group cultured with DMEM medium, the OFR group cultured with DMEM medium containing 0.1 mmol of OFR per liter, the puerarin group 1 and 2 cultured with DMEM medium containing 0.1 mmol of OFR per liter as well as 0.5 mg/ml and 1.0 mg/ml of puerarin respectively. After being incubated for24 h, activity of nitric oxide synthase （NOS）, contents of nitric oxide （NO）, ADMA, endothelin （ET）, and L-cit- rulline （L-cit） in the supernate were measured, and DDAH protein expression in the lysate was detected by Western blotting. Results Compared with those in the blank control group, ADMA and ET contents were higher, while the levels of NO and L-cit and the activity of NOS were lower markedly, but the DDAH expression changed insignificantly in the OFR group. These abnormalities were restored significantly in the puerarin groups. Conclusion The increase of ADMA in OFR injured HUVECs was correlated with the reduction of DDAH activity and irrelevant to DDAH expression. Puerarin could promote ADMA metabolism through increasing DDAH activity, and improve NOS activity, thus to reduce the impairing of OFR on endothelial function.
出处
《中国中西医结合杂志》
CAS
CSCD
北大核心
2006年第12期1103-1106,共4页
Chinese Journal of Integrated Traditional and Western Medicine
基金
江西省自然科学基金科研项目(No.0340102)
