| 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 |
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| 引用本文： | 朱红柳 魏跃钢 闵仲生 高以红 羊剑秋. 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[J]. 中华皮肤科杂志, 2021, 54(10): 869-877. DOI: 10.35541/cjd.20210203 |
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| 作者姓名： | 朱红柳 魏跃钢 闵仲生 高以红 羊剑秋 |
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| 作者单位： | 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 |
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| 摘 要： | 【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。
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| 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 |
| 收稿时间： | 2021-03-09 |
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 |
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| Zhu Hongliu,Wei Yuegang,Min Zhongsheng,Gao Yihong,Yang Jianqiu. 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[J]. Chinese Journal of Dermatology, 2021, 54(10): 869-877. DOI: 10.35541/cjd.20210203 |
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| Authors: | Zhu Hongliu Wei Yuegang Min Zhongsheng Gao Yihong Yang Jianqiu |
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| Affiliation: | 1Department of Dermatology, Jiangyin Hospital Affiliated to Nanjing University of Chinese Medicine, Jiangyin 214400, Jiangsu, China; 2Department of Dermatology, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210029, China |
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| Abstract: | 【Abstract】 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 H2O2 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 H2O2 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 0.03 μg/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 （Keap1）, transforming growth factor （TGF）-β1, 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 H2O2 , 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-siRNA1, Nrf2-siRNA2, Nrf2-siRNA3 groups showed significantly decreased Nrf2 mRNA and protein expression （all P < 0.05）, but significantly increased apoptosis rate （Nrf2-siRNA1, Nrf2-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 H2O2 , 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 Keap1 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. |
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| Keywords: | Alopecia Oxidative stress Human dermal papilla cells Pinus massoniana needle extract Nrf2-ARE signaling pathway |
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