Song Lei, Yang Yue-jin, Dong Qiu-ting, Qian Hai-yan, Xu Hui, Meng Xian-min, Tang Yue
Coronary Heart Disease Center, Fuwai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, China.
Zhonghua Xin Xue Guan Bing Za Zhi. 2011 Nov;39(11):1033-8.
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.
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 µmol/L), AMPK inhibitor-compound C (CC), PI3K inhibitor-LY294002 (LY), Ator + CC and Ator + LY. Cell apoptosis was assessed using Annexin V/Prospidine 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.
MSCs apoptosis in Ator (0.01 - 10 µmol/L) treated H/SF groups was significantly reduced compared with H/SF 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 + LY and 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).
Atorvastatin can protect MSCs from H/SF induced apoptosis through AMPK pathway, which resulting in activation of eNOS.
由于梗死后期环境恶劣，间充质干细胞（MSCs）移植效果不佳。我们之前的研究已证明他汀类药物可提高植入的骨髓间充质干细胞的存活率，但确切机制仍有待阐明。我们推测阿托伐他汀（Ator）可保护间充质干细胞免受缺氧和无血清（H/SF）诱导的凋亡，并研究其潜在机制。
体外培养中国小型猪骨髓来源的间充质干细胞，并将其暴露于缺氧和H/SF环境中，同时加入不同浓度（0.001 - 10 μmol/L）的阿托伐他汀、AMPK抑制剂——化合物C（CC）、PI3K抑制剂——LY294002（LY）、阿托伐他汀 + CC以及阿托伐他汀 + LY。使用Annexin V/碘化丙啶试剂盒通过流式细胞术评估细胞凋亡。通过蛋白质免疫印迹法检测AMPK、Akt、内皮型一氧化氮合酶（eNOS）的磷酸化水平以及eNOS的表达水平。进行实时定量聚合酶链反应（Real Time-PCR）分析AMPK、Akt和eNOS的基因表达。
与H/SF组相比，阿托伐他汀（0.01 - 10 μmol/L）处理的H/SF组中间充质干细胞凋亡显著减少（1.94% - 6.10% 对10.94%，P < 0.01或0.05）。阿托伐他汀 + CC组的凋亡率高于1 μmol/L阿托伐他汀组（4.94% ± 0.98% 对2.59% ± 0.84%，P < 0.01），而阿托伐他汀 + LY组与1 μmol/L阿托伐他汀组相似（2.02% ± 0.45% 对2.59% ± 0.84%，P > 0.05）。阿托伐他汀处理组中AMPK、Akt和eNOS的基因表达显著上调。同时，阿托伐他汀处理的间充质干细胞中AMPK和eNOS的磷酸化增加（P < 0.01或0.05）。eNOS的磷酸化与AMPK的磷酸化显著相关（r = 0.599，P = 0.004），但与Akt的磷酸化无关（P = 0.263）。
阿托伐他汀可通过AMPK途径保护间充质干细胞免受H/SF诱导的凋亡，从而导致eNOS激活。
