淫羊藿苷和淫羊藿次苷Ⅱ对内皮细胞eNOS表达和NOS活性的影响

Effects of icariin and icarisideⅡon eNOS expression and NOS activity in porcine aorta endothelial cells

目的:用过表达表皮生长因子受体(epidermal growth factor receptor,EGFR)的猪动脉内皮细胞(porcineaorta endothelial,PAE)作为研究对象,探讨淫羊藿苷(icariin)和淫羊藿次苷Ⅱ(icarisideⅡ)对PAE中内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)表达和一氧化氮合酶(nitric oxide sythase,NOS)活性的影响及其可能机制。方法:利用PAE中转染EGFR并筛选稳定株,分别用12.5μmol/L的淫羊藿苷和淫羊藿次苷Ⅱ处理PAE和PAE-EGFR细胞48 h,观察各组细胞eNOS的表达变化;另在淫羊藿苷或淫羊藿次苷Ⅱ处理PAE和PAE-EGFR细胞时,分别加入表皮生长因子(epidermal growth factor,EGF)共同处理细胞,观察各组细胞eNOS的表达变化;检测淫羊藿苷和淫羊藿次苷Ⅱ处理前后PAE和PAE-EGFR细胞NOS的酶活性改变,并以西地那非作为对照。结果:Western blot显示PAE-EGFR细胞表达eNOS的基础值要比PAE细胞高,淫羊藿苷和淫羊藿次苷Ⅱ处理时PAE和PAE-EGFR细胞株中都能引起eNOS表达增加,并且在PAE-EGFR稳定株中eNOS的表达量比PAE细胞明显提高(P<0.01);在表达EGFR的PAE-EGFR细胞中,当淫羊藿次苷Ⅱ的浓度达到10-8 mol/L时,NOS活性增加到(15.37±1.49)u/mg,比PAE细胞高4.66 u/mg。在药物浓度达到10-7,10-6和10-5 mol/L时,淫羊藿次苷Ⅱ对PAE-EGFR细胞NOS活性的影响较PAE细胞显著增加(P<0.01);淫羊藿苷也能增加PAE和PAE-EGFR细胞的NOS酶活性,但是其效果比较淫羊藿次苷Ⅱ组平均低20%,而西地那非没有显著影响NOS酶活性。结论:淫羊藿苷和淫羊藿次苷Ⅱ可上调PAE和PAE-EGFR细胞eNOS的表达和提高NOS活性,对血管内皮细胞功能可能具有改善作用,而且淫羊藿次苷Ⅱ的效果优于淫羊藿苷,其作用机制可能与激活PAE细胞的EGF-EGFR信号通路有关。

Objective： To investigate the effects of icariin and icarisideⅡon eNOS expression and NOS activity in endothelial cells and possible mechanisms using EGFR over-expressed porcine aorta endothelial（PAE） cell line.Methods： The EGFR gene was transfected into PAE cells and genetic stable cell line（PAE-EGFR） was selected.12.5 μmol/L of icariin and of icarisideⅡwere used to treat the PAE and PAE-EGFR cells respectively for 48 h,the eNOS expression in each group was observed.EGF was also used to treat the cells to observe the regulatory effects of icariin and icarisideⅡon NOS activity.The re-gulatory effects of icariin and icarisideⅡon NOS activity were also observed,and sildenafil was used as a control.Results： Western blot showed that the basic value of eNOS expression was higher in PAE-EGFR group compared with that in PAE group,both of icariin and icarisideⅡincreased the eNOS expression in PAE and PAE-EGFR group（P〈0.01）,and the value of eNOS expression was higher in PAE-EGFR group than that in PAE group.In the PAE-EGFR cell line,the NOS activity reached（15.37±1.49） u/mg when the concentration of icarisideⅡwas 10^-8 mol/L,which was 4.66 u/mg more than that in the PAE cell line.When the concentration reached 10^-7,10^-6 or 10^-5 mol/L,the change of NOS activity in PAE-EGFR group was greater than that in PAE group（P0.01）.icariin also increased the NOS activity in PAE and PAE-EGFR cells,but the activity was 20% lower compared with icarisideⅡgroup,howe-ver,Sildenafil showed no influence on NOS activity.Conclusion： Icariin and icarisideⅡ may increase the eNOS expression through activating EGF-EGFR pathway in PAE cell,by which endothelial cells function could be regulated and the better effect was noted in icarisideⅡcompared to icariin.