褪黑素调控GPX4/NF-κB p65信号通路干预铁死亡促进小鼠皮肤创面修复  

Melatonin regulates the GPX4/NF-κB p65 signaling pathway to intervene ferroptosis and promote skin wound healing in mice

作　　者：芦周洲 高栋梁[2] 王亚康 LU Zhouzhou;GAO Dongliang;WANG Yakang(Department of Burn and Plastic Surgery,Suzhou First People’s Hospital,Suzhou 234000,China;Department of Burn and Plastic Surgery,Yan’an University Affiliated Hospital,Yan’an 716000,China;Department of Hip Surgery,Xi’an Honghui Hospital,Xi’an 710054,China)

机构地区：[1]宿州市第一人民医院烧伤整形科,安徽宿州234000 [2]延安大学附属医院烧伤整形外科,陕西延安716000 [3]西安市红会医院髋关节外科,陕西西安710054

出　　处：《陕西医学杂志》2025年第2期181-186,共6页Shaanxi Medical Journal

基　　金：陕西省自然科学基础研究计划项目(2019JQ-536)。

摘　　要：目的:探讨褪黑素对人皮肤成纤维细胞(HSF)增殖和铁死亡的作用,并探究其促进小鼠皮肤创面修复的分子机制。方法:采用CCK-8法检测HSF细胞增殖能力,选取细胞增殖率最高的褪黑素浓度进行后续实验。将HSF细胞随机分为对照组、谷氨酸组、谷氨酸+褪黑素组。谷氨酸+褪黑素组用100μmol/L褪黑素预处理细胞60 min,随后除对照组外,另两组加入谷氨酸(10 mmol/L)诱导铁死亡。采用相应试剂盒检测HSF细胞活性氧(ROS)、谷胱甘肽(GSH)、丙二醛(MDA)、Fe^(2+)水平。构建小鼠为全层皮肤损伤模型,并随机分为对照组和褪黑素组,每组10只。褪黑素组在皮肤损伤处外敷含5 mg褪黑素的凡士林乳膏,对照组外敷等量凡士林乳膏,每日清洁换药,连续9 d。计算术后第5、7、9天各组创面愈合率。采用HE和Masson染色观察第5、9天小鼠创面组织病理学变化及胶原纤维生成情况。采用免疫荧光染色法检测术后第9天创面组织中细胞增殖情况,Western blot检测术后第9天创面组织中谷胱甘肽过氧化物酶4(GPX4)、核因子(NF)-κB p65、p-NF-κB p65蛋白表达。结果:褪黑素浓度为100μmol/L时HSF细胞增殖率最高,故选取100μmol/L褪黑素进行后续实验。与对照组比较,谷氨酸组HSF细胞GSH水平降低,ROS、MDA、Fe^(2+)水平升高(均P<0.05)。与谷氨酸组比较,谷氨酸+褪黑素组HSF细胞GSH水平升高,ROS、MDA、Fe^(2+)水平降低(均P<0.05)。与对照组比较,褪黑素组小鼠术后第5、7、9天创面愈合率增加(均P<0.05)。与对照组比较,褪黑素组小鼠术后第5、9天创面组织再上皮化程度更高,胶原纤维生成比例增加(均P<0.05)。与对照组比较,褪黑素组小鼠术后第9天创面组织Ki-67免疫荧光强度增加(P<0.05)。与对照组比较,褪黑素组小鼠术后第9天创面组织GPX4蛋白表达水平升高,NF-κB p65、p-NF-κB p65蛋白表达水平降低(均P<0.05)。结论:褪黑素能够促进皮肤成纤维细胞增殖,Objective:To investigate the effects of melatonin on the proliferation and ferroptosis of human skin fibroblasts(HSF)and to explore its molecular mechanisms in promoting skin wound healing in mice.Methods:The proliferation ability of HSF cells was detected using the CCK-8 assay,and the concentration of melatonin with the highest cell proliferation rate was selected for subsequent experiments.HSF cells were randomly divided into a control group,glutamate group,and glutamate+melatonin group.The glutamate+melatonin group was pretreated with 100μmol/L melatonin for 60 minutes,and then glutamate(10 mmol/L)was added to induce ferroptosis in both the glutamate and glutamate+melatonin groups,except for the control group.The levels of reactive oxygen species(ROS),glutathione(GSH),malondialdehyde(MDA),and Fe^(2+)in HSF cells were detected using corresponding assay kits.A full-thickness skin injury model was constructed in mice,which were randomly divided into a control group and a melatonin group,with 10 mice in each group.The melatonin group applied a vaseline ointment containing 5 mg of melatonin to the skin injury site,while the control group applied an equal amount of vaseline ointment.The wounds were cleaned and dressed daily for 9 consecutive days.The wound healing rate was calculated on postoperative days 5,7 and 9.HE and Masson staining were used to observe the pathological changes and collagen fiber formation in the wound tissue on days 5 and 9.Immunofluorescence staining was used to detect cell proliferation in the wound tissue on day 9,and Western blot was used to detect the expression of glutathione peroxidase 4(GPX4),nuclear factor(NF)-κB p65,and p-NF-κB p65 proteins in the wound tissue on day 9.Results:When the concentration of melatonin was 100μmol/L,the proliferation rate of HSF cells was the highest,so 100μmol/L melatonin was selected for subsequent experiments.Compared with the control group,the GSH level in the glutamate group decreased,while the levels of ROS,MDA,and Fe^(2+)increased(all P<0.05).Co

关 键 词：创面修复 褪黑素 铁死亡 谷胱甘肽过氧化物酶4 核因子-κB p65 小鼠 

分 类 号：R-33[医药卫生]