ABSTRACT
Objective To evaluate the effect of ultraviolet (UV) irradiation and all-trans retinoic acid (ATRA) on expression of Hrd1 in human skin and fibroblasts,and to explore their mechanisms.Methods From December 2017 to June 2018,12 human skin tissue samples were collected from Department of Dermatology,The First Affiliated Hospital of Nanjing Medical University,including 3 sun-exposed and 3 non-sun-exposed skin tissue samples of patients aged 30-40 years,and 3 sun-exposed and 3 non-sun-exposed skin tissue samples of patients aged 60-70 years.Immunohistochemicai examination was performed to determine the expression of Hrd 1 in the above samples.A total of 40 BALB/c mice were randomly classified into 4 groups:UV group treated with UVA irradiation at 10 J/cm2 and UVB irradiation at 30 mJ/cm2 every day,ATRA group topically treated with 0.1 ml of ATRA 0.1％ cream once a day on the shaved back,UV + ATRA group treated with topical ATRA 0.1％ cream before the above UV irradiation,and control group receiving no treatment.After 14 weeks,these mice were sacrificed,skin tissues were excised from the back,and the expression of Hrd 1 was determined by immunohistochemical examination.In vitro cultured human fibroblasts were divided into 4 groups:UV group and ATRA + UV group covered with phosphate buffer saline (PBS) followed by UVA irradiation at 10 J/cm2 or UVB irradiation at 30 mJ/cm2,ATRA group treated with culture media containing 1.μmol/L ATRA for 24 hours,and ATRA + UV group also treated with culture media containing 1 μmol/L ATRA for 24 hours after the ultraviolet irradiation.Western blot analysis was performed to determine the expression of Hrd 1 in fibroblasts in the above groups,fluorescence microscopy to detect the levels of reactive oxygen species (ROS) in the above groups.Statistical analysis was carried out by one-way analysis of variance (ANOVA) for comparison among groups,and least significant difference (LSD)-t test for multiple comparisons.The difference was considered to be statistically significant when the P value was less than the significant level of 0.05.Results In both the groups of 30-40 years and 60-70 years,the expression of Hrd1 was significantly higher in the sun-exposed skin tissues (0.307 ± 0.256,0.486 ± 0.579,respectively) than in the non-sun-exposed skin tissues (0.196 ± 0.330,0.199 ± 0.375,respectively;t =5.486,10.579 respectively,both P ＜ 0.05).In the in vivo experiment,the expression of Hrd1 in the skin tissues of mice significantly differed among the control group,UV group,ATRA group and ATRA + UV group (0.189 ± 0.015,0.288 ± 0.017,0.187 ±0.020,0.226 ± 0.021 respectively,F =19.553,P ＜ 0.001),and the UV group showed significantly higher Hrd1 expression compared with the control group (t =5.337,P =0.033)and ATRA + UV group (t =4.891,P =0.039).In the in vitro experiment,the level of Hrd1 in the fibroblasts significantly differed among the 4 groups after the UVA or UVB irradiation (F =120.704,102.119,both P ＜ 0.001).The effect of the UVA and UVB irradiation on the expression of Hrd1 was basically consistent,and the Hrd1 level was significantly higher in the UV group than in the control group and ATRA + UV group (both P ＜ 0.05).After the UV irradiation,the ROS level was significantly higher in the UV group than in the control group and ATRA + UV group (both P ＜ 0.05).Conclusion ATRA can inhibit ultraviolet-induced Hrd1 expression in skin fibroblasts,likely by inhibiting the generation of cellular ROS.