丹参多酚酸盐修复糖基化终产物引起的晚期EPCs功能障碍及其分子机制

Effect of Depside Salt from Salvia Miltiorrhizae in Repairing Advanced Glycation End Products-induced Late Endothelial Progenitor Cell Dysfunction and Its Molecular Mechanism

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中文关键词: 丹参多酚酸盐糖基化终产物晚期内皮祖细胞血管新生

英文关键词:depside salt from salvia miltiorrhizae advanced glycation end products late endothelial progenitor cell agiogenesis

基金项目:国家自然科学基金资助项目(No.30170370);江苏省自然科学基金资助项目(No.BK2004083)

作者	单位
陈琴	南京大学医学院附属鼓楼医院心内科
黄铭涵	福建中医学院附属第二人民医院
江时森	南京军区南京总医院心血管内科
徐标	南京大学医学院附属鼓楼医院心内科

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中文摘要:

目的观察丹参多酚酸盐对糖基化终产物(advanced glycation end products,AGE)引起的晚期内皮祖细胞(endothelial progenitor cells,EPCs)功能障碍的修复作用,并探讨其中分子机制。方法密度梯度离心法分离脐血中单个核细胞,采用EGM-2-MV培养液培养晚期EPCs;利用牛血清白蛋白及葡萄糖制备晚期糖基化修饰的白蛋白(AGE modified bovine serum albumin,AGE-albumin)。实验分为单独加入200μg/mLAGE-albumin的AGE组;同时加入200μg/mLAGE-albumin及0.1μg/mL、1μg/mL、10μg/mL的丹参多酚酸盐低、中、高剂量组;加入200μg/mL未经修饰白蛋白的对照组。采用AnnexinV/PI双染法检测晚期EPCs凋亡;ECMatirx-gel检测晚期EPCs形成新生血管的能力。采用RT-PCR法检测细胞中糖基化终产物受体(the receptor for AGE,RAGE)、内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)mRNA水平;Western blot法检测RAGE、eNOS及蛋白激酶Akt的蛋白表达水平。结果与对照组比较,AGE组AnnexinV+/PI-细胞比例增加,在ECMatirx-gel上形成新生血管能力降低;晚期EPCs中ragemRNA及RAGE蛋白表达增加,enosmRNA及eNOS及Akt蛋白表达减少。丹参多酚酸盐1.0、10.0μg/mL组,与AGE组比较,晚期EPCs凋亡明显减少(P<0.05,P<0.01),在ECMatirx-gel上形成新生血管能力增加(P<0.01),晚期EPCs中RAGE表达减少(P<0.05,P<0.01),eNOS、Akt表达增加(P<0.05);与对照组比较,丹参多酚酸盐10μg/mL组对晚期EPCs凋亡及在ECMatirx-gel上形成新生血管能力,ragemRNA及RAGE,enosmRNA及eNOS,Akt蛋白表达,差异均无统计学意义(P>0.05)。结论 AGE使EPCs功能受损,表现为凋亡增加、迁移及体外形成新生血管的能力下降,丹参多酚酸盐可修复由AGE-albumin引起的EPCs功能损害。

英文摘要:

Objective To investigate the effects of depside salt from salvia miltiorrhizae (DSSM) in repairing advanced glycation end products (AGE)-induced late endothelial progenitor cell (EPC) dysfunction,and its possible molecular mechanism. Methods Mononuclear cells (MNCs) were separated using density gradient centrifugation from human umbilical cord blood,and cultured with EGM-2-MV culture fluid to late EPCs. Then the EPCs were divided into 5 groups:Group A incubated with 200 μg/mL AGE-modified bovine serum albumin (AGE-albumin) alone (A),Groups B,C and D with equal dosage of AGE-albumin plus DSSM at different dosages (0.1 μg/mL,1 μg/mL,and 10 μg/mL),Group E with 200 μg/mL of unmodified-AGE. The late EPCs apoptosis was detected by Annexin V+/PI double-stain,angiogenic capacity was detected by ECMatrix-gel,mRNA expressions of the receptor for AGE (RAGE) and endothelial nitric oxide synthase (eNOS) were measured by reverse-transcriptase polymerase chain reaction (RT-PCR),and the protein expressions of RAGE,eNOS and protein kinase (Akt) were measured by Western blot. Results Compared with Group E,in Group A,the Annexin V+/PI-ratio and expression of RAGE in EPCs increased,the angiogenic capacity,mRNA and protein expressions of eNOS,and protein expression of Akt decreased significantly. These abnormal changes in Groups C and D were significantly smaller than those in Group A (P<0.05 or P<0.01). And all the indices in Group D were adjacent to those in Group E,showing insignificant difference between the two groups (P>0.05). Conclusions AGE could injure the function of EPCs,revealing increase of cell apoptosis and migration,deprivation of angiogenic capacity in vitro. DSSM could repair the EPCs dysfunction induced by AGE-albumin. Up-regulation of eNOS and Akt in these cells may be involved in the mechanism.

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