自噬促进大鼠肠缺血再灌注损伤的细胞铁死亡

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Fe2+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、Fe2+含量升高（P<0.01），I/R+3-MA组LDH（P<0.05）、Fe2+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、Fe2+（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）、Fe2+（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。

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

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. Fe2+ 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 Fe2+ were increased (P<0.01) in I and I/R, LDH (P<0.05), Fe2+ (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, Fe2+ (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), Fe2+ (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.