Protective effect of Nrf2 protein against ultraviolet B-induced photodamage to HaCaT cells / 中华皮肤科杂志

ABSTRACT

Objective To evaluate the protective effect of nuclear factor E2-related factor 2(Nrf2) protein against ultraviolet B (UVB)-induced photodamage to HaCaT cells,and to explore its mechanisms.Methods Cultured HaCaT cells were divided into 4 groupscontrol group receiving no treatment,UVB group irradiated with 30 mJ/cm2 UVB for 30 s,Nrf2 group transfected with a lentiviral vector overexpressing the Nrf2 gene,and Nrf2 + UVB group transfected with a lentiviral vector overexpressing the Nrf2 gene followed by radiation with 30 mJ/cm2 UVB for 30 s.After the treatment,HaCaT cells in the above 4 groups were cultured for another 24 hours.Then,changes in the morphology of HaCaT cells were observed after UVB radiation,Western blot analysis was performed to determine Nrf2 protein expression,cell counting kit-8 (CCKS) assay to detect survival rates of HaCaT cells,flow cytometry to detect levels of reactive oxygen species (ROS),and a biochemical method to detect superoxide dismutase (SOD) levels in cells,and enzyme-linked immunosorbent assay to detect levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in the culture supematant of HaCaT cells.One-way analysis of variance was used for comparing means in several groups,and least significant difference (LSD)-t test for multiple comparisons.Results Polygonal and clustered HaCaT cells were observed in the control group.After UVB radiation,HaCaT cells became shrunken and round,the number of floating cells increased,and the number of adherent cells markedly decreased.There was a significant difference in Nrf2 protein expression among the control group,UVB group,Nrf2 group and Nrf2 + UVB group (1.84 ± 0.047,0.63 ± 0.082,2.19 ± 0.168 and 1.43 ± 0.069 respectively;F =64.81,P ＜ 0.05),and the Nrf2 protein expression was significantly higher in the Nrf2 group than in the control group (t =14.82,P ＜ 0.05);the survival rates of HaCaT cells also significantly differed among the above 4 groups (98.00％ ± 2.39％,24.40％ ± 2.98％,71.63％ ± 3.39％and 43.38％ ± 3.39％ respectively;F =236.66,P ＜ 0.05),and the UVB group showed significantly decreased cell viability compared with the control group (t =33.34,P ＜ 0.05)and Nrf2 + UVB group (t=10.07,P ＜ 0.05);a significant difference in the ROS level in HaCaT cells was observed among the above 4 groups (1.27 ± 0.10,5.65 ± 0.19,2.10 ± 0.73 and 3.67 ± 0.19 respectively;F =481.39,P ＜ 0.05),and the UVB group showed a significantly increased ROS level compared with the control group (t =33.68,P ＜0.05) and Nrf2 + UVB group (t =12.47,P ＜ 0.05).The SOD level in HaCaT cells significantly differed among the above 4 groups (F =170.76,P ＜ 0.05),and was significantly lower in the UVB group than in the control group (t =11.25,P ＜ 0.05) and Nrf2 + UVB group (t =17.52,P ＜ 0.05).The IL-6 level also significantly differed among the above 4 groups (F =532.34,P ＜ 0.05),and was significantly higher in the UVB group than in the control group (t =28.48,P ＜ 0.05) and Nrf2 + UVB group (t =27.82,P ＜ 0.05).There was no significant difference in the TNF-α level among the above 4 groups (F =2.02,P =0.19).Conclusion Nrf2 can protect HaCaT cells from UVB-induced oxidative damage,by reducing intracellular ROS levels and increasing the activity of the endogenous antioxidant enzyme SOD.