Effects of UVB Irradiation on the Cell Viability
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When the HaCaT cells were irradiated with UVB at the intensity of 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 4.5 J/m2, the mean survival rate of the cells was 100.00%, 80.02%, 78.08%, 67.82%, 55.61%, 49.10%, 42.33%, 39.17%, 33.14%, and 28.96%, respectively (Figure 1A). We selected the intensity of UVB 2 J/m2, the mean survival rate of the cells was 55.61% for the rest of our experiments.
Effects of TAK-242 on the Cell Viability
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When the concentration of TAK-242 was at 0, 0.010, 0.100, 1.000, 2.000, 5.000, 10.000, 12.000, 15.000, and 20.000 μmol/L, the mean survival rate of the cells was 100.00%, 100.81%, 104.64%, 108.75%, 117.31%, 107.57%, 103.71%, 103.77%, 102.75%, and 93.04%, respectively (Figure 1B). We selected the TAK-242 concentration of 2.000 μmol/L, the mean survival rate was 117.31% for our experiment.
Effects of 1,25(OH)2D3 Concentration on Cell Viability
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When the concentration of 1,25(OH)2D3 was at 0, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 1,000, and 10,000 nmol/L, the mean survival rate of the cells was 100.00%, 103.88%, 103.32%, 113.84%, 113.61%, 114.50%, 115.02%, 130.49%, 116.96%, and 106.51%, respectively (Figure 1C). We selected the concentration of 1,25(OH)2D3 at 100 nmol/L, the mean survival rate was 130.49% for our experiment.
Effects of 1,25(OH)2D3 or/and TAK-242 on the Morphology of HaCaT Cells
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HaCaT cells from untreated control, 1,25(OH)2D3 + UVB group, TAK-242 + UVB group or 1,25(OH)2D3 + TAK-242 + UVB group had complete cell membrane, appropriate cell density, even dye of the nuclei with rare nulcear fragmentation, membrane rupture, or unclear sketch of cell structure. HaCaT cells from UVB group had lower cell density, membrane rupture, nuclear fragmentation and unclear cell sketch(Figure 2A). We used Image J software to perform the quantification of the number of cells surviving. As shown in Figure 2B, there were more cells surviving in the untreated control, 1,25(OH)2D3 + UVB group, TAK-242 + UVB group or 1,25(OH)2D3 + TAK-242 + UVB group than those in the UVB group (P < 0.05).
Effects of 1,25(OH)2D3 or/and TAK-242 on the Apoptosis of HaCaT Cells
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As shown in Figure 3A, the cells from UVB group had significantly higher number of apoptotic bodies than those from TAK-242 + UVB group, 1,25(OH)2D3 + UVB, or 1,25(OH)2D3 + TAK-242 + UVB group. In flow cytometry determination, the cells from 1,25(OH)2D3 + TAK-242 + UVB group, 1,25(OH)2D3 + UVB group or TAK-242 + UVB group had significantly lower number of apoptotic cells than those from UVB group (P < 0.05), Figure 3B.
Effects of 1,25(OH)2D3 or/and TAK-242 on the Production of ROS
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Compared with the UVB group cells, the 1,25(OH)2D3 + UVB group cells, TAK-242 + UVB group or 1,25(OH)2D3 + TAK-242 + UVB group cells had significantly lower production of ROS (P < 0.05); moreover, the 1,25(OH)2D3 + TAK-242 + UVB group cells had lower production of ROS than those from 1,25(OH)2D3 + UVB group, or TAK-242 + UVB group (P < 0.05), Figure 4.
Effects of 1,25(OH)2D3 or/and TAK-242 on the Migration of HaCaT Cells
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HaCaT cells from 1,25(OH)2D3 + UVB group, TAK-242 + UVB group, or 1,25(OH)2D3 + TAK-242 + UVB group had significant closure of the scratch than those from UVB group on 24 h, moreover, the 1,25(OH)2D3 group cells or 1,25(OH)2D3 + TAK-242 group cells had significant closure than those from TAK-242 group on 24 h (P < 0.05), but there was no significant difference in the closure of scratch between 1,25(OH)2D3 + UVB and 1,25(OH)2D3 + TAK-242 + UVB group cells, Figure 5.
Effects of 1,25(OH)2D3 or/and TAK-242 on the Expression of Oxidative Stress and ER Stress Related Proteins
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For the expression of NF-κB, HaCaT cells from 1,25(OH)2D3 + UVB group, or TAK-242 + UVB group, or 1,25(OH)2D3 + TAK-242 + UVB group had significantly lower expression of NF-κB than those from UVB group (P < 0.05). In addition, the expression of NF-κB from 1,25(OH)2D3 + TAK-242 + UVB group was significantly lower than that from the TAK-242 + UVB group (P < 0.05), Figure 6.
For the expression of TLR-4, HaCaT cells from 1,25(OH)2D3 + UVB group, or TAK-242 + UVB group, or 1,25(OH)2D3 + TAK-242 + UVB group had significantly lower expression of TLR-4 than those from UVB group (P < 0.05), moreover, the cells from TAK-242 + UVB group had lower expression of TLR-4 than those from 1,25(OH)2D3 + UVB, or 1,25(OH)2D3 + TAK-242 + UVB group (P < 0.05), Figure 6.
For the expression of IkB-ɑ, the cells from UVB group had significantly higher ratio of phosphorylated IkB-ɑ to IkB-ɑ than those from TAK-242 + UVB group, 1,25(OH)2D3 + UVB, or 1,25(OH)2D3 + TAK-242 + UVB group (P < 0.05); the cells from TAK-242 + UVB group had significantly lower ratio of phosphorylated IkB-ɑ to IkB-ɑ than those from 1,25(OH)2D3 + TAK-242 + UVB group (P < 0.05), Figure 6.
For the expression of Bip, HaCaT cells from 1,25(OH)2D3 + UVB group, or TAK-242 + UVB group, or 1,25(OH)2D3 + TAK-242 + UVB group had significantly lower expression of Bip than those from UVB group (P < 0.05); moreover, the cells from 1,25(OH)2D3 + TAK-242 + UVB group had significantly lower expression of Bip than those from the 1,25(OH)2D3 + UVB, or TAK-242 + UVB group (P < 0.05). Figure 7.
For the expression of PERK, HaCaT cells from 1,25(OH)2D3 + UVB group, or TAK-242 + UVB group, or 1,25(OH)2D3 + TAK-242 + UVB group had significantly lower expression of p-PERK than those from UVB group (P < 0.05), moreover, the cells from 1,25(OH)2D3 + UVB group had lower expression of p-PERK than those from TAK-242 + UVB group, or 1,25(OH)2D3 + TAK-242 + UVB group (P < 0.05). Figure 7.
Effects of 1,25(OH)2D3 or/and TAK-242 on the Expression of Apoptosis Related Proteins
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For the expression of apoptosis related proteins, the cells from 1,25(OH)2D3 + UVB group, or TAK-242 + UVB group, or 1,25(OH)2D3 + TAK-242 + UVB group had significantly lower expression of Caspase-3, Caspase-8, and Cytc than those from UVB group (P < 0.05). The cells from 1,25(OH)2D3 + TAK-242 + UVB group had significantly lower expression of Caspase-8, Caspase-3 and Cytc than those from 1,25(OH)2D3 + UVB group, or TAK-242 + UVB group (P < 0.05). Figure 8.