外泌体分泌在1,4-苯醌诱导的HL-60细胞凋亡中起保护作用

慢性苯暴露损害造血系统,可引起再生障碍性贫血,甚至白血病。外泌体(exosomes)是细胞分泌的纳米级膜泡,在许多生理和病理过程中发挥重要作用。然而,苯及其代谢产物对外泌体分泌的影响仍不清楚。本研究旨在观察苯的活性代谢产物1,4-苯醌(1,4-benzoquinone,1,4-BQ)能否引起人早幼粒白血病细胞HL-60外泌体分泌量的变化以及外泌体释放在1,4-BQ诱导的细胞凋亡中的作用。应用不同浓度1,4-BQ处理细胞24 h,超高速离心法提取细胞培养基中的外泌体,结果发现1,4-BQ能促进外泌体分泌,呈剂量反应关系。进一步应用外泌体抑制剂GW4869抑制外泌体分泌,流式细胞仪检测细胞凋亡率、蛋白质免疫印迹法检测抑凋亡蛋白质Bcl-2、凋亡通路关键蛋白质cleaved caspase-9和cleaved caspase-3的表达,探讨外泌体分泌对1,4-BQ所致细胞凋亡的影响。结果显示:与对照组相比,1,4-BQ单独处理组的凋亡率、Bcl-2、cleaved caspase-9和cleaved caspase-3的表达均显著增高(P<0.05),而1,4-BQ+GW4869组的凋亡率及凋亡相关蛋白质的表达均显著高于1,4-BQ单独处理组(P<0.05),表明抑制外泌体分泌可增加1,4-BQ诱导的细胞凋亡。综上表明,1,4-BQ能促进外泌体分泌,并在1,4-BQ诱导的细胞凋亡中起保护作用。本研究为了解苯的毒性效应和毒性机制提供了新的实验证据。

Exosomes Protect HL-60 Cells in 1,4-Benzoquinone-induced Apoptosis

Chronic benzene exposure damages the hematopoietic system and causes aplastic anemia and even leukemia.Exosomes are nanoscale vesicles secreted by cells and play an important role in many physiological and pathological processes.However,the effects of benzene and its metabolites on secretion of exosomes is unclear.The aim of this study was to investigate whether 1,4-benzoquinone(1,4-BQ),an active metabolite of benzene,can cause changes of secretion of exosomes in HL-60 cells and the role of exosome release in 1,4-BQ-induced apoptosis.The cells were treated with different concentrations of 1,4-BQ for 24 hours,and the exosomes were extracted from the cell culture medium by low-temperature ultrahigh speed centrifugation.The results showed that 1,4-BQ promotes the secretion of exosomes in a doseresponse relationship.Furthermore,the exosome inhibitor GW4869 was used to investigate the effect of exosome secretion in 1,4-BQ-induced apoptosis.The apoptosis ratios were analyzed by flow cytometry and expression of apoptosis-related proteins were detected by Western blotting.The apoptosis ratio and the expression of cleaved caspase-9,cleaved caspase-3 and Bcl-2 were increased compared with the control group(P<0.05),and during the co-treatment of 1,4-BQ and GW4869,the apoptotic ratio and expression of apoptosis-related proteins were significantly higher than that in the 1,4-BQ group(P<0.05),indicating that inhibiting exosome secretion could increase 1,4-BQ-induced apoptosis.In summary,1,4-BQ increases exosomes secretion which plays a protective role in 1,4-BQ-induced apoptosis.This study provides new evidence for understanding benzene’s toxic effects and mechanisms.