关键词: 紫檀芪, HaCaT细胞, UVB, Nrf2, 抗氧化物酶

Abstract: Deng Huiyan, Li Huaping, Chen Quan, Li Runxiang, Liang Bihua, Gao Aili, Zhou Xin, Zhu Huilan Department of Dermatology, Guangzhou Institute of Dermatology, Guangzhou 510095, China Corresponding author: Zhu Huilan, Email: zhlhuilan@126.com 【Abstract】 Objective To evaluate the protective effect of pterostilbene against ultraviolet B （UVB）-induced acute damage in HaCaT cells, and to explore related mechanisms. Methods The 3-（4, 5-dimethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2H-tetrazolium （MTS） assay and flow cytometry were performed to estimate the proliferative activity and the apoptosis and necrosis rate of HaCaT cells treated with different concentrations of pterostilbene respectively, so as to screen the non-toxic concentration of pterostilbene. HaCaT cells were randomly divided into several groups: normal control group receiving no treatment, UVB group irradiated with 57 mJ/cm2 UVB, 3 pterostilbene groups treated with 2.44, 4.88 and 9.75 μmol/L pterostilbene respectively for 24 hours, 3 pterostilbene + UVB groups treated with 2.44, 4.88 and 9.75 μmol/L pterostilbene respectively for 24 hours followed by UVB radiation. Western blot analysis was conducted to detect changes of the transcription factor NF-E2-related factor 2 （Nrf2） in cell nuclei and cytoplasm before and after the treatment with pterostilbene and UVB, quantitative PCR to determine the mRNA of catalase and superoxide dismutase in the HaCaT cells, and enzyme-linked immunosorbent assay （ELISA） to evaluate the activity of catalase and superoxide dismutase. Results MTS assay and flow cytometry showed that 2.44, 4.88 and 9.75 μmol/L pterostilbene had non-toxic effect on HaCaT cells. The protein of Nrf2 in the nuclei and cytoplasm in the normal control group was 1.03 ± 0.08 and 1.04 ± 0.11 respectively. Compared with the normal control group, the protein of Nrf2 in the nuclei and cytoplasm experienced no significant changes in the 2.44-, 4.88- and 9.75-μmol/L pterostilbene groups, and the UVB group showed similar protein of Nrf2 in the cytoplasm, but significantly increased protein of Nrf2 in the nuclei （1.77 ± 0.08, q = 17.24, P < 0.01）. Compared with the normal control group and UVB group, the 2.44-, 4.88- and 9.75-μmol/L pterostilbene + UVB groups all showed significantly lower protein of Nrf2 in the cytoplasm （0.86 ± 0.10, 0.87 ± 0.11 and 0.46 ± 0.11 respectively, all P < 0.05）, but significantly higher protein of Nrf2 in the nuclei （2.38 ± 0.11, 2.57 ± 0.11 and 2.07 ± 0.13, all P < 0.01）. As qPCR showed, UVB radiation could significantly inhibit the mRNA of CAT （P < 0.05）, but had no obvious effect on the mRNA of SOD （P > 0.05）. The mRNA of CAT and SOD experienced no significant changes in the 2.44-, 4.88- and 9.75-μmol/L pterostilbene groups compared with the normal control group（P > 0.05）. However, 2.44, 4.88 and 9.75 μmol/L pterostilbene could significantly reduce the inhibitory effect of UVB radiation on the mRNA of CAT （P < 0.05） and up-regulate the mRNA of SOD in the pterostilbene + UVB groups （P < 0.05）. ELISA revealed that UVB radiation could inhibit the activity of CAT and SOD in the HaCaT cells （both P < 0.001）, while 2.44, 4.88 and 9.75 μmol/L pterostilbene could reduce the inhibitory effect of UVB radiation on the activity of CAT and SOD （all P < 0.05）. However, the activity of CAT and SOD were still lower in the 2.44-, 4.88- and 9.75-μmol/L pterostilbene + UVB groups than in the normal control group （P < 0.05）. Conclusion Pterostilbene can prevent UVB-induced acute damage in HaCaT cells by activating the Nrf2 pathway and up-regulating the of the downstream antioxidant enzymes CAT and SOD.

Key words: Pterostilbene, HaCaT cells, UVB, Nrf2, Antioxidant Enzymes