NCOA4介导的铁自噬在小鼠肠缺血再灌注损伤中的作用及其与铁死亡的关系

Role of nuclear receptor coactivator 4-mediated ferritinophagy in intestinal ischemia-reperfusion injury in mice:relationship with ferroptosis

目的评价核受体辅激活因子4(NCOA4)介导的铁自噬在小鼠肠缺血再灌注损伤中的作用及其与铁死亡的关系。方法SPF级健康雄性C57BL/6小鼠30只,8~10周龄,体重21~25 g,采用随机数字表法分为5组(n=6):假手术组(Sham组)、肠缺血再灌注组(I/R组)、肠缺血再灌注+NCOA4沉默组(I/R+NCOA4 shRNA组)、肠缺血再灌注+自噬抑制剂3-甲基腺嘌呤组(I/R+3-MA)和肠缺血再灌注+NCOA4沉默+自噬激活剂雷帕霉素组(I/R+NCOA4 shRNA+RAPA组)。采用夹闭肠系膜上动脉45 min再灌注的方法制备小鼠肠缺血再灌注损伤模型。于造模前2周,I/R+NCOA4 shRNA组和I/R+NCOA4 shRNA+RAPA组尾静脉注射AAV-NCOA4-shRNA 1×10^(11) vp,Sham组、I/R组和I/R+3-MA组注射AAV shCtrl(腺病毒对照)1×10^(11) vp。于造模前7 d I/R+NCOA4 sh-RNA+RAPA组腹腔注射雷帕霉素4 mg/kg,1次/d;于造模前1 h I/R+3-MA组腹腔注射3-甲基腺嘌呤10 mg/kg。于再灌注2 h时,处死取肠组织,行Chiu评分,采用比色法检测MDA、谷胱甘肽(GSH)和Fe^(2+)含量,ELISA法检测TNF-α和IL-1β含量,Western blot法检测NCOA4、微管相关蛋白1轻链3(LC3)、长链脂酰辅酶A合成酶4(ACSL4)、铁蛋白重链1(FTH1)和谷胱甘肽过氧化物酶4(GPX4)表达水平,计算LC3Ⅱ/LC3Ⅰ比值。结果与Sham组比较,I/R组肠组织Chiu评分、MDA、Fe^(2+)、TNF-α和IL-1β含量升高,GSH含量降低,NCOA4和ACSL4表达上调,LC3Ⅱ/LC3Ⅰ比值升高,GPX4和FTH1表达下调(P<0.05);与I/R组比较,I/R+NCOA4 shRNA组和I/R+3-MA组肠组织Chiu评分、MDA、Fe^(2+)、TNF-α和IL-1β含量降低,GSH含量升高,I/R+NCOA4 shRNA组肠组织NCOA4和ACSL4表达下调,GPX4和FTH1表达上调(P<0.05),I/R+3-MA组ACSL4表达下调,LC3Ⅱ/LC3Ⅰ比值降低,GPX4和FTH1表达上调(P<0.05);I/R+NCOA4 shRNA组与I/R+NCOA4 shRNA+RAPA组上述指标比较差异无统计学意义(P>0.05)。结论NCOA4介导的铁自噬可促进铁死亡,参与小鼠肠缺血再灌注损伤的病理生理机制。

Objective To evaluate the role of nuclear receptor coactivator 4(NCOA4)-mediated ferritinophagy in intestinal ischemia-reperfusion(I/R)injury in mice and the relationship with ferroptosis.Methods Thirty SPF-grade healthy male C57BL/6 mice,aged 8-10 weeks,weighing 21-25 g,were divided into 5 groups(n=6 each)using a random number table method:sham operation group(Sham group),intestinal I/R group(I/R group),intestinal I/R+NCOA4 silencing group(I/R+NCOA4 shRNA group),intestinal I/R+autophagy inhibitor 3-methyladenine(3-MA)group(I/R+3-MA group),and intestinal I/R+NCOA4 silencing+autophagy activator rapamycin(RAPA)group(I/R+NCOA4 shRNA+RAPA group).The intestinal I/R injury model was developed by clamping the superior mesenteric artery for 45 min followed by reperfusion in anesthetized animals.At 2 weeks before developing the model,AAV-NCOA4-shRNA 1×10^(11) vp was injected via the tail vein in group I/R+NCOA4 shRNA and group I/R+NCOA4 shRNA+RAPA,and AAV shCtrl(adenovirus control)1×10^(11) vp was injected in Sham,I/R and I/R+3-MA groups.Rapamycin 4 mg/kg was intraperitoneally injected once a day starting from 7 days before developing the model in group I/R+NCOA4 shRNA+RAPA.In group I/R+3-MA,3-MA 10 mg/kg was intraperitoneally injected at 1 h before developing the model.The animals were sacrificed at 2 h of reperfusion,and intestinal tissues were obtained for determination of contents of malondialdehyde(MDA),glutathione(GSH)and Fe^(2+)(by colorimetry),contents of tumor necrosis factor-alpha(TNF-α)and interleukin-1beta(IL-1β)(by enzyme-linked immunosorbent assay),and expression of NCOA4,microtubule-associated protein 1 light chain 3(LC3),long-chain fatty acyl-CoA synthase 4(ACSL4),ferritin heavy chain 1(FTH1)and glutathione peroxidase 4(GPX4)(by Western blot).The LC3Ⅱ/LC3Ⅰratio was calculated.Intestinal damage was assessed and scored according to Chiu.Results Compared with group Sham,the Chiu′s score and contents of MDA,Fe^(2+),TNF-αand IL-1βwere significantly increased,GSH content was decreased,the expression of NCOA4 and ACSL4 was up-regulated,LC3Ⅱ/LC3Ⅰratio was increased,and the expression of GPX4 and FTH1 was down-regulated in group I/R(P<0.05).Compared with group I/R,the Chiu′s score and contents of MDA,Fe^(2+),TNF-αand IL-1βwere significantly decreased,and GSH content was increased in I/R+NCOA4 shRNA and I/R+3-MA groups,the expression of NCOA4 and ACSL4 was significantly down-regulated,and the expression of GPX4 and FTH1 was up-regulated in group I/R+NCOA4 shRNA(P<0.05),and ACSL4 expression was significantly down-regulated,LC3Ⅱ/LC3Ⅰratio was decreased,and the expression of GPX4 and FTH1 was up-regulated in group I/R+3-MA(P<0.05).Conclusions NCOA4-mediated ferritinophagy can promote ferroptosis,which is involved in the pathophysiological mechanism of intestinal I/R injury in mice.