Abstract:

Objective To observe the effects of autophagy induced by different doses of ultraviolet B （UVB） irradiation on the apoptosis 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, 5.0 and 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/L following 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 annexinⅤ 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/cm2 alone （（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/cm2 alone （（7.100 ± 0.781） % vs. （10.133 ± 0.681） %, t = 6.29, P < 0.05）. Conclusions 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: 3-MA