结合珠蛋白通过抑制ERK1/2减轻肝细胞铁死亡

Haptoglobin suppresses hepatocyte ferroptosis via inhibition of the ERK1/2 signaling pathway

目的·探究结合珠蛋白(haptoglobin,Hp)在肝细胞铁死亡(ferroptosis)中的作用及机制。方法·给予8周龄的C57BL/6J雄性小鼠正常饲料(normal iron diet,NID组,n=5)和高铁饲料(high iron diet,HID组,n=8)饲喂12周后,收集2组小鼠的肝脏和血清。利用血清学生化指标、肝脏苏木精-伊红(hematoxylin-eosin,H-E)染色、天狼星红染色、普鲁士蓝染色和4-羟基壬烯醛(4-hydroxynonenal,4-HNE)染色明确2组小鼠肝脏损伤、病理学改变、纤维化、铁沉积和脂质过氧化反应程度;利用RNA测序(RNAsequencing,RNA-seq)技术和生物信息学分析高铁饮食对小鼠肝脏转录组表达的调控,并筛选新的调控铁死亡的候选基因。在AML-12小鼠肝细胞中过表达Hp或利用siRNA敲减Hp后,给予铁死亡诱导剂RAS合成致死分子3(RAS-selective lethal small molecule 3,RSL3)和/或细胞外信号调节激酶1/2(extracellular regulated protein kinases 1/2,ERK1/2)活性抑制剂SCH772984,利用细胞计数、real-time PCR和C11-BODIPY581/591免疫荧光及流式细胞术等实验明确Hp在肝细胞铁死亡中的作用及机制。结果·相较于NID组小鼠,HID组小鼠血清中谷丙转氨酶(glutamic-pyruvic transaminase,GPT)和谷草转氨酶(glutamic-oxalacetic transaminase,GOT)水平明显升高,肝脏出现明显的肝细胞死亡、纤维化、铁沉积和脂质过氧化产物累积。RNA-seq及生物信息学分析结果分析发现,HID组小鼠肝脏中急性期反应、氧化还原反应、催化反应及血红蛋白结合等信号通路发生了明显的变化,而Hp mRNA在HID组显著下调。而且,HID组小鼠肝组织及RSL3处理的AML-12肝细胞中的Hp mRNA和蛋白表达水平均明显降低。重要的是,在AML-12肝细胞中,Hp过表达可明显减轻RSL3引起的肝细胞脂质过氧化和铁死亡。反之,Hp敲减可加重RSL3引起的肝细胞铁死亡。机制上,Hp明显抑制RSL3引起的肝细胞ERK1/2磷酸化的增强;而且,SCH772984可明显恢复Hp敲减导致的肝细胞脂质过氧化和铁死亡的加重。结论·Hp是一种新的肝细胞铁死亡抑制分子,而且Hp主要通过抑制ERK1/2活性减轻肝细胞铁死亡及其引起的肝损伤。

Objective·To explore the role and mechanism of haptoglobin(Hp)in hepatocyte ferroptosis.Methods·Thirteen 8-week-old C57BL/6J male mice were randomly divided into two groups:one group of mice were fed with normal iron diet(NID group,n=5)and the other were fed with high iron diet(HID group,n=8).After 12 weeks,the murine serums and livers were collected from both groups.Liver injury,histopathological changes,hepatic fibrosis,iron deposition and peroxidation in murine liver tissues were determined by serum index test,hematoxylin-eosin(H-E)staining,Sirius red staining,Prussian blue staining and 4-hydroxynonenal(4-HNE)staining,respectively.RNA-sequencing(RNA-seq)and bioinformatics were used to analyze the effect of high iron diet on the expression of transcriptome in the murine livers and to screen new candidate genes that might regulate ferroptosis.After Hp was overexpressed or knocked down in mouse liver cells(AML-12),RAS-selective lethal small molecule 3(RSL3)or/and extracellular regulated protein kinases 1/2(ERK1/2)inhibitor SCH772984 was/were added to cells.The role and mechanism of Hp in hepatocyte ferroptosis were determined by cell counting,real-time PCR,C11-BODIPY581/591 immunofluorescence and flow cytometry.Results·After 12 weeks of feeding,the levels of glutamic-pyruvic transaminase(GPT)and glutamic-oxalacetic transaminase(GOT)in the HID group significantly increased compared with the NID group.The high iron diet induced hepatocytes death,hepatic fibrosis,iron deposition and lipid peroxidation products accumulation.RNA-seq analysis and bioinformatics results showed that acute phase response,catalytic activity response,antioxidant activity and hemoglobin binding signaling pathways were significantly regulated in the livers of HID mice,and Hp mRNA decreased significantly.Furthermore,the mRNA and protein expression levels of Hp in liver tissues of HID mice and in AML-12 hepatocytes treated with RSL3 obviously decreased.Importantly,Hp significantly alleviated lipid peroxidation and ferroptosis induced by RSL3 in AML-12 hepatocytes.Meanwhile,Hp suppressed RSL3-induced phosphorylation of ERK1/2 in AML-12 hepatocytes.Moreover,ERK1/2 inhibitor SCH772984 totally reversed the aggravation of hepatocyte ferroptosis induced by Hp knockdown.Conclusion·Hp is a novel inhibitory molecule in hepatocytes ferroptosis,which alleviates hepatocyte ferroptosis and liver injury by inhibiting ERK1/2 signaling pathway.