硫化氢对甲醛诱导HT22细胞铁自噬-铁死亡的拮抗作用

发布时间：2020-09-25 20:15

　　【研究背景与目的】甲醛(Formaldehyde,FA)具有神经毒性,我们课题组以往研究发现硫化氢(Hydrogen sulfide,H_2S)可拮抗FA诱导神经细胞毒性,但是H_2S对FA诱导神经细胞毒性保护机制远未阐述清楚,前期我们实验室发现FA诱导的HT22细胞毒性与铁死亡密切相关。铁蛋白是机体重要的铁储存蛋白,其可通过铁自噬来释放铁实现对机体的调控,然而过度铁自噬可诱发铁死亡。本课题拟观察FA是否能诱导HT22细胞铁自噬-铁死亡以及H_2S是否能够拮抗FA引起的HT22细胞铁自噬-铁死亡,从铁自噬-铁死亡途径探讨H_2S拮抗FA神经毒性的保护机制。【方法】荧光双标法观察NCOA4与LC3B或FTH1的共定位;mRFP-GFP-LC3转染荧光显微镜分析GFP-LC3荧光斑;台盼蓝染色拍照观察细胞活力、FITC/PI或者7AAD/PE染色流式细胞仪检测细胞死亡;酶联免疫吸附法(Enzyme linked Immunosorbent Assay,Elisa)检测细胞内丙二醛(Malondialdehyde,MDA)、谷胱甘肽(Glutathione,GSH)和4-羟基壬烯醛(4-Hydroxynonenal,4-HNE)的含量;BODIPY~(TM)581/591 C11检测细胞脂质活性氧含量;钙黄绿素(Calcein-acetoxymethlester,Ca-AM)染色流式检测细胞Fe~(2+)含量;Western blot分别检测微管结合蛋白1A与1B的第三条轻链(Microtubule-associated,LC3II/I)、泛素结合蛋白(Sequestosome-1,p62/SQSTMI)、铁蛋白重链-1(Ferritin heavy polypeptide 1,FTH1)、以及核受体辅酶激活蛋白4(Nuclear receptor coactivator,NCOA4)的表达。【结果】1.将FA(50、100、200μM)与HT22细胞共孵育24 h后,FA能剂量依赖性地增加HT22细胞死亡率,降低细胞内GSH含量,增加细胞内脂质活性氧、MDA、4-HNE和游离Fe~(2+)的含量,表明FA可引发HT22细胞铁死亡。2.去铁草酰胺(Desferrioxamine,DFO),一种铁离子螯合剂10μM预处理30 min能拮抗FA诱导的HT22细胞死亡率增加,细胞内GSH含量降低,脂质活性氧、MDA、4-HNE和游离Fe~(2+)含量的增加,表明铁离子的过度释放介导了FA诱导的HT22细胞铁死亡。3.FA(50、100、200μM,24 h)能促进NCOA4与LC3B或FTH1共定位、GFP-LC3荧光斑增加、自噬相关蛋白LC3II/I上调和p62下调,同时使FTH1下降和NCOA4的上调,表明FA可引发HT22细胞铁自噬。4.NCOA4 shRNA能取消FA诱导的HT22细胞死亡率增加、细胞内GSH含量的降低、细胞内脂质活性氧、MDA、4-HNE和游离Fe~(2+)含量的增加,表明铁自噬参与了FA引发的HT22细胞铁死亡。5.H_2S(100、200、400μM,30 min)预处理后使能取消NCOA4与LC3B或FTH1共定位、GFP-LC3荧光斑增加、自噬相关蛋白LC3II/I上调和p62下调以及FTH1下降和NCOA4的上调,表明H_2S可明显拮抗FA诱导的HT22细胞铁自噬。6.H_2S(100、200、400μM,30 min)预处理能取消FA诱导的HT22细胞死亡率增加、细胞内GSH含量降低、细胞内脂质活性氧、MDA、4-HNE和游离Fe~(2+)的含量增加,表明H_2S可拮抗FA诱导的HT22细胞铁死亡。【结论】1.甲醛可通过促进铁自噬诱导HT22细胞铁死亡;2.H_2S可抑制甲醛诱导的HT22细胞铁自噬-铁死亡。
【学位单位】：南华大学
【学位级别】：硕士
【学位年份】：2019
【中图分类】：R741
【部分图文】：

细胞,拮抗作用

图 3.1. FA 对 HT22 细胞铁死亡的影响Fig 3.1. Effect of FA on the Ferroptosis in HT22 cells. A, After HT22 cells wereexposed to FA（50, 100, 200 μM）for 24 h, the cell viability was measured by Trypanblue stain. B, The cell death was determined with flow cytometry after FITC/PIstaining. C, The cell lipid ROS was determined with flow cytometry after BODIPYTM581/591 C11 staining. The cellular GSH (D), MDA (E), and 4-HNE (F) levels weremeasured using Elisa kit. G, The content of cellular Fe2+was determined with flowcytometry after Ca-AM staining. Mean Fe2+fluorescence =(Mean Ca-AM+2,2′-BIPfluorescence) - Mean Ca-AM fluorescence. Values are the mean±S.E.M., n=3,*P<0.05,**P<0.01,***P<0.001, versus control group.3.2 DFO 对 FA 诱导 HT22 细胞铁死亡的拮抗作用

图 3.2. DFO 对 FA 引起 HT22 细胞铁死亡的影响Fig 3.2. Effect of DFO on FA-induced Ferroptosis in HT22 cells. Afterpretreatment with DFO (10 μM) for 30 min, HT22 cells were co-cultured with FA( 100 μM）for 24 h, A. The cell viability was measured by Trypan blue stain. B, Thecell death was determined with flow cytometry after FITC/PI staining. C, The the celllipid ROS was determined with flow cytometry after BODIPYTM581/591 C11staining.The cellular GSH (D), MDA (E), and 4-HNE (F) levels were measured usingElisa kit. G, The content of cellular Fe2+was determined with flow cytometry afterCa-AM staining. Mean Fe2+fluorescence =(Mean Ca-AM+2,2′-BIP fluorescence) -Mean Ca-AM fluorescence. Values are the mean±S.E.M., n=3,*P<0.05,**P<0.01,***P<0.001, versus control group,#P<0.05,##P<0.01,###P<0.001, versus FA-treatedalone group.

自噬,细胞

28图 3.3. FA 对 HT22 细胞铁自噬的影响Fig 3.3. Effect of FA on the ferritinophagy in HT22 cells. A,B After HT22 cellswere exposed to FA（50, 100, 200 μM）for 24 h, the cellular distribution ofendogenous NCOA4, LC3B, and FTH1 was evaluated by confocal microscopy. Eachphotomicrograph represents 20× magnification of the distinct region of interest. C-E,HT22 cells expressing mRFP GFP LC3 were treated overnight with the indicatedcompounds, fixed and imaged by automated acquisition. Representative images of anoverlay of GFP (green) and mCherry (red) are shown with a scale bar equivalent to 10

【相似文献】