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.

Key words: Alopecia, Oxidative stress, Human dermal papilla cells, Pinus massoniana needle extract, Nrf2-ARE signaling pathway