Autophagy In Skin Fibroblast And Keratinocyte Which Were Exposed To Ultraviolet Irradiation

For investigating the interaction between apoptosis and autophagy which induced by different doses of ultraviolet B radiation in human skin fibroblast, the first part of this study has been designed. Inhibition effect by3-MA incubation was confirmed by MDC staining and LC3expression calibrated by immunofluorescence. The method of inhibition of autophagy was used by incubation4h with0.5mmol/L3-MA after different doses of UVB radiation immediately. The qualitative and quantitive methods of apoptosis assay were performed by fluorescence staining with Hoechst accompanied with PI and flow cytometry stained with Annexin V-FITC and PI. Fibroblast incubated with0.5mmol/L3-MA presented obvious inhibition of autophagy and low impairment on cell viability. The percentage of autophagic cell descended from63.037±5.876, which were induced by starvation, to34.425±5.183. The intensity of fluorescence which reflected expression of LC3increased gradually followed by increase of the UVB radiation doses, however the increase of intensity of fluorescence attenuated after inhibition autophagy by3-MA. Under radiation of50mJ/cm2dose, inhibition of fibroblast autophagy resulted in elevation of intermedial and non-viable apoptosis. Hoechst and PI staining showed increase of cells which showed both strong blue and red fluorescence. Flow cytometry assay revealed that increase of cells which stained with Annexin V-FITC and PI, from7.267±0.473to10.933±0.839. The difference has statistical significance (t=-5.197, P=0.035<0.05). On the contrary, under radiation of100mJ/cm2dose, inhibition of autophagy resulted in descent of intermedial and non-viable apoptosis. Hoechst and PI assay showed that decrease of cells which showed both strong blue and red fluorescence. Flow cytometry assay revealed that decrease of cells which stained with Annexin V-FITC and PI, from10.133±0.681to7.100±0.781. The difference has statistical significance (t=6.286, P=0.024<0.05). Fibroblast autophagy induced by50mJ/cm2UVB radiation has the pro-survival effect by inhibition of apoptosis.100mJ/cm2UVB radiation on fibroblast maybe induce autophagic cell death.The aim of the second part of this study is to investigate the expression of Bax,Bcl-2, Caspase-3and Beclin-1, which were regulatory elements involved in cross-talk between apoptosis and autophagy, within keratinocytes of mice skin exposed chronic ultraviolet irradiation. We have constituted the model of chronic UV-damaged mice, which were irradiated UVA plus UVB simulated solar light. The irradiation began form the MED (UVB0.07J/cm2, UVA0.7J/cm2), and increased0.5MED per week. The protocol contained1irradiation per day,5irradiation per week, total9weeks, as well as UVB9.45J/cm2and UVA94.5J/cm2of total dose. The expression of Bax,Bcl-2,Caspase-3and Beclin-1was investigated in epidermal keratinocytes of two groups through immunohistochemical study and analyzed contrastively between the starting point and end point of this research. Wilcoxon signed ranks text was performed for statistics. The average value for Bax,Bcl-2,Caspase-3and Beclin-1was0.30(0-2),0.25(0-2),0.35(0-2),0.25(0-1) respectively in chronic UV-damaged group before UV irradiation. The average value for Bax,Bcl-2,Caspase-3and Beclin-1was2.70(2-6),3.30(2-9),3.35(2-6) and0.25(0-1) respectively after UV irradiation. There was significance difference in expression of Bax,Caspase-3and Beclin-1(P<0.05), but no statistical difference in expression of Bel-2(P>0.05). The average value for Bax,Bcl-2,Caspase-3and Beclin-1was0.20(0-1),0.20(0-1),0.30(0-1),0.20(0-1) respectively in control group at the beginning of study, and the average value for Bax,Bcl-2,Caspase-3and Beclin-1was0.30(0-1),0.20(0-1),0.30(0-1),0.10(0-1) respectively at the end of study. There was no statistical difference in expression of Bax,Bcl-2,Caspase-3and Beclin-1(P>0.05). Chronic UV irridiation can up-regulate the expression of Beclin-1and Bax which both are involved in cross-talk between apoptosis and autophagy. It may play a role in cross-talk of apoptosis and autophagy in chronic skin photodamage.The third part of this study was designed to investigate the regulation of autophagy in human keratinocyte cell line, HaCaT suffering from UVB irradiation. At the point of fourth hour after three different doses of UVB irradiation from low to high, HaCaT cells were performed ultrastructural analysis through transmission electron microscopy and investigation of LC3-Ⅱ expression by immunoblotting. Lots of double-membrane structures, the content of which was similar to cytoplasm, were observed in the cytoplasm exposed to low dose of UVB. Degraded organelles can be found in some of these structures. The quantity of these structures in cells exposed to medium dose of UVB also can be found but not as more as that in cells exposed to low dose. However, this structure was hardly seen in cells exposed to high dose of UVB. Accordingly, expression of LC3-Ⅱ was up-regulated in HaCaT suffering from low dose of UVB irradiation, and down-regulated by high dose of UVB. These findings demonstrated that autophagy in keratinocyte was induced by low dose of UVB irradiation, but inhibited by high dose of UVB irradiation.