Effects of ultraviolet B-induced autophagy on apoptosis in human skin fibroblasts: a preliminary study

Abstract

[Objective] To observe the effects of autophagy induced by different doses of ultraviolet B (UVB) irradiation on the apeptosis in human skin fibroblasts.[Methods] Skin fibroblasts were isolated from the circumcision specimen of a 23-year-old healthy male,and subjected to a primary culture.After 3 to 10 passages,the cells were collected and applied in the following experiment.Methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferation of some fibroblasts treated with different concentrations (0,0.5,2.0,5.0and 10.0 mmol/L) of 3-methyladenine (3-MA).To qualitatively and quantitatively detect the autophagy in fibroblasts treated with different concentrations of 3-MA and in fibroblasts treated with 3-MA of 0.5 mmol/Lfollowing UVB irradiation,monodansylcadaverine (MDC) staining was carried out,and immunofluorescence was used to detect the expression of microtubule-associated protein 1 light chain (LC3).Some fibroblasts were classified into 8 groups to remain untreated,be irradiated with UVB of 30,50 and 100 mJ/cm2 alone,treated with 3-MA of 0.5 mmol/L alone,or treated with 0.5 mmol/L 3-MA following irradiation with UVB of 30,50 and 100 mJ/cm2,respectively,then,cell apoptosis was qualitatively detected by Hoechst and propidium iodide (PI)staining,and quantitatively detected by flow cytometry with annexin V and PI.[Results] The percentage of autophagic cells was (63.037 ± 5.876) ％ in fibroblasts treated with starvation condition,significantly decreased to (34.425 ± 5.183) ％ in fibroblasts treated with 3-MA of 0.5 mmol/L.The expression of LC3 showed a gradually increasing trend from untreated fibroblasts,to fibroblasts irradiated with UVB of 30,50 and 100 mJ/cm2,while the increase was attenuated by the 4-hour treatment with 3-MA immediately after the irradiation.Compared with the other concentrations,the 3-MA of 0.5 mmol/L showed the least influence on the viability of fibroblasts.The addition of 3-MA of 0.5 mmol/L increased the percentage of cells both positive for Hoechst and PI staining in fibroblasts irradiated with UVB of 50 mJ/cm2,but decreased that in fibroblasts irradiated with UVB of 100 mJ/cm2.Similarly,the percentage of middle and late apoptotic cells was significantly higher in fibroblasts irradiated with UVB of 50 mJ/cm2 followed by treatment with 3-MA of 0.5 mmol/L than in those irradiated with UVB of 50 mJ/cm2alone ((10.933 ± 0.839) ％ vs.(7.267 ± 0.473) ％,t =5.20,P＜ 0.05),but lower in fibroblasts irradiated with UVB of 100 mJ/cm2 followed by treatment with 3-MA of 0.5 mmol/L than in those irradiated with UVB of 100 mJ/cm2alone ( (7.100 ± 0.781 ) ％ vs.( 1 0.133 ± 0.681 ) ％,t =6.29,P ＜ 0.05 ).[Conclusion]s The irradiation with UVB of 50 mJ/cm2 may protect fibroblasts by inducing autophagy and suppressing apoptosis,while the high level of autophagy induced by UVB of 100 mJ/cm2 may lead to autophagic cell death in fibroblasts. Key words: Ultraviolet rays; Fibroblasts; Autophagy; Apoptosis; 3-MA