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Objective:To evaluate the protective effect of exogenous biliverdin on ultraviolet B (UVB) -induced photodamage to keratinocytes, and to explore its mechanisms.Methods:HaCaT cells were divided into 5 groups: UVB group irradiated with 30 mJ/cm 2 UVB alone, 0.1-, 1- and 10-μmol/L UVB groups treated with 0.1, 1 and 10 μmol/L biliverdin respectively and irradiated with 30 mJ/cm 2 UVB, and control group receiving no treatment. After irradiation, cells in the above groups continued to be cultured for 24 hours. Then, the reactive oxygen species (ROS) level, superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were detected in HaCaT cells, and enzyme-linked immunosorbent assay (ELISA) was performed to detect levels of the inflammatory factors interleukin 6 (IL-6) and IL-8 in the culture supernatants of HaCaT cells. One-way analysis of variance was used for comparison of means among multiple groups, and least significant difference (LSD) - t test was used for multiple comparisons. Results:Significant differences were observed among the UVB group, 0.1-, 1- and 10-μmol/L UVB groups and control group in the ROS levels (3 613.33 ± 206.61, 2 958.67 ± 193.87, 2 678.33 ± 178.24, 2 274.67 ± 118.81, 1 905.67 ± 250.25, respectively, F = 34.02, P < 0.05), SOD activity (24.41 ± 1.78, 28.96 ± 2.21, 29.75 ± 1.75, 30.19 ± 2.29, 37.52 ± 2.31, respectively, F = 57.36, P < 0.05), MDA contents (5.61 ± 0.32, 5.46 ± 0.55, 4.65 ± 0.22, 2.55 ± 0.93, 1.31 ± 0.05, respectively, F = 214.09, P < 0.05), and supernatant levels of IL-6 ( F = 29.73, P < 0.05) and IL-8 ( F = 11.40, P < 0.05). The UVB group showed significantly increased levels of ROS, IL-6 and IL-8, and MDA contents compared with the other 4 groups (all P < 0.05), but significantly decreased SOD activity compared with the other 4 groups ( P < 0.05) . Conclusion:Exogenous biliverdin has some protective effect on UVB-induced photodamage, likely by reducing oxidative damage to cells, attenuating inflammatory reactions and suppressing lipid peroxidation.
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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 groups:control 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.
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Objective To evaluate the protective effect of exogenous biliverdin on ultraviolet B (UVB)-radiated HaCaT cells,and to explore its mechanism.Methods Cultured HaCaT cells were divided into 5 groups:control group receiving no treatment,UVB group irradiated with 30 mJ/cm2 UVB,3 biliverdin + UVB groups treated with 100 nmol/L,1 μmol/L and 10 μmol/L biliverdin respectively for 1 hour followed by 30 mJ/cm2 UVB radiation.After 24-hour treatment,changes in the morphology of HaCaT cells were observed,and cell counting kit 8 (CCK8) assay was performed to determine cell survival rates in the above groups.Western blot analysis was conducted to measure the protein expression of antioxidant signaling molecule NF-E2-related factor-2 (Nrf-2) and the photodamage signaling molecules matrix metalloproteinase-1 (MMP-1) and MMP-3.Results CCK8 assay showed that the survival rate of HaCaT cells was significantly lower in the UVB group than in the control group (P ＜ 0.05),but significantly higher in the 100-nmol/L,1-μmol/L and 10-μmol/L biliverdin + UVB groups than in the UVB group (all P ＜ 0.05).Western blot analysis showed that the protein expression of MMP-1 and MMP-3 was significantly higher in the UVB group (1.150 ± 0.187,0.979 ± 0.054 respectively) than in the control group (0.116 ± 0.018,0.636 ± 0.035 respectively;both P ＜ 0.01),but was significantly lower in the 100-nmol/L,1-μmol/L and 10-μmol/L biliverdin + UVB groups (MMP-1:0.825 ± 0.139,0.313 ± 0.047 and 0.286 ± 0.036 respectively;MMP-3:0.888 ± 0.017,0.672 ± 0.042 and 0.569:±:0.037 respectively) than in the UVB group (all P ＜ 0.05).Moreover,the protein expression of Nrf-2 was significantly lower in the UVB group (0.906 ± 0.034) than in the control group (1.242 ± 0.141,P ＜ 0.05),but significantly higher in the 100-nmol/L,1-μmol/L and 10-μmol/L biliverdin + UVB groups (1.556 ± 0.112,1.897 ± 0.234 and 2.035 ±0.274) than in the UVB group (all P ＜ 0.01).Conclusion Exogenous biliverdin protects against UVB-induced photodamage in HaCaT cells,which may be associated with Nrf-2 antioxidant signaling pathway.