马尾松针提取物通过核因子E2相关因子2-抗氧化反应元件通路对人毛乳头细胞氧化应激损伤保护作用的研究  

Protective effect of Pinus massoniana needle extract against oxidative stress in human dermal papilla cells via the nuclear factor-erythroid 2-related factor 2-antioxidant responsive element signaling pathway

作　　者：朱红柳[1] 魏跃钢 闵仲生 高以红 羊剑秋[1] Zhu Hongliu;Wei Yuegang;Min Zhongsheng;Gao Yihong;Yang Jianqiu(Department of Dermatology,Jiangyin Hospital Affiliated to Nanjing University of Chinese Medicine,Jiangyin 214400,Jiangsu,China;department of Dermatology,Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210029,China)

机构地区：[1]南京中医药大学江阴附属医院皮肤科,江苏江阴214400 [2]南京中医药大学附属医院皮肤科,210029

出　　处：《中华皮肤科杂志》2021年第10期869-877,共9页Chinese Journal of Dermatology

基　　金：国家自然科学基金(81804100)。

摘　　要：目的探讨马尾松针提取物(PMNE)对人毛乳头细胞(HDPC)抗氧化应激的保护作用及机制。方法以HDPC为研究对象,分别采用0(对照组)、0.1、0.2、0.4、0.8、1.0 mmol/L H2O2处理HDPC,建立体外HDPC氧化应激的最佳条件;在HDPC中转染核因子E2相关因子2(Nrf2)干扰片段siRNA1、siRNA2、siRNA3或过表达质粒pCMV6-XL5-Nrf2,实时荧光定量PCR和Western印迹法分别检测Nrf2 mRNA及蛋白的表达;H2O2条件下检测转染后各组细胞活性及凋亡率。常规培养HDPC并分组处理,对照组:正常培养,不予其他处理;双氢睾酮组:加入0.03 μg/ml双氢睾酮;原花青素组:加入0.03 μg/ml双氢睾酮和6.00 μg/ml原花青素B2处理;不同浓度PMNE组:加入0.03 μg/ml双氢睾酮培养液,同时分别给予1、5、25、100 μg/ml PMNE处理;分别检测各组细胞活性及凋亡率、细胞内活性氧(ROS)相对荧光强度和丙二醛(MDA)含量,Nrf2、醌氧化还原酶1(NQO1)、血红素加氧酶1(HO-1)、Kelch样ECH相关蛋白1(Keap1)、转化生长因子(TGF)-β1、Sma和Mad相关蛋白2/3(Smad2/3)、p-Smad2/3的表达水平。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 HDPC细胞活力为75% ~ 85%时,选择0.4 mmol/L H2O2作为体外HDPC氧化应激最适处理浓度。Nrf2-siRNA1、Nrf2-siRNA2、Nrf2-siRNA3组Nrf2 mRNA和蛋白表达均显著低于空白组和对照组(均P < 0.05),细胞凋亡率(12.50% ± 0.05%、26.07% ± 0.05%、58.44% ± 1.03%)均显著高于空白组(10.38% ± 0.64%)和对照组(13.05% ± 0.12%),均P < 0.05。Nrf2-siRNA2组的Nrf2蛋白表达量最低,选择Nrf2-siRNA2为最佳干扰片段进行后续实验。Nrf2过表达组Nrf2 mRNA和蛋白表达均显著高于空白组和对照组(均P < 0.05),其细胞凋亡率明显低于空白组和对照组(均P < 0.05)。0.4 mmol/L H2O2处理条件下,Nrf2过表达组细胞凋亡率显著低于过表达空载组(t = 3.66,P < 0.001),细胞活性高于过表达空载组(t = 40.40,P < 0.001);Nrf2-siRNA2组细胞凋亡率显�Objective To investigate protective effect of Pinus massoniana needle extract(PMNE)against oxidative stress in human dermal papilla cells(HDPC),and to explore its mechanisms.Methods As research objects,some cultured HDPC were treated with H_(2)O_(2) at different concentrations of 0(control group),0.1,0.2,0.4,0.8 and 1.0 mmol/L,in order to establish the optimal condition for in vitro oxidative stress in HDPC;some other HDPC were transfected with nuclear factor-erythroid 2-related factor 2(Nrf2)specific small interfering RNAs(siRNA1,siRNA2,siRNA3)or a Nrf2-overexpressing plasmid(pCMV6-XL5-Nrf2),the HDPC transfected with a scrambled-siRNA and an empty plasmid pCMV6-XL5 served as the control siRNA group and control plasmid group respectively,and HDPC subjected to conventional culture served as the blank group;after the above treatment,real-time fluorescence-based quantitative PCR and Western blot analysis were performed to determine Nrf2 mRNA and protein expression,respectively;cell viability and apoptosis were detected in the above transfected cells after the treatment with H_(2)O_(2) at an optimal concentration.In the subsequent experiment,some HDPC were divided into several groups:control group subjected to conventional culture,dihydrotestosterone group treated with 0.03μg/ml dihydrotestosterone,proanthocyanidin group treated with 0.03 μg/ml dihydrotestosterone and 6.00 μg/ml proanthocyanidin B2,PMNE groups treated with 0.03 μg/ml dihydrotestosterone and PMNE at different concentrations of 1,5,25 and 100 μg/ml;after the above treatment,cell viability and apoptosis were detected,relative fluorescence intensity of intracellular reactive oxygen species(ROS),malondialdehyde(MDA)content,mRNA and protein expression of Nrf2,quinone oxidoreductase 1(NQO1),heme oxygenase 1(HO-1),Kelch-like ECH-related protein 1(Keapl),transforming growth factor(TGF)-pi,Sma-and Mad-related protein 2/3(Smad2/3),phosphorylated Smad2/3(p-Smad2/3)were determined in HDPC.One-way analysis of variance was used for comparisons among multiple g

关 键 词：秃发 氧化性应激 人毛乳头细胞 马尾松针提取物 Nrf2-ARE信号通路 

分 类 号：R285.5[医药卫生—中药学]