| Objective: To investigate the effects of autophagy and apoptosis and its interaction regulatory elements beclin-1-bcl-2-bax induced by low to moderate UVB exposure on melanoma cells(A375).Methods:(1).A375 cells were irradiated with UVB at low dose(10 m J/cm2)and moderate dose(15 m J/cm2),and the difference of autophagosome formation was observed by MDC staining at 3,6,9,and 12 hours after irradiation.(2)A375 cells were irradiated with UVB at low dose and moderate dose,and the differences in apoptosis of cells were detected by AO/EB staining at 3,6,9,and 12 hours after irradiation.(3)A375 cells were irradiated with UVB at low dose and moderate dose,then the Cell viability was measured by CCK-8 at time points of 18 h,24h,36 h,and 48h;(4)A375 cells were irradiated with low dose,moderate dose of UVB,then the total cellular protein was extracted at 3,6,9 and 12 h after irradiation.and the A375 cells was irradiated with the dose of 2.5、5.0、7.5、10.0、15.0 m J/cm2 of UVB,then the total cellular protein was extracted at 9h point after irradiation.Then the changes of autophagy-related proteins Beclin-1 and LC3,anti-apoptosis protein Bcl-2 and pro-apoptotic protein Bax were detected by Western blot.Results:(1)The results of MDC staining showed that autophagy induced by moderate dose and low dose of UVB exposure was gradually increased with the extension of time.However,At 12 h,autophagy has been observed to decrease in the moderate dose;And comparing the autophagy at the same time point with different doses at moderate and low doses,it was shown that the UVB-induced autophagy effect was stronger than the low dose.(2)The results of AO / EB staining showed that the apoptotic effect induced by moderate and low doses was showed the increase of apoptotic cells with the extension of time;And comparing the apoptotic at the same time point with different doses at moderate and low doses,it was shown that the UVB-induced apoptotic effect was stronger than the low dose.(3)The cell viability assay by CCK-8 showed that compared with control group,moderate and low dosage groups had a significant difference at the same time from 18 h to 48h(p <0.05),and the order is: control group> low dose group> moderate dose group;After 24 h with UVB exposure,there was a significant difference between moderate and low dosage groups(p <0.05);(4)Western blot detection of autophagy and apoptosis related proteins showed that 10 mj / cm2 of UVB irradiated A375 cells within 12 h,as time goes by,autophagy-related protein Beclin 1,LC3 and pro-apoptotic protein Bax increased gradually,while the expression of anti-apoptotic protein Bcl-2 decreased gradually.After A375 cells were treated with 15 mj / cm2 UVB,Beclin-1,LC3 and Bax expression increased gradually to 9h,but at 12 h the expression of Beclin 1,LC3 were observed decreased,while Bax continued to rise 12 h after irradiation;A375 cells with different dose UVB exposure,At 9h time point,Beclin-1,LC3 increased gradually up to 10 mj / cm2 and peaked at 15 mj / cm2;the expression of Bax increased with increasing dose,while the expression of Bcl-2 gradually decreased with the increase of dose.Conclusion:(1)Moderate and low doses of UVB irradiation can inhibit the viability of A375 cells by inducing autophagy and apoptosis.(2)the cell viability iuduced by low and moderate doses UVB exposure was different: at low doses,the cells was induced relatively weak autophagy to partially resist the induction of apoptosis by UVB,so the cell viability in the low dose irradiation was higher than that in the moderate dose.When irradiated with moderate doses in the early,both autophagy and apoptosis play a synergistic role,Together to promote apoptosis,and at the later-stage,apoptosis gradually dominates the cell’s fate.(3)The autophagic and apoptotic interactive regulatory element Beclin-1-Bcl-2-Bax plays a role in A375 cells with UVB exposure,which together determine the cell’s fate. |
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