Abstract:

Zhu Longfei, Tian Jun, Jian Zhe, Liu Bangmin, Xiao Qian, Li Chunying, Gao Tianwen. Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi′an 710032, China Corresponding author: Gao Tianwen, Email: gaotw@fmmu.edu.cn 【Abstract】 Objective To explore the effects of NF-E2-related factor 2 （Nrf2） overexpression on mitochondrial biosynthesis and function in melanocytes. Methods An immortalized human vitiligo melanocyte cell line PIG3V was used in this study. An overexpression plasmid Nrf2-pEX-1 containing the full-length Nrf2 gene was constructed. PIG3V cells were divided into 3 groups: blank group receiving no treatment, control group transfected with the pEX-1 plasmid, overexpression group transfected with the Nrf2-pEX-1 plasmid. After transfection, real-time quantitative reverse transcription-PCR （RT-PCR） and Western blot were performed to determine the mRNA and protein levels of mitochondrial biosynthesis-related factors （including Nrf2, nuclear respiratory factor 1 （NRF1） and mitochondrial transcription factor A （TFAM）） respectively; RT-PCR was also conducted to measure the copy number of mitochondrial DNA （mtDNA）, and flow cytometry to estimate mitochondial membrane potential （MMP）; luciferase reporter system was used to estimate the intracellular adenosine triphosphate （ATP） level. Statistical analysis was carried out by using a two-sample t-test. Results After transfection, a significant increase was observed in the mRNA expression levels of Nrf2 and NRF1 at 24 hours （both P < 0.001） and in those of Nrf2 and TFAM at 48 hours （both P < 0.05）, but no significant change was noted in the mRNA expression level of TFAM at 24 hours （P > 0.05） or in that of NRF1 at 48 hours （P > 0.05） in the overexpression group compared with the control group. In the case of Nrf2, NRF1 and TFAM protein levels, the overexpression group showed significant increases compared with the control group at 48 hours after transfection （all P < 0.05）, while no significant difference was noted between the two groups at 24 hours. Compared with the control group, MMP in the overexpression group increased by 2.313% at 24 hours （t = 5.546, P = 0.005） and by 14.872% at 48 hours （t = 8.537, P = 0.001） after transfection. Both the relative copy number of mtDNA and ATP level were similar between the overexpression group and control group at 24 hours after transfection （both P > 0.05）, but significantly higher in the overexpression group than in the control group at 48 hours （t = 5.760, P = 0.005; t = 22.040, P = 0.008）. Conclusion Up-regulation of Nrf2 pathway can improve mitochondrial function and biosynthesis in PIG3V cells likely by promoting the expressions of mitochondrial biosynthesis-related genes and proteins.