褪黑素通过激活Nrf2抑制铁死亡改善随意皮瓣存活  

Melatonin activates Nrf2 to inhibit ferroptosis and improves the survival of random skin flaps

作　　者：楼承浩 卢颖枫 王健[1,2,3] LOU Chenghao;LU Yingfeng;WANG Jian(Department of Wound Repair,the First Affiliated Hospital of Wenzhou Medical University,Wenzhou 325015,China;Department of Hand Surgery and Peripheral Neurosurgery,the First Affiliated Hospital of Wenzhou Medical University,Wenzhou 325015,China;School of Pharmaceutical Science,Wenzhou Medical University,Wenzhou 325035,China)

机构地区：[1]温州医科大学附属第一医院创面修复科,浙江温州325015 [2]温州医科大学附属第一医院手外科·周围神经外科,浙江温州325015 [3]温州医科大学药学院,浙江温州325035

出　　处：《温州医科大学学报》2023年第7期526-531,539,共7页Journal of Wenzhou Medical University

基　　金：国家自然科学基金项目(8217051594)。

摘　　要：目的:探讨褪黑素(MLT)是否能通过减轻随意皮瓣的铁死亡促进随意皮瓣存活。方法:将20只6~8周龄的雄性C57BL/6小鼠随机分为对照组和MLT组,观察术后3、7 d各组皮瓣存活情况及血流动力学。取皮瓣组织评估水肿情况,通过病理学染色、透射电子显微镜观察、免疫印迹等检测方法探讨MLT对随意皮瓣存活的作用及对铁死亡的调节作用。为探讨核红细胞因子2相关因子2(Nrf2)在MLT抑制随意皮瓣铁死亡中起到的作用,使用Nrf2抑制剂ml385作为抑制剂,随机将45只6~8周龄的C57BL/6小鼠分为对照组、MLT组和MLT+ml385组,观察形态学变化并检测相关生化指标变化。结果:各组在术后第3、7天都出现远端皮瓣坏死。MLT组较对照组增加了皮瓣存活面积(P<0.05)及皮瓣血流灌注(P<0.05)。HE染色可见MLT组皮瓣内小血管含量多;Masson染色见真皮层纤维排列规整;免疫组织化学染色可见MLT组较对照组CD34阳性小血管增多(P<0.05),凋亡相关蛋白减少(P<0.05),抗氧化蛋白含量增加(P<0.05)。透射电镜可见皮瓣组织中铁死亡特征性改变。组织脂质过氧化物(LPO)检测、组织Fe含量检测、谷胱甘肽(GSH)水平以及Western blot结果表明,与对照组相比,MLT组GSH上调(P<0.05),组织Fe积累降低(P<0.05),LPO减少(P<0.05)。联合使用ml385后,Western blot结果表明,与MLT组比,MLT+ml385组中Nrf2下调(P<0.05),抗氧化指标HO-1下调(P<0.05),抗脂质过氧化指标GPX4下调(P<0.05),Fe结合蛋白轻链(FTL)下调(P<0.05)。结论:MLT治疗有利于小鼠随意皮瓣的存活;铁死亡是影响随意皮瓣存活的因素之一,抑制铁死亡能够促进随意皮瓣存活;MLT抑制铁死亡依赖于激活Nrf2提高抗氧化能力减轻脂质过氧化并促进Fe代谢。Objective:To investigated whether melatonin(MLT)can promote the survival of random flaps by reducing ferroptosis in random flaps.Methods:Twenty male C57BL/6 mice aged 6-8 weeks were randomly divided as the control group and MLT group.The flap survival and hemodynamics after 3 and 7 days in both groups were observed.The flap tissue was taken to evaluate edema,and the effect of melatonin on the survival of random flaps and the regulation of ferroptosis was investigated by pathological staining,transmission electron microscope and immunoblotting.In order to explore the role of nuclear factor erythroid 2-related factor 2(Nrf2)in the inhibition of ferroptosis by melatonin,45 male C57BL/6 mice aged 6-8 weeks were randomly divided into control group,MLT group,and MLT+ml385 group.Morphological changes were observed and changes in relevant biochemical indicators were detected.Results:Distal flap necrosis occurred in all groups on day 3 and day 7.Compared with the control group,MLT group had the increased flap survival area(P<0.05)and flap blood perfusion(P<0.05).HE staining showed that MLT group had more small blood vessels in the flap,and Masson staining showed that fibres in the dermis were regularly arranged in MLT group.Immunohistochemical staining showed that MLT group had more CD34-positive vessels(P<0.05),reduced apoptosis-related proteins(P<0.05)and increased content of antioxidant protein(P<0.05),compared with the control group.Characteristic changes of ferroptosis in the flap tissue were observed by transmission electron microscopy.Tissue lipid peroxide detection,tissue iron content detection,glutathione level and western blot results showed that MLT group up-regulated GSH(P<0.05),decreased tissue iron accumulation(P<0.01),and reduced lipid oxidation level(P<0.05).After the combined use of ml385,the results of western blot showed that compared with the MLT group,Nrf2(P<0.05),the antioxidant protein HO-1(P<0.05),the anti-lipid peroxidation protein GPX4(P<0.05),and the iron-binding protein light chain FTL(P<0.05)w

关 键 词：褪黑素 铁死亡 随意皮瓣 缺血再灌注 核红细胞因子2相关因子2 

分 类 号：R622.1[医药卫生—整形外科]