二甲双胍激活单磷酸腺苷活化蛋白激酶/核转录因子E2相关因子2信号通路抑制长波紫外线诱导的HaCaT细胞光老化  

Metformin inhibits ultraviolet A-induced photoaging of HaCaT cells by activating the adenosine monophosphate-activated protein kinase/nuclear factor-erythroid 2-related factor 2 signaling pathway

作　　者：李华平 高爱莉 梁碧华 邓蕙妍 陈教全 邹荟 林天一 张三泉 朱慧兰 Li Huaping;Gao Aili;Liang Bihua;Deng Huiyan;Chen Jiaoquan;Zou Hui;Lin Tianyi;Zhang Sanquan;Zhu Huilan(Department of Dermatology,Guangzhou Institute of Dermatology,Institute of Dermatology,Guangzhou Medical University,Guangzhou 510095,China)

机构地区：[1]广州市皮肤病防治所广州医科大学皮肤病研究所,广州510095

出　　处：《中华皮肤科杂志》2023年第12期1123-1130,共8页Chinese Journal of Dermatology

基　　金：广州市科技计划项目(202002030474、2023A03J0470);广州市临床重大技术项目(2019ZD20)。

摘　　要：目的研究二甲双胍对长波紫外线(UVA)所致人永生化角质形成细胞系(HaCaT)光老化的影响及机制。方法采用细胞计数(CCK8)法测定不同浓度(0~100 mmol/L)二甲双胍对HaCaT细胞活力的影响,选择10 mmol/L二甲双胍进行后续实验。将培养的HaCaT细胞分为空白对照组、二甲双胍组、UVA照射组和二甲双胍+UVA组(用含10 mmol/L二甲双胍的培养基处理24 h后采用UVA照射细胞),其中UVA每天以10 J/cm^(2)照射1次,连续照射3 d。共处理4 d后收集细胞,β半乳糖苷酶法测定各组衰老细胞比例,2,7-二氯二氢荧光素二乙酸酯法测定胞内活性氧(ROS)水平,彗星实验测定DNA损伤水平。另将培养的HaCaT细胞分为空白对照组、二甲双胍组、1.25μmol/L多索吗啡(dorsomorphin)+二甲双胍组、2.5μmol/L dorsomorphin+二甲双胍组,其中后两组分别采用1.25、2.5μmol/L dorsomorphin处理细胞2 h后再用10 mmol/L二甲双胍处理24 h,Western印迹法检测核转录因子E2相关因子2(Nrf2)的细胞定位及磷酸化水平。使用小干扰RNA(siRNA)法将siRNA-Nrf2转染至HaCaT细胞以敲低Nrf2表达(siRNA-Nrf2组),对2.5μmol/L dorsomorphin处理的HaCaT细胞以及Nrf2敲低的HaCaT细胞进行二甲双胍孵育和UVA照射(dorsomorphin+二甲双胍+UVA组、siRNA-Nrf2+二甲双胍+UVA组),进一步分析各组衰老细胞比例。统计分析采用单因素方差分析及两因素方差分析,组间两两比较采用LSD-t检验。结果不同浓度二甲双胍处理24 h可不同程度地影响HaCaT细胞活力(F=5206.31,P<0.001),其中10~20 mmol/L二甲双胍组细胞相对存活率与对照组(0 mmol/L二甲双胍组)相比差异无统计学意义(均P>0.05),而25~100 mmol/L二甲双胍组细胞相对存活率显著低于对照组(均P<0.05)。UVA照射后HaCaT细胞发生明显皱缩且变狭长,细胞间隙增大,UVA照射组细胞相对存活率(76.13%±1.03%)显著低于空白对照组(100.00%±1.24%,LSD-t=14.86,P<0.001),而二甲双胍+UVA组细胞存活率(106.69%±2.45%Objective To evaluate the effect of metformin on ultraviolet A(UVA)-induced photoaging of an immortalized human keratinocytes cell line(HaCaT),and to explore its potential mechanisms.Methods Cell counting kit 8(CCK8)assay was performed to evaluate the effect of metformin at different concentrations(0-100 mmol/L)on the viability of HaCaT cells,and 10 mmol/L metformin was selected for subsequent experiments.Cultured HaCaT cells were divided into a blank control group(conventional culture),a metformin group(treated with culture medium containing 10 mmol/L metformin),a UVA irradiation group(conventional culture for 24 hours followed by 10 J/cm^(2) UVA irradiation)and a metformin+UVA group(treated with culture medium containing 10 mmol/L metformin for 24 hours followed by 10 J/cm^(2) UVA irradiation);UVA irradiation was performed at a dose of 10 J/cm^(2) once a day for 3 consecutive days.After 4-day treatment,cells were collected,theβ-galactosidase assay was performed to determine the proportion of senescent cells in each group,2′,7′-dichlorodihydrofluorescein diacetate assay to detect levels of intracellular reactive oxygen species(ROS),and the comet assay to detect DNA damage levels.Additionally,some HaCaT cells were divided into the blank control group,metformin group,1.25μmol/L dorsomorphin(an adenosine monophosphate-activated protein kinase[AMPK]inhibitor)+metformin group,and 2.5μmol/L dorsomorphin+metformin group,and cells in the latter two groups were treated with 1.25 and 2.5μmol/L dorsomorphin respectively for 2 hours,followed by the treatment with 10 mmol/L metformin for 24 hours.Western blot analysis was performed to determine the cellular localization and phosphorylation levels of nuclear factor-erythroid 2-related factor 2(Nrf2).By using the small-interfering RNA(siRNA)-mediated silencing method,siRNA-Nrf2 was transfected into HaCaT cells to knock down Nrf2 expression(siRNA-Nrf2 group);2.5μmol/L dorsomorphin-treated HaCaT cells or Nrf2-knockdown HaCaT cells were treated with metformin and UVA irr

关 键 词：皮肤衰老 细胞衰老 紫外线 二甲双胍 光老化 长波紫外线 核因子E2相关因子2 AMP活化蛋白激酶 

分 类 号：R751[医药卫生—皮肤病学与性病学]