回心草对脐静脉内皮细胞的保护作用及对分泌一氧化氮和一氧化氮合酶的影响

The Protective Effect of Rhodobryum giganteum (Schwaegr.) Par.on Umbilical Vascular Endothelial Cells and the Influence on Secretion of NO and NOS

目的:研究回心草水提取液及其含药血清对人脐静脉内皮活细胞数、内皮细胞一氧化氮(NO)及一氧化氮合酶(NO合酶)的影响,探讨回心草抗动脉硬化的药理作用机制。方法:采用H2O2建立体外培养的人脐静脉血管内皮细胞(HUVEC)损伤模型。采用MTT法测定回心草提液对H2O2损伤的内皮细胞的吸光度(OD);采用经典Griess Reagent检测各剂量组细胞上清液中NO浓度,采用一氧化氮荧光检测探针DAF-FMDA(3-amino,4-aminomethyl-2′,7′-difluorescein,diacetate)检测细胞内NOS的活性。结果:H2O212.5 mmol.L-1为最合适的细胞刺激浓度;回心草药液与H2O2损伤的血管内皮细胞共同孵育24 h后,2.50 mg.mL-1、3.33 mg.mL-1浓度组与模型组比较有显著差异(P<0.05,P<0.01);回心草水提液作用于内皮细胞,进行NO,NOS含量的测定,其中回心草2.50 mg.mL-1剂量组有显著作用(P<0.05)。结论:12.5 mmol.L-1的H2O2作用24 h,可成功建立内皮细胞氧化应激损伤模型;回心草水提液直接作用于H2O2损伤的内皮细胞,2.50,3.33 mg.mL-1两个浓度组可以减轻内皮细胞的损伤;回心草2.50 mg.mL-1可增加NOS活性,促进NO的分泌。

Objective：To study the effect and mechanism of Rhodobryum giganteum （Schwaegr.） Par. ＇s antiatherosclerotic effect. Methods：Vascular endothelial cells were cultivated and the H2 O2 were used to induce the oxidative stress injury of human umbilical vascular endothelial ceils （HUVEC）. Water extract of Rhodobryum giganteum （Schwaegr.） Par. was added to cultivated HUVEC, and the activity of the cells was carefully determined by OD value with MTT method. The Griess Reagent was used to detect the NO concentration of different groups. At the same time, the NO fluorescence analysis probe, DAF-FM DA （3-amino,4-aminomethyl-2＇, 7＇-difluorescein, diacetate）, was used to determine the activity of NO synthase. Results：The most suitable stimulating concentration of H2 O2 on HUVEC is 12.5 mmol· L^-1. Water extract of Rhodobryum giganteum（Schwaegr. ）Par. was co-cultured with HUVEC damaged by H2O2. The OD values indicate that 3.33,2.50 mg· mL^-1 water extract groups increase activity of the ceils significantly compared with the model group （ P 〈 0.05）, and 3.33 mg· mL^-1 is much better than 2.50 mg· mL^-1 dose group. To determine the content of NO and NOS, the 2.50 mg · mL^-1 dose has significant effect （ P 〈 0.05 ）. Conclusions： The H2 O2 concentration of 12.5 mmol· L^-1 could be used to establish the injured model of endothelial cell successfully. 2.50 and 3.33 mg· mL^-1 water extract of Rhodobryum giganteum （Schwaegr.） Par. have protective effect on ECV304 injured by H2 O2 · 2.50 mg· mL^-1 water extract of Rhodobryum giganteum （ Schw aegr. ） Par could increase the activity of NOS and promote the synthesis and secretion of NO.