刺囊酸对于氧化型低密度脂蛋白诱导的血管内皮祖细胞损伤的保护作用及机制  

Echinocystic Acid,Extracted from Gleditsia sinensis Fruit,Protects Endothelial Progenitor Cell from Damage Caused by OxLDL,through the Akt / eNOS pathway

作　　者：赖朋[1] 刘怡欣[2] 

机构地区：[1]西华大学生物工程学院,成都610039 [2]四川大学华西医院老年病科,成都610041

出　　处：《天然产物研究与开发》2015年第5期785-792,798,共9页Natural Product Research and Development

基　　金：四川省教育厅自然科学重点项目(14ZB0134);西华大学人才引进项目(Z1220529)

摘　　要：血管内皮祖细胞(endothelial progenitor cells,EPCs)是一种具有分化为内皮细胞能力的前体细胞,对于维持血管内皮完整性和血管生成具有极其重要的作用。高血脂状态下的氧化型低密度脂蛋白(ox LDL)增加严重影响EPCs的作用与功能,这也是高血脂诱发动脉粥样硬化(AS)的机制之一。在此之前的体内研究表明,从皂荚中提取的刺囊酸(echinocystic acid,EA)有较好的抗粥样硬化作用,但具体机制尚未得到阐述。鉴于EPCs在AS发病过程中的重要性,本论文研究了EA对于EPCs的保护作用。本研究采用ox LDL造成体外培养分化的EPCs数量和功能损伤,给药组经不同剂量的EA处理,通过TUNEL、Transwell小室等手段观察凋亡、粘附、迁移、一氧化氮(NO)释放等数量与功能指标。通过设置药理抑制组和Western blot考察了EA对于PI3K/Akt/e NOS通路的作用。结果显示ox LDL明显增加EPCs的凋亡,TUNEL阳性率为35.2%,为正常组的5.5倍;ox LDL还抑制了EPCs的粘附(200倍视野下从正常的24个减少到14个),使细胞迁移率从11%降至6.6%、NO的合成从18.37μM降到7.97μM;ox LDL抑制e NOS的表达和Akt及e NOS的磷酸化,抑制率均在50%以上。高剂量的EA显著改善了上述结果,TUNEL阳性率为14.7%、EPCs的粘附恢复为20个、细胞迁移率为10.2%、NO的合成为19.28μM,EA虽然对e NOS的表达没有影响,但却显著激活了e NOS(p-e NOS/e NOS为1.33,模型组为0.82),并将ox LDL抑制的Akt磷酸化恢复至正常的76%。另外,PI3K抑制剂能部分抵消EA的作用。结果表明刺囊酸对于ox LDL诱导的EPCs的损伤具有保护作用,机制为直接增加Akt和e NOS的磷酸化。Our previous studies revealed that echinocystic acid（ EA） showed obvious attenuation of atherosclerosis in rabbits fed a high-fat diet. However,the underlying mechanisms remain to be elucidated. Considering the importance of endothelial progenitor cells（ EPCs） in atherosclerosis,we hypothesise that EPCs may be one of the targets for the antiatherosclerotic potential of EA. After in vitro cultivation,EPCs were exposed to 100 μg / m L oxidised low-density lipoprotein（ ox LDL） and incubated with or without EA（ 5 and 20 μM） for 48 h. An additional two groups of EPCs（ ox LDL ＋20 μM EA） were pre-treated with either wortmannin,an inhibitor of the phosphoinositide 3-kinase（ PI3K） pathway,or nitro-l-arginine methyl ester（ l-NAME）,an endothelial nitric oxide synthase（ e NOS）-specific inhibitor. Assessment of EPC apoptosis,adhesion,migration and nitric oxide（ NO） release was performed using terminal deoxynucleotidyl transferase-mediated d UTP nick-end labelling（ TUNEL） staining,cell counting,caspase-3 activity assay,transwell chamber assay and Griess reagent,respectively. The protein expression of protein kinase B（ Akt） and e NOS was detected using western blot. Treatment of EPCs with ox LDL induced significant apoptosis（ Model,35. 2%,vs. Control,6. 4%） and impaired adhesion（ Model,14,vs. Control,24）,migration（ Model,6. 6%,vs. Control,11%） and NO production（ Model,7. 97 μM,vs. Control,18. 37 μM）. The deleterious effects of ox LDL on EPCs were attenuated by EA（ apoptosisratio： 14. 7%,adhesion cells： 20,migration ratio： 10. 2%,NO production： 19. 28μM）. However,when EPCs were pretreated with wortmannin or l-NAME,the effects of EA were abrogated. Additionally,ox LDL significantly downregulated e NOS protein expression,as well as repression of e NOS and Akt phosphorylation. The inhibitory effect of ox LDL on Akt /e NOS phosphorylation was attenuated by EA. Taken together,the results indicated that EA protects EPCs from damage caused by ox LDL,via the

关 键 词：血管内皮祖细胞 刺囊酸 氧化型低密度脂蛋白 Akt/eNOS通路 

分 类 号：R966[医药卫生—药理学]