METHODS: We first, used a Cell Counting Kit-8 (CCK-8) to detect the viability of BMCs after they were exposed to different doses of FA (50, 100, 200 μmol/L) for different exposure time (12, 24, 48 h), then chose 24 h as an exposure time to detect the expression of PrxIII for exposing different doses of FA by Quantitative reverse transcription-PCR (qRT-PCR) and Western blot analysis. Based on our preliminary experimental results, we chose 100 μmol/L FA as an exposure dose to expose for 24 h, and used a small interfering RNA (siRNA) to silenced PrxIII to examine the cell viability by CCK-8, reactive oxygen species (ROS) level by DCFH-DA, apoptosis by Annexin V/PI double staining and cell cycle by flow cytometry (FCM) so as to explore the possible regulatory effect of PrxIII silencing on FA-induced bone marrow toxicity.
RESULTS: High expression of PrxIII occurred in the process of FA-induced oxidative stress. Silencing of PrxIII prevented FA from inducing oxidative stress, thus increasing cell viability, decreasing ROS level, rescuing G0 -G1 and G2 -M arrest, and reducing cell apoptosis.
CONCLUSIONS: PrxIII silencing might be a potential target for alleviating FA-induced oxidative damage.
方法:我们首先,使用细胞计数试剂盒-8(CCK-8)检测BMC暴露于不同剂量的FA(50、100、200μmol/L)不同暴露时间(12、24、48h)后的活力,然后选择24小时作为暴露时间,通过定量逆转录PCR(qRT-PCR)和Westernblot分析检测暴露不同剂量FA的PrxIII的表达。根据我们的初步实验结果,我们选择100μmol/LFA作为暴露剂量,暴露24小时,并使用小干扰RNA(siRNA)沉默PrxIII,通过CCK-8检查细胞活力,通过DCFH-DA检查活性氧(ROS)水平,通过膜联蛋白V/PI双重染色和流式细胞术(FCM)检测细胞周期,以探讨PrxIII沉默对FA诱导的骨髓毒性的可能调节作用。
结果:PrxIII的高表达发生在FA诱导的氧化应激过程中。沉默PrxIII可防止FA诱导氧化应激,从而增加细胞活力,降低ROS水平,拯救G0-G1和G2-M逮捕,减少细胞凋亡。
结论:PrxIII沉默可能是减轻FA诱导的氧化损伤的潜在目标。