异鼠李素对H2O2诱导的HaCaT细胞线粒体结构及功能损伤的保护作用研究  

Protective effects of isorhamnetin against H2O2-induced mitochondrial structural and functional damage in HaCaT cells

作　　者：戴乐恒 胡雯[1] 张坤杰 康晓静[1] Dai Leheng;Hu Wen;Zhang Kunjie;Kang Xiaojing(Department of Dermatology and Venereology,People′s Hospital of Xinjiang Uygur Autonomous Region,Xinjiang Clinical Research Center for Dermatologic Diseases,Xinjiang Key Laboratory of Dermatology Research(XJYS1707),Urumqi 830001,China)

机构地区：[1]新疆维吾尔自治区人民医院皮肤性病科,新疆皮肤病临床医学研究中心,新疆皮肤病研究重点实验室(XJYS1707),乌鲁木齐830001

出　　处：《中华皮肤科杂志》2023年第9期857-861,共5页Chinese Journal of Dermatology

基　　金：国家自然科学基金(82173406)

摘　　要：目的探究异鼠李素对氧化应激状态下HaCaT细胞线粒体结构及功能的保护作用。方法以HaCaT细胞为研究对象,实验分为4组:对照组(常规培养HaCaT细胞)、异鼠李素组(仅加入60μmol/L异鼠李素处理)、H2O2组(仅加入600μmol/L H2O2处理)、异鼠李素+H2O2组(加入60μmol/L异鼠李素预处理12 h后换液加入600μmol/L H2O2处理12 h)。采用流式细胞仪检测细胞活性氧(ROS)水平,电镜观察线粒体超微结构,共聚焦荧光显微镜观察线粒体膜电位,ATP检测试剂盒检测线粒体ATP含量,实时荧光定量PCR(qRT-PCR)测定线粒体DNA拷贝数。多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果HaCaT细胞经H2O2处理后呈氧化应激状态,H2O2组细胞内ROS水平(10725.0±845.8)显著高于对照组(1708.0±69.4,t=18.4,P<0.001),线粒体膜电位荧光强度、ATP含量、线粒体DNA特征性基因ND-1表达量均显著低于对照组(t值分别为4.58、4.48、6.11,P值分别为0.01、0.01、0.003)。异鼠李素预处理后,异鼠李素+H2O2组ROS水平(7640.0±922.7)显著低于H2O2组(t=4.27,P=0.013),线粒体膜电位荧光强度、ATP含量、线粒体DNA ND-1基因表达量均显著高于H2O2组(t值分别为4.59、4.58、5.61,P值分别为0.01、0.01、0.005)。电镜下,异鼠李素+H2O2组线粒体结构较H2O2组清晰、完整,线粒体嵴无肿胀或轻度肿胀,未见线粒体空泡化;可见自噬小体吞噬受损的线粒体。结论异鼠李素可能通过诱导自噬降低ROS水平,对H2O2诱导的HaCaT细胞线粒体结构及功能损伤有保护作用。Objective To evaluate protective effects of isorhamnetin on mitochondrial structure and function in HaCaT cells under oxidative stress.Methods HaCaT cells served as the research object,and were divided into 4 groups:control group receiving conventional culture,isorhamnetin group treated with 60μmol/L isorhamnetin,H2O2 group treated with 600μmol/L H2O2,and isorhamnetin+H2O2 group pretreated with 60μmol/L isorhamnetin for 12 hours,followed by medium replacement and 12-hour treatment with 600μmol/L H2O2.Flow cytometry was performed to detect cellular reactive oxygen species(ROS)levels,transmission electron microscopy to observe mitochondrial ultrastructure,confocal fluorescence microscopy to evaluate mitochondrial membrane potential,real-time fluorescence-based quantitative PCR(qRT-PCR)to determine the mitochondrial DNA copy number,and adenosine triphosphate(ATP)assay kit was used to determine the mitochondrial ATP content.One-way analysis of variance was used for comparisons among multiple groups,and least significant difference-t test for multiple comparisons.Results Oxidative stress was provoked in HaCaT cells after the treatment with H2O2.Compared with the control group,the H2O2 group showed significantly increased ROS levels(10725.0±845.8 vs.1708.0±69.4,t=18.4,P<0.001),but significantly decreased fluorescence intensity of mitochondrial membrane potential,mitochondrial ATP content,and expression of ND-1(a characteristic gene of mitochondrial DNA)(t=4.58,4.48,6.11,P=0.01,0.01,0.003,respectively).After the pretreatment with isorhamnetin followed by H2O2 treatment,the isorhamnetin+H2O2 group showed significantly decreased ROS levels(7640.0±922.7)compared with the H2O2 group(t=4.27,P=0.013),but significantly increased fluorescence intensity of mitochondrial membrane potential,mitochondrial ATP content,and expression of ND-1 compared with the H2O2 group(t=4.59,4.58,5.61,P=0.01,0.01,0.005,respectively).Under the electron microscope,the mitochondrial structure was clearer and more complete in the isorhamnetin+H2

关 键 词：白癜风 氧化性应激 线粒体 活性氧 膜电位 腺苷三磷酸 异鼠李素 HACAT细胞 

分 类 号：R758.41[医药卫生—皮肤病学与性病学]