摘要目的 确定脂质抗氧化酶Peroxiredoxin 6(Prdx6)对急性肺损伤的抗氧化保护作用.方法应用100% O2吸入诱导雄性小鼠,建立急性肺损伤模型,用H2O2干预诱导,建立细胞损伤模型.分别观察Prdx6基因敲除(Prdx6-/-)小鼠、Prdx6基因过度表达(Tg Prdx6)小鼠、胞浆谷胱甘肽过氧化物酶(Gpxl)基因敲除(Gpxl-/-)小鼠、野生(WT)小鼠及其肺Ⅱ型上皮细胞的生存率,支气管肺泡灌洗液(BALF)中蛋白和细胞总数;Western Blot法检测Prdx6或Gpx1蛋白表达水平;观察肺Ⅱ型上皮细胞凋亡情况;采用硫代巴比妥酸反应物法和双苯酯-1磷酸芘荧光测定法分别检测肺组织和肺Ⅱ型上皮细胞膜质脂氧化水平.结果 Prdx6-/-小鼠生存时间较WT小鼠缩短约24 h,TgPrdx6小鼠的生存时间较其他组延长;在细胞损伤模型中,Prdx6对细胞生存的保护作用与H2O2干预浓度相关;高氧吸入72 h后,Prdx6-/-小鼠的肺内Gpx1蛋白表达水平下调7倍;Gpx1-/-小鼠的肺内Prdx6蛋白表达水平则无明显下调.H2O2干预处理后,肺Ⅱ型上皮细胞凋亡细胞数及百分率的增加与H2O2剂量正相关,并在Prdx6-/-小鼠表现最为明显,而Tg Prdx6小鼠组则呈现了持续稳定的10%的凋亡细胞数;Prdx6-/-小鼠组的肺Ⅱ型上皮细胞的细胞膜脂质氧化水平升高,约为WT小鼠2倍,为Tg Prdx6组4倍;Prdx6-/-小鼠肺组织的脂质氧化水平较其他组明显增高.结论 Prdx6具有重要的抗氧化性肺损伤的保护作用,特别是Pxdx6抗细胞凋亡和抗细胞膜脂质氧化的功能提示了其以细胞为单位独特的保护机制.

abstractsObjective To confirm the antioxidant protective effect of peroxiredoxin 6 (Prdx6) in acute lung injury in mice.Methods Lung injury or lung alveolar type Ⅱ epithelial cell (AEC Ⅱ) injury models were induced in mice by 100% O2 exposure or H2O2 treatments.Mice and AEC Ⅱ cell survival rate or BALF analysis were applied for evaluating the degree of acute lung injury.Western Blot assay was used to determine Prdx6 or Gpxl protein expression in lung.Annexin V staining method was applied to detect cell apoptosis on cultured AEC Ⅱ cell,and thibarbituric acid reactive substance (TBARS) measurement and dephenyl-1-pyrenyl phosphoine (DPPP) assays were separately used to measure the level of lipid peroxidation in mice lung and AEC Ⅱ cell membrane.Results Under 100% O2 exposure,prdx6-/-mice presented 24 h shorter survival time compared to wild type (WT) mice,on the contrary,Prdx6 gene over-expressed (Tg Prdx6) mice showed enhanced mice survival;meanwhile,the degree of AEC Ⅱ cell injury had H2O2-dose dependent pattern with interactive relationship of Prdx6 protection.Under 100% O2 exposure for 72 h,it caused 7-fold decreased Gpx1 expression in Prdx6-/-mouse lung with no remarkable decrease of Prdx6 expression in Gpx1-/-mice.The percentage of apoptotic cells was significantly increased in AEC Ⅱ cells from Prdx6-/-mice,and the percentage of AEC Ⅱ apoptotic cells from Tg Prdx6 kept consistently around 10% under H2O2 treatments;also,the lipid peroxidation level of AEC Ⅱ cell membrane was the highest in the group of Prdx6-/-mice,which was about 2 or 4-fold increased compared to the groups of WT or Tg Prdx6,separately;meanwhile,the lipid pemxidation level in Prdx6-/-mice,was also the highest compared to the other groups.Conclusions Prdx6 plays a critical role in defending acute oxidative lung injury and it's function of defending cell apoptosis and cell membrane lipid peroxidation suggests its unique cell-based protective effect.