抑制铁死亡可减轻脓毒症小鼠的心肌损伤:脂钙蛋白-2的作用

Inhibiting ferroptosis attenuates myocardial injury in septic mice:the role of lipocalin-2

目的观察铁死亡在盲肠结扎与穿孔(CLP)法诱导的脓毒症小鼠心肌损伤中的作用,并探讨脂钙蛋白-2(Lcn2)在铁死亡中的可能作用。方法选取8周龄雄性C57BL/6小鼠,采用CLP法诱导脓毒症心肌损伤模型。小鼠随机分为3组(10只/组):假手术组、脓毒症组(CLP,小鼠接受CLP手术)、脓毒症+铁死亡抑制剂Ferrostain-1组(CLP+Fer-1,小鼠腹腔注射浓度为5 mg/mL的Fer-15 mg/kg,1 h后接受CLP手术)。各组小鼠术后24 h通过超声心动图检测小鼠心功能。H&E染色观察心肌损伤,透射电镜观察心肌纤维细微结构和线粒体的变化;ELISA法测定血清中炎症因子肿瘤坏死因子-α(TNF-α)水平;组织铁试剂盒测定心肌组织铁含量的变化;Westernblot法检测心肌组织中Lcn2蛋白和铁死亡相关蛋白谷胱甘肽过氧化物酶4(GPX4)和铁死亡抑制蛋白1(FSP1)的表达变化。结果与假手术组相比,CLP术后24 h,脓毒症小鼠心脏收缩与舒张功能减弱,左心室射血分数(LVEF%)、左心室缩短分数(LVFS%)和左心室舒张末期内径(LVIDd)降低(P<0.05);左心室收缩期末期内径(LVIDs)升高(P<0.05)。与CLP组相比,CLP+铁死亡抑制剂Fer-1组LVEF%、LVFS%和LVIDd升高(P<0.05);LVIDs降低(P<0.05)。光镜下观察到CLP小鼠心肌纤维排列不整齐,部分变性,有炎症细胞浸润,间质水肿,横纹模糊,红细胞渗出。透射电镜观察到部分线粒体嵴减少,外膜破裂,部分线粒体变小,膜密度增高。CLP+Fer-1组小鼠心肌形态学有改善,线粒体损伤减轻。与假手术组相比,CLP组血清TNF-α水平、心肌组织铁含量、Lcn2蛋白表达升高(P<0.01),GPX4、FSP1蛋白表达降低(P<0.01)。与CLP组相比,CLP+Fer-1组血清TNF-α水平、心肌组织铁含量、和Lcn2蛋白表达降低(P<0.05);GPX4、FSP1蛋白表达升高(P<0.05)。结论来源于GPX4、FSP1不同途径的铁死亡参与CLP引起的脓毒症性心肌损伤的发生,抑制铁死亡可减轻脓毒症心肌损伤,Lcn2可能参与其中。

Objective To explore the contribution of ferroptosis to myocardial injury in mouse models of sepsis and the role lipocalin-2(Lcn2)in ferroptosis.Methods Adult male C57BL/6 mice were randomized equally into sham-operated group,cecal ligation and puncture(CLP)-induced sepsis group,and CLP+Fer-1 group where the mice received intraperitoneal injection of 5 mg/mL Fer-1(5 mg/kg)1 h before CLP.The left ventricular functions(including LVEF%,LVFS%,LVIDd and LVIDs)of the mice were assessed by echocardiography at 24 h after CLP.Myocardial injury in the mice was observed with HE staining,and the changes of myocardial ultrastructure and mitochondria were observed using transmission electron microscopy(TEM).Serum TNF-αlevel was measured with ELISA,and the changes of myocardial iron content were detected using tissue iron kit.The protein expressions of myocardial Lcn2,glutathione peroxidase 4(GPX4)and ferroptosis suppressor protein 1(FSP1)were determined with Western blotting.Results The septic mice showed significantly decreased LVEF%,LVFS%and LVIDd and increased LVIDs at 24 h after CLP(P<0.05),and these changes were significantly improved by Fer-1 treatment.Sepsis caused obvious myocardial pathologies and changes in myocardial ultrastructure and mitochondria,which were significantly improved by Fer-1 treatment.Fer-1 treatment also significantly ameliorated sepsis-induced elevations of serum TNF-αlevel,myocardial tissue iron content,and Lcn2 protein expression and the reduction of GPX4 and FSP1 protein expression levels(P<0.05).Conclusion GPX4-and FSP1-mediated ferroptosis are involved in myocardial injury in mice with CLP-induced sepsis,and inhibition of ferroptosis can attenuate septic myocardial injury,in which Lcn2 may play a role.