N-乙酰半胱氨酸对过氧化氢诱导的骨髓间充质干细胞凋亡的保护及作用机制研究

Effect and mechanisms of N-acetyl-L-cysteine on H_2O_2-induced apoptosis in BMSCs

目的研究N-乙酰半胱氨酸(NAC)对过氧化氢(H2O2)诱导的骨髓间充质干细胞(BMSCs)凋亡的影响,并探讨其作用机制。方法采用全骨髓贴壁培养法培养SD大鼠BMSCs,随机分成5组:正常对照组、H2O2处理组、NAC组、NAC+H2O2组和LY294002干预组。流式细胞术检测各组的细胞凋亡率及细胞内活性氧水平;RT-PCR检测各组rBMSCs中FOXO1、FOXO3、FOXO4基因水平的表达;Western blot检测各组rBMSCs中Akt、p-Akt、Bcl-2的表达。结果与正常组相比,H2O2组诱导的rBMSCs凋亡数明显增高,细胞内ROS含量明显增高,FOXO1、FOXO3、FOXO4基因水平的表达量明显下降;NAC明显抑制了rBMSCs凋亡和ROS产生,上调了FOXO1、FOXO3、FOXO4基因水平的表达,增加了细胞内p-Akt和Bcl-2的表达,但LY294002抑制剂明显抑制了NAC对氧化应激后rBMSCs的上述功能。结论 N-乙酰半胱氨酸可通过激活PI3K-Akt信号途径抑制细胞内活性氧的产生、上调FOXO1、FOXO3、FOXO4基因水平及抗凋亡Bcl-2的表达来保护BMSCs免受氧化应激引起的凋亡。

Aim To investigate the role of N-acetyl-L- cysteine （NAC） in hydrogen peroxide （ H2O2 ） -induced apoptosis in bone marrow-derived mesenchymal stem cells （BMSCs） and its underlying mechanism. Meth- ods BMSCs isolated from SD rats by whole bone mar- row adherence culture method were randomly divided into normal control group, H2O2 group, NAC group, H202 plus NAC group, H2O2 and NAC plus LY294002 group. Both apoptosis of rBMSCs and ROS production were detected by flow cytometry. The mRNA levels of FOXO1, FOXO3 and FOXO4 in rBMSCs were deter- mined by RT-PCR, and the expressions of p-Akt, Bcl-2 and Akt were examined by Western blot. Results Compared with the control group, the production of ROS and the number of cell apoptosis in H2O2 group significantly increased, and the expression of FOXO1, FOXO3 and FOXO4 mRNA levels decreased in rBMSCs; NAC treatment inhibited H2O2 -induced ROS pro- duction and cell apoptosis, but increased p-Akt and Bcl-2 expression, and upregulated FOXO1, FOXO3 and FOXO4 mRNA levels. LY294002, a PI-3Kinse in- hibitor,not only decreased the expression of FOXO1, FOXO3 and FOXO4 mRNA levels and the expression of p-Akt and Bcl-2, but also markedly increased ROS production and cell apoptosis. Conclusion N-acetyl- L-cysteine can protect BMSCs against apoptosis in- duced by oxidative stress through decreasing reactive oxygen species production and increasing the expres- sion of FOXO1, FOXO3 and FOXO4 mRNA levels and anti-apoptotic protein Bcl-2 expression in BMSCs by activation of PI3K-Akt signaling pathways.