乙酰化修饰下的自噬在苯致造血损伤中的作用机制

Benzene poisoning-induced hematopoietic injury is one of the serious occupational diseases. However, its underlying pathogenic mechanism is not yet fully understood. Our previous studies have found that benzene exposure increased autophagy in cultured bone marrow mononuclear cells and in the clinical samples of patients with benzene poisoning. And also the number of live cells increased when treated with autophagy inhibitor (3-MA). These results suggest that autophagy may play an important role in benzene-induced hematopoietic injury. Our studies further demonstrated that autophagy decreased ,hematopoietic recoveried after treatment with histone deacetylase inhibitor, suggesting that histone acetylation modification may be implicated in changes of autophagy during benzene exposure. This proposed project is aimed to evaluate the role of histone acetylation modification in regulating autophagy during benzene-induced hemopoietic injury. The study will be carried out on the in vitro cultured 34+ cells as well as the CD34+ bone marrow cells from benzene poisoning aplastic anemia model mice and the patients with benzene poisoning. Modification of histone acetylation will be achieved with acetyl transferase (HAT) and deacetylase inhibitors (TSA and Chinese medicine bitter melon protein MCP30), overexpression and shRNA interference. Autophagy will be assessed Western blot, immunofluorescence and electron microscopy and etc. This project from the perspective of autophagy elucidates a novel mechanism for benzene induced-hematopoietic injury, which could provide new ideas and new targets for the prevention and control of benzene poisoning and has important basic research and clinical implications.

苯中毒诱发的造血系统损伤是严重危害从业者健康的职业疾病之一，其致病机制尚未完全阐明。本课题组前期研究发现骨髓单个核细胞染苯后、临床苯中毒病人中自噬水平升高，而自噬抑制剂（3-MA）处理后活细胞数增多，提示自噬在苯致造血损伤中起重要作用。课题组进一步研究发现组蛋白去乙酰化酶抑制剂(HDACI)抑制苯致造血损伤中引起的自噬增加，保护造血，说明组蛋白乙酰化修饰可能通过自噬参与苯致造血损伤。本项目拟采用组蛋白乙酰基转移酶（HAT）、HDACI（TSA及我们独创的中药苦瓜蛋白MCP30），在体外CD34+细胞培养，苯中毒再障小鼠模型及临床苯中毒病人骨髓CD34+细胞中，采用过表达、shRNA 干扰、western blot、电镜等方法全面评估乙酰化修饰下的自噬在苯致造血损伤中的机制。本项目从自噬出发阐明苯致造血损伤的新机制，为苯中毒血液病的防治提供新思路和新靶点，具有重要的基础研究及临床应用价值。

苯中毒诱发的造血系统损伤是严重危害从业者健康的职业疾病之一，其致病机制尚未完全阐明，且目前尚无较好的苯中毒的防治药物或方案。迄今为止，苯中毒的治疗仍以环境脱离及支持对症治疗为主，尚未研发出苯中毒的“有效药”，缺乏特异疗法。本课题组研究发现骨髓单个核细胞染苯后、临床苯中毒病人中自噬水平升高，而加用自噬激动剂雷帕霉素，细胞死亡增多；加用自噬抑制剂 3MA，细胞死亡减少，活细胞数增多，提示自噬在苯致造血损伤中起重要作用。氢醌可降低细胞内乙酰化水平，氢醌降低乙酰化 H3，抑制 p300 活性。p300 特异性抑制剂 C646 或 siRNA 干扰 p300，均可降低蛋白乙酰化，促进细胞自噬水平，与氢醌作用类似。且氢醌可下降 p300 的蛋白水平及 mRNA 水平。MAP30，一种苦瓜蛋白，可逆转氢醌所致的 p300 低表达，从而抑制自噬减少细胞死亡。而在体内通过皮下注射苯构建苯致骨髓毒性小鼠模型，苯组小鼠的血三系明显下降，而加用 MAP30 及自噬抑制剂氯喹组，外周血三系、股骨病理及骨髓涂片均显示骨髓毒性有部分的逆转作用，而与体外结果类似的，苯组小鼠自噬水平增高，乙酰化水平下降，同时 p300 mRNA 及蛋白水平均明显下降。加用 MAP30 组和加用氯喹组均有不同程度逆转。结论：苯/氢醌通过抑制 p300，促进骨髓细胞自噬，这是苯致造血毒性的重要机制。而 MAP30 可提高 p300 表达，从而抑制自噬，保护骨髓细胞。由此可见，MAP30有希望成为治疗苯致血液毒性的靶向药物之一。本项目从自噬出发阐明苯致造血损伤的新机制，为苯中毒血液病的防治提供新思路和新靶点，具有重要的基础研究及临床应用价值。