摘要
目的观察不同浓度脂多糖(lippopolysaccharide,LPS)对肺上皮细胞坏死性凋亡和线粒体动力学相关蛋白1(dynamin related protein 1,Drp1)依赖的线粒体自噬的影响,探讨LPS制作脓毒症急性肺损伤模型的合适浓度。方法对数生长期A549细胞分为对照组、5 mg/L LPS组、10 mg/L LPS组、25 mg/L LPS组、50 mg/L LPS组,5 mg/L LPS组、10 mg/L LPS组、25 mg/L LPS组、50 mg/L LPS组分别采用PBS稀释的5、10、25、50 mg/L LPS处理16 h,对照组给予等体积PBS培养16 h。采用Western blot法检测各组细胞坏死性凋亡标志蛋白p-RIP3、RIPK1、p-MLKL和肿瘤坏死因子(tumor necrosis factor,TNF)-α以及线粒体自噬标志蛋白PINK1、LC3B、Drp1、p-Mfn2相对表达量,并采用JC-1线粒体探针检测线粒体膜电位。结果10、25、50 mg/L LPS组细胞p-RIP3、RIPK1、p-MLKL、TNF-α、PINK1、Drp1、LC3B蛋白相对表达量依次增高(P<0.05),且均高于5 mg/L LPS组和对照组(P<0.05)。10、25、50 mg/L LPS组p-Mfn2蛋白相对表达量(1.043±0.085、0.844±0.085、0.204±0.040)和细胞线粒体膜电位(7.323±0.331、6.429±0.456、4.661±0.456)依次降低(P<0.05),且均低于5 mg/L LPS组(1.288±0.035、9.013±0.648)和对照组(2.497±0.440、14.392±1.216)(P<0.05)。5 mg/L LPS组细胞p-RIP3、RIPK1、p-MLKL、TNF-α、PINK1、LC3B蛋白相对表达量高于对照组(P<0.05),p-Mfn2蛋白相对表达量和细胞线粒体膜电位低于对照组(P<0.05),Drp1蛋白相对表达量与对照组比较差异无统计学意义(P>0.05)。结论10~50 mg/L LPS可诱导肺上皮细胞发生坏死性凋亡,可能与Drp1依赖的线粒体自噬有关;10~50 mg/L LPS可能是建立脓毒症急性肺损伤细胞模型的较合适浓度。
Objective To investigate the suitable concentration of lippopolysaccharide(LPS)for sepsis induced acute lung injury models by observing the effects of LPS at different concentrations on the necroptosis and dynamic related protein 1(Drp1)-dependent mitophagy of lung epithelial cells.Methods The A549 cells in logarigathmic growth phase were divided into 5,10,25 and 50 mg/L LPS groups cultured with 5,10,25 and 50 mg/L LPS diluted by PBS respectively,and control group was treated with the equivalent volume of PBS,for 16 h.Western blot was used to detect the relative expressions of necroptosis marker proteins as p-RIP3,RIPK1,p-MLKL and tumor necrosis factor(TNF)-αas well as mitophagy marker proteins as PINK1,LC3 B,Drp1 and p-Mfn2 in each group.The mitochondrial membrane potential was examined by JC-1.Results The relative expressions of p-RIP3,RIPK1,p-MLKL and TNF-αincreased,and cell mitochondrial membrane potential(14.392±1.216,9.013±0.648,7.323±0.331,6.4286±0.456,4.661±0.456)decreased gradually in turn in control group,5 mg/L LPS group,10 mg/L LPS group,25 mg/L LPS group and50 mg/L group(P<0.05).The relative expressions of PINK1,Drp1 and LC3 Bproteins increased,and the relative expressions of p-Mfn2 and the mitochondrial membrane potential decreased gradually in turn in control group,5 mg/L LPS group,10 mg/L LPS group,25 mg/L LPS group and 50 mg/L group(P<0.05),except the relative expression of Drp1 protein showed no significant differences between 5 mg/L LPS group and control group(P>0.05).Conclusion LPS at concentration of 10 to 50 mg/L would induce the necroptosis of lung epithelial cells probably by Drp1-dependent mitophagy,and it might be the suitable concentration for establishing the septic acute lung injury cell model.
作者
陈祝桂
彭志勇
张智豪
梁青春
刘瑞蒙
张良清
刘友坦
CHEN Zhugui;PENG Zhiyong;ZHANG Zhihao;LIANG Qingchun;LIU Ruimeng;LIU Youtan(Department of Anesthesiology,Affiliated Hospital of Guangdong Medical University,Zhanjiang524023,China;Department of Anesthesiology,Shenzhen Hospital of Southern Medical University,Shenzhen518100,China)
出处
《中华实用诊断与治疗杂志》
2020年第4期330-333,共4页
Journal of Chinese Practical Diagnosis and Therapy
基金
国家自然科学基金(81670075)。
作者简介
通信作者:刘友坦,E-mail:youtanliuhao@163.com;通信作者:张良清,E-mail:zhanglq1970@163.com。
