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组细胞凋亡率显著高于对照组(t = 13.13,P < 0.001),而细胞活性显著低于对照组(t = 67.37,P < 0.001)。PMNE处理实验中,原花青素组和不同浓度PMNE组细胞活性均显著高于双氢睾酮组(均P < 0.01),而细胞凋亡率显著低于双氢睾酮组(均P < 0.01);原花青素和不同浓度PMNE均能显著抑制双氢睾酮诱导的HDPC细胞内ROS和MDA的过度表达(均P < 0.01);原花青素组和5、25、100 μg/ml PMNE组Nrf2、NQO1、HO-1蛋白表达显著高于双氢睾酮组(均P < 0.05),原花青素组和25、100 μg/ml PMNE组Keap1、TGF-β1蛋白表达及Smad2/3磷酸化水平显著低于双氢睾酮组(均P < 0.05)。结论 Nrf2在抵抗HDPC的氧化应激损伤中起重要作用,PMNE可能通过激活Nrf2抗氧化反应元件通路而对HDPC产生明显的保护作用。
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 groups,and least significant difference-t test for multiple comparisons.Results The viability of HDPC ranged from 75%to 85%after the treatment with 0.4 mmol/L H_(2)O_(2),which was selected as the optimal condition for in vitro oxidative stress in HDPC.Compared with the blank group and control siRNA group,the Nrf2-siRNAl,Nrf2-siRNA2,Nrl2-siRNA3 groups showed significantly decreased Nrf2 mRNA and protein expression(all P<0.05),but significantly increased apoptosis rate(Nrf2-siRNAl,Nif2-siRNA2,Nrf2-siRNA3 groups,blank group and control group:12.50%±0.05%,26.07%±0.05%,58.44%±1.03%,10.38%±0.64%,13.05%±0.12%,respectively;all P<0.05).Nrf2 protein expression was the lowest in the Nrf2-siRNA2 group,so Nrf2-siRNA2 was selected as the optimal interfering fragment for subsequent experiments.Compared with the blank group and control plasmid group,the Nrf2 overexpression group showed significantly increased Nrf2 mRNA and protein expression(both P<0.05),but a significantly decreased apoptosis rate(all P<0.05).After the treatment with 0.4 mmol/L H_(2)O_(2),the Nrf2 overexpression group showed a significantly decreased apoptosis rate,but significantly increased cell viability compared with the empty vector group(t=3.66,40.40,respectively,both P<0.001);the Nrf2-siRNA2 group showed a significantly increased apoptosis rate,but significantly decreased cell viability compared with the control group(t=13.13,67.37,respectively,both P<0.001).In the PMNE treatment experiment,the proanthocyanidin group and PMNE groups showed significantly increased cell viability,but significantly decreased apoptosis rates compared with the dihydrotestosterone group(all P<0.01);proanthocyanidin and PMNE at different concentrations could significantly inhibit dihydrotestosterone-induced overexpression of ROS and MDA in HDPC(all P<0.01);the protein expression of Nrf2,NQO1 and HO-1 was significantly higher in the proanthocyanidin group,5-,25-and 100-μg/ml PMNE groups than in the dihydrotestosterone group(all P<0.05),while the protein expression of Keapl and TGF-β1,and the Smad2/3 phosphorylation level were significantly lower in the proanthocyanidin group,25-and 100-μg/ml PMNE groups than in the dihydrotestosterone group(all P<0.05).Conclusion Nrf2 plays an important role in protecting against oxidative damage in HDPC,and PMNE may exert marked protective effect on HDPC by activating the Nrf2-antioxidant responsive element signaling pathway.
作者
朱红柳
魏跃钢
闵仲生
高以红
羊剑秋
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)
出处
《中华皮肤科杂志》
CAS
CSCD
北大核心
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)。
作者简介
通信作者:朱红柳,Email:17768318386@163.com。
