阿托伐他汀主要经AMP蛋白激酶而非PI3K／Akt信号通路抑制猪骨髓间充质干细胞凋亡  

Atorvastatin protects swine bone marrow mesenchymal stem cells from apoptosis through AMPK but not PI3K/Akt pathway

作　　者：宋雷[1] 杨跃进[1] 董秋婷[1] 钱海燕[1] 徐辉[1] 孟宪敏[2] 唐跃[3] 

机构地区：[1]北京协和医学院中国医学科学院阜外心血管病医院冠心病诊治中心,100037 [2]北京协和医学院中国医学科学院阜外心血管病医院中心实验室,100037 [3]北京协和医学院中国医学科学院阜外心血管病医院动物实验中心,100037

出　　处：《中华心血管病杂志》2011年第11期1033-1038,共6页Chinese Journal of Cardiology

基　　金：首都医学发展科研基金（2007-2018）;中国医药卫生事业发展基金（2008-zhfj2）

摘　　要：目的骨髓间充质干细胞移植治疗急性心肌梗死的效果受梗死后局部恶劣微环境限制，前期研究已证实他汀类药物可通过改良心肌微环境提高移植疗效，但具体机制不详。本研究旨在观察阿托伐他汀（Ator）对体外缺氧无血清（H／SF）诱导的猪骨髓间充质干细胞凋亡的影响并探索其作用机制及通路。方法猪骨髓间充质干细胞分正常对照组、H／SF组、浓度梯度（0．001～10μmol／L）Ator处理组、AMP蛋白激酶（AMPK）通路抑制剂compoundC组（CC组），Ator＋CC组、磷脂酰肌醇3激酶（P13K）／丝氨酸苏氨酸激酶（Akt）通路拮抗剂LY294002组（LY组）、Ator＋LY组。流式细胞仪检测各组细胞凋亡比例，Westernblot检测AMPK、Akt、内皮型一氧化氮合酶（eNOS）蛋白及其磷酸化水平，实时聚合酶链反应（Real Time—PCR）检测AMPK、Akt和eNOS基因表达水平。结果Ator0．01—10μmo]／L组间充质干细胞凋亡比例显著低于H／SF对照组（1．94％～6．10％比10．94％，P〈0．01或0．05），Ator＋cc组问充质干细胞凋亡比例显著高于1μmol／LAtor组（4．94％±0．98％比2．59％±0．84％，P〈0．01），而Ator＋LY组细胞凋亡比例与1μmol／L Ator组比较差异无统计学意义（2．02％±0．45％比2．59％±0．84％，P〉0．05）。Ator浓度梯度组AMPK、Akt和eNOS基因表达增加伴AMPK和eNOS磷酸化水平提高（P〈0．01或0．05）。eNOS的磷酸化水平与AMPK磷酸化显著相关（r=0．599，P=0．004），而与Akt磷酸化水平无显著相关（P=0．263）。结论阿托伐他汀可抑制H／SF诱导的猪骨髓间充质干细胞凋亡，该效应主要与AMPK信号通路介导的eNOS活化有关，本实验条件下P13K／Akt信号通路不起主要作用。Objective The effect of mesenchymal stem cells （MSCs） transplantation is poor because of the harsh environment post infarction. Our previous studies have proven that Statins could enhance the implanted bone marrow MSCs survival, but the exact mechanism remained to be clarified. We hypothesized that atorvastatin （Ator） could protect MSCs from hypoxia and serum-free （H/SF） induced apoptosis and investigated the potential mechanisms. Methods Chinese mini-swine＇s bone marrow derived MSCs were cultured in vitro and exposed to hypoxia and H/SF, Ator of various concentrations （0. 001 -10 p^mol/L）, AMPK inhibitor-compound C （CC）, PI3K inhibitor-LY294002 （LY）, Ator ＋ CC and Ator ＋ LY. Cell apoptosis was assessed using Annexin V/Propidine Iodine kit by flow cytometry. Phosphorylation of AMPK, Akt, endothelial nitric oxide synthase （eNOS） level and phosphorylation were tested with Western blot. Real Time-PCR was performed to analyze the gene expression of AMPK, Akt and eNOS. Results MSCs apoptosis in Ator （0. 01 - 10 μmol/L） treated H/SF groups was significantly reduced compared with HISF group （ 1.94% - 6. 10% vs. 10. 94%, P 〈 0. 01 or 0. 05）. Apoptosis was higher in Ator ＋ CC group than in 1 μmol/L Ator group （4. 94% ± 0. 98% vs. 2. 59% ± 0. 84%, P 〈 0.01 ） and similar between Ator＋LYand 1 μmol/L Ator group （2.02%±0.45% vs. 2.59% ±0.84%, P〉0.05）. The gene expressions of AMPK, Akt and eNOS were significantly upregulated in atorvastatin treated groups. Meanwhile, phosphorylation of AMPK and eNOS increased in MSCs treated with atorvastatin （ P 〈 0. 01 or 0. 05 ）. Phosphorylation of eNOS significantly correlated with AMPK phosphorylation （r = 0. 599, P = 0. 004）, but not with Akt phosphorylation （ P = 0. 263 ）. Conclusions Atorvastatin can protect MSCs from H/SF induced apoptosis through AMPK pathway, which resulting in activation of eNOS.

关 键 词：间质干细胞 细胞凋亡 阿托伐他汀 

分 类 号：R965[医药卫生—药理学]