自噬促进大鼠肠缺血再灌注损伤的细胞铁死亡

Involvement of autophagy in iron ion regulation promotes ferroptosis in cells undergoing intestinal ischemia-reperfusion injury

目的:探讨自噬对肠缺血再灌注损伤中细胞铁死亡的影响。方法:采用随机数字表法将24只SPF级Wistar大鼠(体质量200~220 g)分为4组(n=6):伪手术组(sham组)、缺血组(I组)、缺血再灌注组(I/R组)、缺血再灌注+自噬抑制剂组(I/R+3-MA组)。夹闭肠系膜上动脉1 h构建缺血模型,再灌注2 h建立大鼠肠缺血再灌注损伤模型;采用HE染色光镜下观察肠黏膜病理改变并行Chiu评分;比色法检测Fe^(2+)含量;ELISA法检测乳酸脱氢酶(LDH)、过氧化脂质(LPO)含量;Western Blot检测铁蛋白重肽(ferritin heavy polypeptide,FTH)1、核受体共激活子(nuclear receptor coactivator,NCOA)4、LC3-II/I表达,透射电镜检测线粒体形态。结果:与sham组比较,其余3组小肠组织Chiu评分均增高(P<0.01),LC3II/I表达上调,FTH1、NCOA4表达下调(P<0.01),I组、I/R组LDH、Fe^(2+)含量升高(P<0.01),I/R+3-MA组LDH(P<0.05)、Fe^(2+)含量升高(P<0.01),I/R组LPO含量升高(P<0.01);与I组比较,I/R组Chiu评分增高(P<0.01)、LDH、Fe^(2+)(P<0.05)、LPO(P<0.01)含量升高,FTH1、NCOA4表达下调、LC3II/I表达上调(P<0.01);与I/R组比较,I/R+3-MA组Chiu评分下降,LDH、LPO(P<0.01)、Fe^(2+)(P<0.05)含量下降,FTH1、NCOA4表达上调,LC3II/I表达下调(P<0.01)。线粒体形态学变化:sham组可见肠组织结构完整,线粒体数量较多结构完整;I组线粒体嵴数量减少,双层膜结构不完整;I/R组线粒体嵴大量减少,细胞器损伤严重。3-MA抑制后线粒体数量和内嵴有所增多,膜逐渐恢复完整。结论:肠I/R损伤中,自噬参与细胞铁离子的调控,促进细胞铁死亡的发生,抑制自噬可以减轻I/R肠损伤。

AIM:To investigate the effect of autophagy on cell ferroptosis in intestinal ischemia-reperfusion injury.METHODS:Twenty-four SPF grade Wistar rats weighing 200-220 g were divided into 4 groups(n=6):sham operation group(sham group),ischemia group(I group),ischemia-reperfusion group(I/R group),and ischemia-reperfusion+autophagy inhibitor group(I/R+3-MA group).The ischemia model was established by clamping the superior mesenteric artery for 1 hour,and the intestinal ischemia-reperfusion injury model was established by reperfusion for 2 hours.HE staining was used to observe the pathological changes of intestinal mucosa and Chiu score under light microscope.Fe^(2+)contents was measured by Colorimetric and LDH,LPO contents were measured by ELISA.FTH1,NCOA4,and LC3II/I expression by WB,and mitochondrial morphology was measured by transmission electron microscopy.RESULTS:Compared with the sham group,the remaining three groups had higher Chiu scores,FTH1 and NCOA4 were downregulated and LC3II/I was upregulated(P<0.01),LDH and Fe^(2+)were increased(P<0.01)in I and I/R,LDH(P<0.05),Fe^(2+)(P<0.01)were increased in I/R+3-MA group,the LPO content was elevated(P<0.01)in I/R;Compared with group I,the Chiu score(P<0.01),LDH,Fe^(2+)(P<0.05)and LPO(P<0.01)content were increased in group I/R(P<0.01),FTH1,NCOA4 was downregulated and LC3II/I was upregulated(P<0.01);Compared with the I/R,The Chiu score was decreased,the LDHand LPO(P<0.01),Fe^(2+)(P<0.05)content were decreased in I/R+3-MA,FTH1,NCOA4 was up-regulated and LC3II/I was down-regulated(P<0.01).Mitochondrial morphological changes:in sham group,intestinal tissue and mitochondria were intact,I group the mitochondrial cristae is reduced,and the double membrane structure is incomplete;in I/R group,the mitochondrial cristae was greatly reduced,and the organelle damage was serious.Increincrease in mitochondrial number and internal cristae were observed after 3-MA inhibition,and the membrane gradually became intact.CONCLUSION:In intestinal ischemia-reperfusion injury,autophagy participates in the regulation of cellular iron ions and promotes the occurrence of cell ferroptosis,and the inhibition of autophagy can reduce I/R intestinal injury.