小胶质细胞组蛋白去乙酰化酶3在低压低氧诱导的氧化应激中的作用

Role of microglial histone deacetylases 3 in hypobaric hypoxia-induced oxidative stress

目的探究低压低氧对脑内氧化应激的影响及小胶质细胞HDAC3在其中的作用。方法将野生型小鼠置于模拟海拔高度6000米的低压舱中连续饲养7 d,随后取小鼠海马组织并提取RNA,实时定量聚合酶链反应(real-time polymerase chain reaction,real-time PCR)检测氧化应激标志物诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)和超氧化物歧化酶(superoxidase dismutase,SOD)的mRNA,以及缺氧诱导因子-1α(hypoxia inducible factors-1α,HIF-1α)和组蛋白去乙酰化酶3(histone deacetylases 3,HDAC3)的mRNA。利用低氧培养箱[0.1%(体积分数)O2]对小胶质细胞系BV2细胞进行低氧处理,蛋白质免疫印迹和real-time PCR法检测iNOS、SOD、HDAC3和HIF-1α的蛋白及mRNA水平,以及利用活性氧(reactive oxygen species,ROS)检测试剂盒和流式细胞术检测氧化应激水平。然后敲低或抑制小胶质细胞系HDAC3表达或活性,并进行低氧处理,检测上述相关因子及ROS的变化。最后,利用小胶质细胞特异性敲除HDAC3的小鼠进行低压低氧[0.1%(体积分数)O2]处理,检测干预小胶质细胞HDAC3表达之后iNOS的表达水平。结果体内实验结果显示低压低氧处理显著增加小鼠脑内iNOS和SOD的mRNA;体外实验结果显示低氧处理导致BV2细胞iNOS、HDAC3和HIF-1α的蛋白水平升高,且iNOS和SOD的mRNA也增加。流式分析结果显示低氧处理导致BV2细胞ROS水平上升。敲低或抑制HDAC3则可以显著抑制低氧诱导的ROS产生和iNOS上调。特异性敲除小胶质细胞HDAC3可以显著抑制低压低氧处理导致的小鼠脑组织中iNOS上调。结论低压低氧可引起脑组织及小胶质细胞氧化应激水平升高,抑制小胶质细胞HDAC3可减轻低压低氧诱导的氧化应激。

Objective To investigate the effect of hypobaric hypoxiaon the brain tissue oxidative stress level and whether microglial HDAC3 is involved in this process.Methods First,wild type mice were treated in a hypobaric chamber at a simulated altitude of 6000 meters for 7 days.The mRNA levels of hypoxia inducible factors-1α(HIF-1α),inducible nitric oxide synthase(iNOS),superoxidase dismutase(SOD)and histone deacetylases 3(HDAC3)in the hippocampus were analyzed by real-time quantitative polymerase chain reaction(real-time PCR).Then,BV2 cells were treated in atmosphere with 0.1%O2 in a hypoxia incubator and the mRNA and protein levels of iNOS,SOD,HDAC3 and HIF-1αwere determined by Western blotting and real-time PCR.The levels of reactive oxygen species(ROS)were analyzed by flow cytometry.Furthermore,HDAC3-silenced and wild type BV2 cells were cultured in 0.1%O2 atmosphere and the levels of ROS,iNOS and SOD were analyzed as above.Last,microglial HDAC3 conditional knockout mice and wild type mice were treated in hypoxia atmosphere,and the mRNA level of iNOS were analyzed by real-time PCR.Results The mRNA levels of iNOS,SOD and HDAC3 were upregulated in the hippocampus from mice underwent hypobaric hypoxia.The in vitro experiments indicated that the protein levels of iNOS,HDAC3 and HIF-1αwere increased in BV2 cells treated 0.1%O2,and the mRNA levels of iNOS and SOD were increased as well.By flow cytometry,we found the level of ROS was increased when BV2 cells were cultured in hypoxia atmosphere.Inhibition of HDAC3 significantly reduced the production of hypoxia-induced ROS,as well as decreased expression of SOD and iNOS.In addition,knockout of HDAC3 in microglia inhibited the hypoxia-induced upregulation iNOS.Conclusion The hypoxia treatment resulted in increased oxidative stress in the hippocampus and microglia,and inhibition of microglial HDAC3 ameliorated hypoxia-induced oxidative stress.