STAT3/NCOA4介导的铁自噬在红景天苷减轻小鼠肠缺血再灌注损伤中的作用:与铁死亡的关系

Role of STAT3/NCOA4-mediated ferritinophagy in salidroside-induced attenuation of intestinal ischemia-reperfusion injury in mice: relationship with ferroptosis

目的评价信号转导和转录激活因子3(STAT3)/核受体共激活因子4(NCOA4)介导的铁自噬在红景天苷减轻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小鼠36只, 6~8周龄, 体质量20~25 g, 采用随机数字表法分为6组(n=6):假手术组(S组)、假手术+红景天苷组(SS组)、肠缺血再灌注组(IR组)、肠缺血再灌注+红景天苷组(IS组)、肠缺血再灌注+红景天苷+自噬激活剂雷帕霉素组(ISR组)和肠缺血再灌注+红景天苷+STAT3活化剂Colivelin组(ISC组)。IR组、IS组、ISR和ISC组采用夹闭肠系膜上动脉45 min、再灌注30 min的方法建立小鼠肠缺血再灌注损伤模型, S组和SS组仅分离肠系膜上动脉, 不夹闭。于造模前1周时, SS组、IS组、ISC组和ISR组连续7 d腹腔注射红景天苷40 mg/kg, 1次/d;ISR组连续7 d腹腔注射雷帕霉素4 mg/kg, 1次/d;ISC组连续7 d腹腔注射Colivelin 1 mg/kg, 1次/d;其余2组连续7 d腹腔注射等量生理盐水, 1次/d。于再灌注30 min时处死小鼠, 取小肠组织, HE染色后光镜下观察病理学结果, 并行Chiu评分, 检测MDA、Fe^(2+)、谷胱甘肽(GSH)、ROS含量及SOD活性, 采用Western blot法检测p-STAT3、STAT3、谷胱甘肽过氧化物酶4(GPX4)、NCOA4和铁蛋白重链1(FTH1)的表达。结果与S组比较, IR组小肠组织Chiu评分、MDA、Fe^(2+)和ROS含量升高, GSH含量降低, SOD活性降低, 小肠组织p-STAT3和NCOA4表达上调, GPX4和FTH1表达下调, p-STAT3/STAT3比值升高(P<0.05), 小肠组织发生病理学损伤, SS组上述各指标差异无统计学意义(P>0.05);与IR组比较, IS组小肠组织Chiu评分、MDA、Fe^(2+)和ROS含量降低, GSH含量升高, SOD活性升高, p-STAT3和NCOA4表达下调, GPX4和FTH1表达上调, p-STAT3/STAT3比值降低(P<0.05), 小肠组织病理学损伤减轻;与IS组比较, ISR组和ISC组小肠组织Chiu评分、MDA、Fe^(2+)和ROS含量升高, GSH含量降低, SOD活性降低, p-STAT3和NCOA4表达上调, GPX4和FTH1表达下调, p-STAT3/STAT3比值升高(P<0.05), 小肠组织病理学损伤加重。5组小肠组织STAT3表达比较差异无统计学意义(P>0.05)。结论 STAT3/NCOA4介导的铁自噬参与了红景天苷减轻小鼠肠缺血再灌注损伤的过程, 可能与抑制铁死亡有关。

Objective To evaluate the role of signal transducer and activator of transcription 3(STAT3)/nuclear receptor coactivator 4(NCOA4)-mediated ferritinophagy in salidroside-induced attenuation of intestinal ischemia-reperfusion(I/R)injury in mice and the relationship with ferroptosis.Methods Thirty-six SPF-grade healthy male C57BL mice,aged 6-8 weeks,weighing 20-25 g,were divided into 6 groups(n=6 each)by a random number table method:sham operation group(S group),sham operation+salidroside group(SS group),intestinal I/R group(IR group),intestinal I/R+salidroside group(IS group),intestinal I/R+salidroside+autophagy activator rapamycin group(ISR group)and intestinal I/R+salidroside+STAT3 activator colivelin group(ISC group).The intestinal I/R injury model was established by clamping the superior mesenteric artery for 45 min followed by 30-min reperfusion in IR,IS,ISR and ISC groups,while the superior mesenteric artery was only isolated without clipping in S and SS groups.At 1 week before developing the model,salidroside 40 mg/kg was intraperitoneally injected once a day for 7 consecutive days in SS,IS,ISC and ISR groups,rapamycin 4 mg/kg was intraperitoneally injected once a day for 7 consecutive days in group ISR,colivelin 1 mg/kg was intraperitoneally injected once a day for 7 consecutive days in group ISC,while the equal volume of normal saline was given instead in the rest two groups.The mice were sacrificed at 30 min of reperfusion,and intestinal tissues were obtained for examination of the pathological changes after HE staining(with a optical microscope)which were scored according to Chiu and for determination of contents of malondialdehyde(MDA),Fe,glutathione(GSH)and reactive oxygen species(ROS),activity of superoxide dismutase(SOD)and expression of p-STAT3,STAT3,glutathione peroxidase 4(GPX4),NCOA4 and ferritin heavy chain 1(FTH1)in intestinal tissues(by Western blot).Results Compared with group S,the Chiu′s score and contents of MDA,Feand ROS were significantly increased,the content of GSH was decreased,the activity of SOD was decreased,the expression of p-STAT3 and NCOA4 was up-regulated,the expression of GPX4 and FTH1 was down-regulated,the p-STAT3/STAT3 ratio was increased(P<0.05),pathological injury was found in intestinal tissues,and no significant change was found in the aforementioned indexes in group IR(P>0.05).Compared with group IR,the Chiu′s score and contents of MDA,Feand ROS were significantly decreased,GSH content was increased,SOD activity was increased,the expression of p-STAT3 and NCOA4 was down-regulated,the expression of GPX4 and FTH1 was up-regulated,p-STAT3/STAT3 ratio was decreased(P<0.05),and the pathological injury was significantly alleviated in intestinal tissues in group IS.Compared with group IS,the Chiu′s score and contents of MDA,Feand ROS were significantly increased,GSH content was decreased,SOD activity was decreased,the expression of p-STAT3 and NCOA4 was up-regulated,the expression of GPX4 and FTH1 was down-regulated,p-STAT3/STAT3 ratio was increased(P<0.05),and the pathological injury was aggravated in intestinal tissues in ISR and ISC groups.There was no statistically significant difference in the expression of STAT3 among the five groups(P>0.05).Conclusions STAT3/NCOA4-mediated ferritinophagy is involved in the process of salidroside-induced reduction of intestinal I/R injury in mice,which may be related to inhibiting ferroptosis.