铁死亡在重症急性胰腺炎引起的急性肾损伤中的作用及其机制

Effects of ferroptosis on acute renal injury induced by severe acute pancreatitis and underlying mechanism

目的探讨铁死亡在重症急性胰腺炎(SAP)诱发急性肾损伤(AKI)的作用及其机制。方法胰腺炎建模后24 h,将36只SD大鼠(购自青岛大学医学部实验室动物中心)采用完全随机分组法分为3组(n=12):假手术组(SO组)、重症急性胰腺炎组(SAP组)、铁死亡抑制剂Ferrostatin-1干预组(SAP+Fer组)。建立SAP模型24 h后,检测血清淀粉酶(AMY)、肌酐(Cr)和血尿素氮(BUN)水平;检测肾组织中铁含量、丙二醛(MDA)、活性氧(ROS)、谷胱甘肽(GSH)变化及谷胱甘肽过氧化物酶4(GPX4)活性;苏木精-伊红(HE)染色观察胰腺和肾脏组织学改变;透射电镜(TEM)观察铁死亡的形态学特征;蛋白质印迹法(Western blot)、反转录-聚合酶链反应(RT-PCR)和免疫荧光染色等方法分析长链脂酰CoA合成酶4(ACSL4)、谷胱甘肽过氧化物酶4(GPX4)和铁蛋白重链1(FTH1)等铁死亡相关蛋白和基因的表达。组间比较采用方差分析,进一步两两比较采用LSD-t检验。结果SAP组大鼠血清AMY[(6276.5±562.2)比(1086.6±192.3)U/L]、Cr[(98.0±16.2)比(19.5±5.2)μmol/L]、BUN[(17.1±2.8)比(7.2±1.7)mmol/L]水平明显高于SO组(t=30.314、15.927、10.375,P值均<0.01),差异均有统计学意义;肾组织铁[(0.65±0.13)比(0.44±0.12)mg/g]、MDA[(7.25±0.81)比(1.84±0.24)μmol/g]、ROS[(68.54±7.04)比(15.67±5.21)×104/mg]高于SO组(t=3.855、22.167、11.496,P值均<0.01),GSH[(358.38±41.59)比(518.64±55.72)nmol/g]和GPX4[(7.75±1.09)比(14.72±2.56)U/mg]明显低于SO组(t=7.984、8.674,P值均<0.01),差异有统计学意义;胰腺和肾组织病理损伤程度增加;线粒体出现明显萎缩;肾脏ACSL4蛋白(0.84±0.03比0.34±0.02)表达高于SO组(t=7.876,P<0.01),GPX4、FTH1蛋白(0.69±0.03比1.04±0.06、0.26±0.02比0.83±0.04)表达低于SO组(t=5.651、8.134,P值均<0.01),差异有统计学意义;ACSL4、铁响应元件结合蛋白2(IREB2)mRNA(0.87±0.06比0.24±0.03、1.23±0.05比0.32±0.02)表达高于SO组(t=12.864、15.163,P均<0.01),差异有统计学意义。SAP+Fer组大鼠血清AMY[(5124.3±483.5)比(6276.5±562.2)U/L]、Cr[(55.2±13.7)比(98.0±16.2)μmol/L]、BUN[(9.8±2.1)比(17.1±2.8)mmol/L]水平显著低于SAP组(t=5.287、6.989、7.359,P值均<0.01),差异有统计学意义;肾组织铁[(0.54±0.07)比(0.65±0.13)mg/g]、MDA[(4.67±0.73)比(7.25±0.81)μmol/g]、ROS[(28.39±6.36)比(68.54±7.04)×104/mg]低于SAP组(t=2.639、7.176、8.247,P值均<0.05),GSH[(448.21±45.51)比(358.38±41.59)nmol/g]和GPX4[(11.31±1.89)比(7.75±1.09)U/mg]高于SAP组(t=5.048、5.639,P值均<0.01),差异有统计学意义;胰腺和肾组织病理损伤程度减轻;线粒体轻度萎缩;肾脏ACSL4蛋白(0.49±0.02比0.84±0.03)表达低于SAP组(t=2.781,P<0.05),GPX4、FTH1蛋白(0.69±0.03比1.04±0.06、0.26±0.02比0.83±0.04)表达高于SAP组(t=2.427、2.876,P值均<0.05),差异有统计学意义;ACSL4、IREB2 mRNA(0.52±0.04比0.87±0.06、0.74±0.04比1.23±0.05)表达低于SAP组(t=5.843、6.781,P值均<0.01),差异均有统计学意义。结论铁死亡参与SAP引起的AKI,抑制铁死亡能改善肾功能,减轻肾组织脂质过氧化和病理损伤。

Objective We aimed to investigate effects and mechanisms of ferroptosis on acute kidney injury(AKI)induced by severe acute pancreatitis(SAP).Methods The rats were randomly divided into three groups(n=12):sham operation(SO)group,SAP group,Ferrostatin-1 intervention(SAP+Fer)group.After 24 hours of inducing SAP,the levels of serum amylase(AMY),creatinine(Cr)and blood urea nitrogen(BUN)in rats were assessed;the content of iron,malondialdehyde(MDA),glutathione(GSH)and glutathione peroxidase 4(GPX4)activity in renal tissue were measured.Hematoxylin and eosin(HE)staining was used to examine pancreatic and renal histological changes.Morphological characteristics of ferroptosis were observed by transmission electron microcope(TEM).Western blotting,reverse transcriptase-polymerase chain reaction(RT-PCR),and immunofluorescence staining were performed to analyze the expression of ferroptosis-related proteins and genes such as Acyl-CoA synthetase long-chain family member 4(ACSL4),GPX4 and ferritin heavy chain 1(FTH1).The variance analysis and least significant difference-t(LSD-t)test were used in the data analysis.Results As compared with SO group,the serum levels of AMY[(6276.5±562.2)vs.(1086.6±192.3)U/L],Cr[(98.0±16.2)vs.(19.5±5.2)μmol/L],BUN[(17.1±2.8)vs.(7.2±1.7)mmol/L]were significantly increased(t=30.314,15.927,10.375,P<0.01);Iron[(0.65±0.13)vs.(0.44±0.12)mg/g],MDA[(7.25±0.81)vs.(1.84±0.24)μmol/g]and ROS[(68.54±7.04)vs.(15.67±5.21)×104/mg]in renal tissues were increased(t=11.496,P<0.01),GSH[(358.38±41.59)vs.(518.64±55.72)nmol/g]and GPX4[(7.75±1.09)vs.(14.72±2.56)U/mg]were decreased significantly(t=3.855,22.167,11.496,P<0.01);the severity of pathological injury in the pancreas and kidney was aggravated;typical shrunken mitochondria were observed;the expression of ACSL4[(0.84±0.03)vs.(0.34±0.02)]was elevated(t=7.876,P<0.01)and the expression of GPX4 and FTH1[(0.69±0.03)vs.(1.04±0.06),(0.26±0.02)vs.(0.83±0.04)]was down-regulated(t=5.651,8.134,P<0.01);and the expression of ACSL4 and iron responsive element binding protein 2(IREB2)mRNA[(0.87±0.06)vs.(0.24±0.03),(1.23±0.05)vs.(0.32±0.02)]was up-regulated(t=12.864,15.163,P<0.01)in SAP group.As compared with SAP group,the serum levels of AMY[(5124.3±483.5)vs.(6276.5±562.2)U/L],Cr[(55.2±13.7)vs.(98.0±16.2)μmol/L],BUN[(9.8±2.1)vs.(17.1±2.8)mmol/L]were reduced(t=5.287,6.989,7.359,P<0.01);Iron[(0.54±0.07)vs.(0.65±0.13)mg/g],MDA[(4.67±0.73)vs.(7.25±0.81)μmol/g]and ROS[(28.39±6.36)vs.(68.54±7.04)×104/mg]were decreased(t=2.639,7.176,8.247,P<0.05),GSH[(448.21±45.51)vs.(358.38±41.59)nmol/g]and GPX4[(11.31±1.89)vs.(7.75±1.09)U/mg]were increased(t=5.048,5.639,P<0.05);the severity of pathological injury in the pancreas and kidney injury was alleviated;mild mitochondrial shrinkage was observed;the expression of ACSL4[(0.49±0.02)vs.(0.84±0.03)]was down-regulated(t=2.781,P<0.05)and the expression of GPX4 and FTH1[(0.69±0.03)vs.(1.04±0.06),(0.26±0.02)vs.(0.83±0.04)]was up-regulated(t=2.427,2.876,P<0.05);and the expression of ACSL4 and iron responsive element-binding protein 2(IREB2)mRNA[(0.52±0.04)vs.(0.87±0.06),(0.74±0.04)vs.(1.23±0.05)]was down-regulated in SAP+Fer group(t=5.843,6.781,P<0.01).Conclusion Ferroptosis is involved in SAP-induced AKI and inhibition of ferroptosis in rats can ameliorate renal dysfunction,attenuate lipid peroxidation and pathological damage of renal tissues.