The Effects Of Sirolimus Inducing Autophagy On The Proliferation Of And Apoptosis In HaCaT Cells Irradiated By UVB

IntroductionUltraviolet B (UVB) mainly targets keratinocytes of epidermis, resulting in photo relative skin injuries including DNA damage, oxidative stress and cell apoptosis. Autophagy,as a physiological phenomenon, extensively exists in eukaryotic cells. Autophagy can be triggered by radiation, hunger and other stresses. Autophagy performs a fundermental role of degeneration of denatured protains and injured organelles. Also, autophagy is of significance to maintain cell homeostasis and benefits cells survival undergoing inferior environment. Scientific researches have demonstrated that autophagy can be activated by cell injuries and autophagy supply a protective effect for host. Moreover, it is be suggested that autophagy exsits in keratinocytes and alleviates inflammation in keratinocytes. Whereas, it remains unclear whether antophagy will be induced in keratinocyte exposed to UVB. The role of autophagy is uncertain concerning damages in keratinocytes irradiated by UVB. Whether sirolimus inducing autophagy can alleviate UVB-injuries in keratinocytes is still unknown.Based on the above statements, we attempted to investigate the relative researches.ObjectiveTo detect whether autophagy can be induced by UVB irradiation in HaCaT cells. To explore the effects of sirolimus on autophagy level in UVB-irradiated HaCaT cells. To determine the effects of sirolimus on the proliferation of and the apoptosis in HaCaT cells irradiated by UVB.MethodsHaCaT cells were irradiated by UVB containing doses of0、25、50mJ/cm2and followed by MDC staining to detect the autophagosome. Subsequently, HaCaT cells were divided into6groups as follows:untreated cells; cells treated with DMSO; cells irradiated by25mJ/cm2UVB; cells irradiated by25mJ/cm2UVB and treated with sirolimus; cells irradiated by50mJ/cm2UVB; cells irradiated by50mJ/cm2UVB and treated with sirolimus. Then the autophagosome in HaCaT cells of6groups was examined by MDC staining, and the percentages of the autophagosome positive cells of6groups were counted and compared. Furthermore, the proliferation of HaCaT cells of6groups were determined by MTT assay. The apoptosis in HaCaT cells of6groups were assessed by Hoechest staining and flow cytometry using AnnexinV-FITC and PI fluorescein.ResultsAutophagy was detected in HaCaT cells after UVB exposure. The percentages of the autophagosome positive cells in HaCaT cells receiving UVB irradiation containing doses of0,25,50mJ/cm2respectively were1.07%±1.85%,9.37%±1.14%,16.29%±3.03%, and were of significant difference (F=37.34, n=2,.P<0.01) among three UVB doses. Subsequently, the percentages of the autophagosome positive cells in HaCaT cells of6groups respectively were0.56%±0.79%,1.61%±2.27%,10.00%±0.47%,21.18%±3.03%,15.82%±4.13%,29.45%±1.24%, and similarly were of significant difference (F=45.23, n=5, P<0.01) among6groups. In details, the percentages of the autophagosome positive cells in HaCaT cells irradiated by UVB and sirolimus were higher than in cells irradiated by UVB only.The inhibition ratio of proliferation of HaCaT cells of6groups respectively were0.01%±4.70%、8.40%±12.43%、9.43%±2.87%、6.22%±5.88%、12.12%±1.91%、14.17%±4.30%, and were different significantly (F=9.836, n=5,P<0.01). UVB irridiation containing doses of both25mJ/cm2and50mJ/cm2inhibited the proliferation of HaCaT cells. The ratio of suppression to cell proliferation of HaCaT cells irradiated by25mJ/cm2UVB was9.43%±2.87%, and was6.22%±5.88%of HaCaT cells irradiated by25mJ/cm2UVB and treated with sirolimus, suggesting that sirolimus relieved the suppression to proliferation of HaCaT cells irradiated by25mJ/cm2UVB. However, sirolimus aggravated inhibition ratio of proliferation of HaCaT cells irradiated by50mJ/cm2UVB.Apoptotic cells and apoptotic bodies were examined obviously by Hoechest staining in groups of cells irradiated by25mJ/cm2or50mJ/cm2UVB, and the number of apoptotic cells and apoptotic bodies in groups of cells irradiated by50mJ/cm2UVB were more than in groups of cells irradiated by25mJ/cm2UVB. The number were decreased notabley in those groups of cells treated with sirolimus.Data of flow cytometry using Annexin V-FITC and PI indicated that the percentages of prophase apoptotic cells in6groups respectively were2.18%±1.30%、2.33%±1.96%、 8.55%±8.44%、8.36%±0.70%、11.43%±1.81%、5.18%±5.83%, and the percentages of anaphase apoptotic cells in6groups respectively were1.72%±0.33%、2.15%±1.11%、1.72%±1.84%、12.48%±5.04%、14.24%±4.10%、8.97%±6.01%. Consistently, the percentages of both prophase apoptotic cells and anaphase apoptotic cells in groups treated with50mJ/cm2UVB and sirolimus were lower than treated with50mJ/cm2UVB only, allowing sirolimus reduce apoptotic cells and apoptotic bodies in HaCaT cells irradiated by50mJ/cm2UVB.ConclusionsAutophagy occurred in HaCaT cells irradiated by UVB. The percentages of the autophagosome positive cells in HaCaT cells were improved by sirolimus. Sirolimus relieved the suppression to cell proliferation of cells irradiated by25mJ/cm2UVB. However, sirolimus aggravated the suppression to cell proliferation of cells irradiated by50mJ/cm2UVB. Sirolimus reduced apoptotic cells in HaCaT cells irradiated by50mJ/cm2UVB. Sirolimus inducing autophagy benefited the proliferation of and decreased the apoptosis cells in HaCaT Cells irradiated by UVB specificly.