乳脂球表皮生长因子8通过抑制铜死亡关键调控因子铁氧还原蛋白表达减轻移植肺缺血再灌注损伤

Protective effects of milk fat globule epidermal growth factor 8 on ischemic reperfusion injury of lung grafts by inhibiting the expression of Ferridoxin 1

目的:从铜死亡及其关键调控因子铁氧还原蛋白(FDX1)表达探讨乳脂球表皮生长因子8(MFG-E8)减轻肺移植缺血再灌注损伤的机制。方法:健康雄性SD大鼠50只,随机分为移植组、对照组和MFG-E8组,改良袖套法建立大鼠左肺移植模型。MFG-E8组受体大鼠术前30 min经尾静脉注射rhMFG-E820μg/kg,对照组和移植组受体鼠尾静脉注射同等量生理盐水。肺移植再灌注3 h后阻断右肺门5 min,全自动血气分析检测仪检测动脉血中氧分压(PaO 2)和二氧化碳分压(PaCO 2)。苏木精-伊红(HE)染色观察移植肺组织显微病理改变,组织髓过氧化物酶(MPO)试剂盒检测移植肺MPO活力,原位末端标记(TUNEL)法检测肺细胞凋亡,蛋白免疫印迹(Western blot)法检测肺组织铜蓝蛋白(CER)和铜死亡相关蛋白FDX1表达。组间比较使用t检验,多组比较采取单因素方差分析。结果:再灌注3 h后,HE染色可见移植组肺泡结构破坏明显、肺水肿伴大量炎性细胞浸润;MFG-E8组肺泡结构基本完整,炎性反应明显减轻,仅少量炎性细胞浸润。再灌注3 h后,MFG-E8组动脉PaO 2/FiO 2显著高于移植组[(264.65±47.23)mmHg比(208.36±32.45)mmHg,1 mmHg=0.133 kPa,t=3.106,P<0.05],肺组织MPO活力和细胞凋亡指数均明显低于移植组[(0.75±0.13)U/g prot比(0.98±0.17)U/g prot,t=3.399,P<0.05;(25.21±5.68)%比(34.16±4.75)%,t=3.822,P<0.05]。移植组大鼠肺组织FDX1蛋白水平表达明显高于对照组(0.52±0.18比0.29±0.14,t=3.190,P<0.05);MFG-E8组大鼠肺组织FDX1蛋白水平表达显著低于移植组(0.37±0.12比0.52±0.18,t=2.193,P<0.05);移植组大鼠肺组织CER蛋白水平表达低于对照组(0.26±0.11比0.32±0.14,t=1.066,P>0.05),但差异无统计学意义;MFG-E8组大鼠肺组织CER蛋白水平高于移植组(0.33±0.15比0.26±0.11,t=1.190,P>0.05),但差异无统计学意义。结论:MFG-E8可能通过抑制氧化应激和铜死亡相关蛋白FDX1表达发挥抗肺移植缺血再灌注损伤作用。

Objective To investigate the effects of milk fat globule epidermal growth factor 8(MFG-E8)on the expression of ferredoxin 1(FDX1),a key regulator of cuproptosis,in lung grafts ischemic reperfusion injury.Methods Fifty male Sprague Dawley(SD)rats were randomly divided into transplant group,control group and MFG-E8 group.A modified"cuff-like"technique was used to establish rat left lung transplantation model.Rats in MFG-E8 group were given rhMFG-E820μg/kg via tail vein 30 min before lung transplantation,while in the transplant group and control group,rats were treated with the same amount of normal saline.The pathological symptoms of lung grafts were determined by Hematoxylin-eosin(HE)staining.TUNEL assay was used to detect the apoptosis index of lung grafts.The lung graft’s oxygenation and gas exchange were measured by an automatic blood gas analyzer under occlusion of the right main pulmonary artery and bronchus for 5 minutes after 3 h reperfusion,as well as measurement of myeloperoxidase(MPO)activity.Western blotting was used to detect the expressions of ceruloplasmin(CER)and FDX1.The t-test was used to compare between two groups,and ANOVA was used to compare between multiple groups.Results HE staining showed that the alveolar structure was destroyed obviously and accompanied by extensive neutrophil infiltration in the transplant group,and no obvious alveolar exudates and neutrophil infiltration observed in the MFG-E8 group.compared to the transplant group,the PaO2/FiO2 was improved significantly in the MFG-E8 group[(264.65±47.23)mmHg vs.(208.36±32.45)mmHg,1 mmHg=0.133 kPa,t=3.106,P<0.05].The activity of MPO and apoptotic index(AI)in the MFG-E8 group were significantly lower than those of transplant group[(0.75±0.13)U/g prot vs.(0.98±0.17)U/g prot,t=3.399,P<0.05;(25.21±5.68)%vs.(34.16±4.75)%,t=3.822,P<0.05].compared to the control group,the expression of FDX1 was increased significantly in the transplant group(0.52±0.18 vs.0.29±0.14,t=3.190,P<0.05).compared to the transplant group,the expression of FDX1 was decreased significantly in the MFG-E8 group(0.37±0.12 vs.0.52±0.18,t=2.193,P<0.05).compared to the control group,the expression of CER was decreased in the transplant group,but there was no statistical difference(0.26±0.11 vs.0.32±0.14,t=1.066,P>0.05).compared to the transplant group,the expression of CER was increased in the MFG-E8 group,but no statistical significance was attained(0.33±0.15 vs.0.26±0.11,t=1.190,P>0.05).Conclusion MFG-E8 can alleviate lung graft ischemic reperfusion injury by against oxidative stress response and inhibiting the expression of FDX1.