1,25?OH?₂D₃ And TAK-242 Alleviate The Damage Caused By UVB To HaCaT Cells

Due to the destruction of the ozone layer,the medium-wave ultraviolet?UVB?that reaches the surface is significantly increased,which can cause human dermatitis,skin aging,immunosuppression,and skin cancer.The damage effect of the body has gradually become the focus of attention.Studies have found that the process of UV-induced keratinocyte damage can activate Toll-like receptors?TLRs?in the immune system,which in turn activates the NF-?B signaling pathway,causing the release of inflammatory factors.UVB radiation can also lead to the production and accumulation of a large number of reactive oxygen species?ROS?,causing lipid peroxidation of tissues and cells in the body,causing ERK1/2,p38 MAPK,JNK,and NF-?B signaling pathway abnormal activation.Resatorvid?TAK-242?is a specific inhibitor of NF-?B upstream signaling molecule TLR4;The active form of vitamin D can regulate pro-inflammatory and anti-inflammatory factors and immune cells through the NF-?B and PI3K signaling pathways,and can also play an anti-inflammatory role.It can also regulate Bcl-2 family proteins,FADD and cysteum related to the programmed cell death Caspase expression.Objective:The protective effect of the combined application of calcitriol?1,25?OH?₂D₃?and TAK-242 on HaCaT cell damage caused by UVB was analyzed,and the mechanism of action was analyzed.Method:HaCaT cells were cultured in vitro and given different doses of UVB radiation.After 24 hours of continuous culture,the cell survival rate was detected by MTT method to determine the UVB radiation dose in this experiment;Give different concentrations of 1,25?OH?₂D₃ pretreatment for 24h,and then give different concentrations of TAK-242 for pretreatment for 1h,after UVB irradiation for 24h,detect by MTT method,confirm this experiment 1,25?OH?₂D₃ and TAK-242 Processing concentration.Divide HaCaT cells into 7 groups:normal group without any treatment;1,25?OH?₂D₃ group was given 1,25?OH?₂D₃ alone for 24h;The UVB group was given UVB irradiation alone;TAK-242 group was given TAK-242 alone for 1h;The TAK-242+UVB group was given TAK-242 pretreatment for 1 hour and then UVB irradiation;the1,25?OH?₂D₃+UVB group was given 1,25?OH?₂D₃ pretreatment for 24 hours and then UVB irradiation;The 1,25?OH?₂D₃+TAK-242+UVB group was given 1,25?OH?₂D₃pretreatment for 24h,then TAK-242 pretreatment for 1h,and then given UVB irradiation.The different drug pretreatment groups were irradiated with UVB and continued to cultivate for 24 hours.Cell morphology was observed by HE staining;DAPI staining was used to detect apoptosis,and Annexin-V-PE apoptosis kit was used to detect the level of apoptosis;Western blot method was used to detect the expression of Caspase-8,Caspase-3 and Cytc apoptotic proteins;the cell scratch method was used to detect the migration ability of each group of cells;Use active oxygen detection kit to detect ROS generated in cells;Western blot was used to detect the expression of NF-?B,TLR4,PI3K,I?B?,P-I?B?,Bip,PERK,P-PERK oxidative stress and endoplasmic reticulum stress protein.Statistical analysis was performed using SPSS 22.0.Result:1.Determination of irradiation dose and drug treatment concentration?1?Determination of UVB exposure dose:After UVB irradiation,the HaCaT cell survival rate gradually decreases with the increase of UVB irradiation dose,Compared with the normal group,the UVB irradiation dose was 5mJ/cm²,10mJ/cm²,15mJ/cm²,20mJ/cm²,25mJ/cm²,30mJ/cm²,35mJ/cm²,40mJ/cm²,and 45mJ/cm² group Cell survival rate is significantly reduced,The difference was statistically significant?P<0.05?.When the UVB irradiation dose was 20mJ/cm²,the cell survival rate was 55.61%.And the difference was statistically significant compared with other dose groups?P<0.05?.Therefore,in this experiment,20mJ/cm² was determined as the optimal irradiation dose of UVB.?2?Determination of calcitriol?1,25?OH?₂D₃?concentration:HaCaT cells treated with different concentrations of 1,25?OH?₂D₃,Under the same UVB irradiation dose,the cell survival rate increased first and then decreased with the increase of treatment concentration,Compared with other dose-treated groups,the concentration of1,25?OH?₂D₃ treated with 100nM significantly increased the cell survival rate,and the difference was statistically significant?P<0.05?,Therefore,this experiment determined that 100nM was the optimal treatment concentration of 1,25?OH?₂D₃.?3?TAK-242 concentration determination:Under the same UVB irradiation dose,the HaCaT cell survival rate increased first and then decreased with the increase of TAK-242 treatment concentration,Compared with the other concentration-treated groups,the TAK-242 treatment group at a concentration of 2?M significantly increased the cell survival rate,and the difference was statistically significant?P<0.05?.Therefore,this experiment determined that 2?M was the optimal concentration for TAK-242 treatment.2.Cell morphological changesAfter HE staining in normal group,1,25?OH?₂D₃ group and TAK-242 group,the cell membrane nucleus was uniformly stained,and the cell membrane nucleus was intact.Rarely visible cell fragmentation,cell membrane rupture,cell structure outline was unclear,visible Uniform cell density,no significant decrease in live cell density.In the UVB group,obvious cell membrane rupture and nuclear fragmentation occurred,the outline of the cell structure was unclear,and the density of living cells decreased significantly.Compared with the UVB group,cell membrane rupture,nuclear fragmentation,and cell structure appeared in the 1,25?OH?₂D₃+UVB group,TAK-242+UVB group,and1,25?OH?₂D₃+TAK-242+UVB group.Unclear contours,reduced density of living cells.3.ApoptosisAfter normal cell staining with DAPI,the nuclei were uniformly stained,the cell membrane was intact,and the cells adhered well,showing a polygonal or spindle shape;Compared with the normal group,the number of adherent cells in the UVB group was significantly reduced,the cells became rounded,the gaps became larger,the cell membrane was damaged,and the nuclei were fragmented.Apoptotic features appeared,and the formation of apoptotic bodies was visible.Compared with the UVB group,cell damage,nuclear fragmentation,and apoptotic bodies appeared in1,25?OH?₂D₃+UVB group,TAK-242+UVB group,and 1,25?OH?₂D₃+TAK-242+UVB group.4.Changes in cell migration abilityCompared with the UVB irradiation group,after the cells of the normal group and other treatment groups were scratched for 12 h and 24 h,the relative healing width of the cell scratches was significantly higher than that of the UVB group,and the difference was statistically significant?P<0.05?,After 12h of scratches,compared with other treatment groups,the relative healing width of cells treated with1,25?OH?₂D₃ alone was significantly increased,and the difference was statistically significant?P<0.05?;During the scratch period of 12²4 hours,compared with other treatment groups,the relative healing width of the cells treated with TAK-242 alone was significantly increased,and the difference was statistically significant?P<0.05?.After 12h and 24h of scratches,the relative healing width of cell scratches in the1,25?OH?₂D₃+UVB group and the 1,25?OH?₂D₃+TAK-242+UVB group were higher than those in the TAK-242+UVB group.It has statistical significance?P<0.05?.5.Cellular Reactive Oxygen LevelCompared with the normal group,the levels of ROS produced in all the cells exposed to UVB were significantly increased,and the difference was statistically significant?P<0.05?;Compared with the UVB group,the level of ROS produced in the cells of the TAK-242+UVB group was significantly reduced,and the difference was statistically significant?P<0.05?;Compared with the TAK-242+UVB group,the level of ROS produced in the 1,25?OH?₂D₃+UVB group was significantly reduced,and the difference was statistically significant?P<0.05?;Compared with the 1,25?OH?₂D₃+UVB group and the TAK-242+UVB group,the level of ROS production in the1,25?OH?₂D₃+TAK-242+UVB group was the most significant,and the difference was statistically significant?P<0.05?.6.Apoptosis levelCompared with the normal group,the level of apoptosis in all UVB-irradiated groups was significantly increased,and the difference was statistically significant?P<0.05?;Compared with the UVB cells,the apoptosis levels in the 1,25?OH?₂D₃+UVB group,TAK-242+UVB group,and 1,25?OH?₂D₃+TAK-242+UVB group were significantly reduced,and the difference was statistically significant.Academic significance?P<0.05?;There was no significant difference in apoptosis levels between the three groups of 1,25?OH?₂D₃+UVB group,TAK-242+UVB group,and1,25?OH?₂D₃+TAK-242+UVB group?P>0.05?.7.Oxidative stress related protein expression levelCompared with the normal group,the expression levels of TLR4 and NF-?B protein and the ratio of P-I?B?/I?B?in the UVB group significantly increased,and the differences were statistically significant?P<0.05?;Compared with the normal group,the expression levels of I?B?and PI3K proteins in the UVB group cells were significantly reduced,and the difference was statistically significant?P<0.05?.Compared with the normal group,the expression level of NF-?B protein in the1,25?OH?₂D₃ group cells was significantly reduced,and the difference was statistically significant?P<0.05?;Compared with the normal group,the expression of TLR4protein in the TAK-242 group cells was significantly reduced,and the difference was statistically significant?P<0.05?.Compared with UVB Group,1,25?OH?₂D₃+UVB group,TAK-242+UVB group and 1,25?OH?₂D₃+TAK-242+UVB group of cells TLR4,NF-?B protein expression And the ratio of P-I?B?/I?B?significantly decreased,the expression level I?B?,PI3K protein was significantly increased,the difference was statistically significant?P<0.05?.8.Endoplasmic reticulum stress-related protein expression levelsCompared with the normal group,the expression levels of P-PERK and Bip protein and the ratio of P-PERK/PERK in the UVB group were significantly increased,and the differences were statistically significant?P<0.05?;Compared with the normal group,the expression level of PERK protein in the UVB group cells was significantly reduced,and the difference was statistically significant?P<0.05?.Compared with the UVB group,P-PERK,Bip protein expression levels and P-PERK/PERK ratios in the1,25?OH?₂D₃+UVB group,TAK-242+UVB group,1,25?OH?₂D₃+TAK-242+UVB group were significantly reduced,The difference was statistically significant?P<0.05?.Compared with the TAK-242+UVB group,the expression level of Bip protein in the 1,25?OH?₂D₃+UVB group and the 1,25?OH?₂D₃+TAK-242+UVB group was significantly reduced,and the difference was statistically significant?P<0.05?.9.Apoptosis-related protein expression levelCompared with the normal group,the expression levels of Cytc,Caspase-8 and Caspase-3 in the UVB group cells were significantly increased,and the difference was statistically significant?P<0.05?;Compared with the UVB group,the expression levels of Caspase-3 and Cytc proteins in the 1,25?OH?₂D₃+UVB group cells were significantly reduced,and the difference was statistically significant?P<0.05?;Compared with UVB group,the expression levels of Cytc,Caspase-8 and Caspase-3 in TAK-242+UVB group and 1,25?OH?₂D₃+TAK-242+UVB group were significantly reduced,and the difference was statistically significant?P<0.05?;Compared with the TAK-242+UVB group,the expression levels of Caspase-3 and Cytc proteins in the1,25?OH?₂D₃+UVB group and the 1,25?OH?₂D₃+TAK-242+UVB group were significantly reduced.Significance?P<0.05?;Compared with the 1,25?OH?₂D₃+UVB group and TAK-242+UVB group,the Cytc,Caspase-8,and Caspase-3 protein expression levels in the 1,25?OH?₂D₃+TAK-242+UVB group were the most significantly reduced.The difference was statistically significant?P<0.05?.Conclusion:1.UVB can cause the increase of intracellular ROS level,cause oxidative stress and endoplasmic reticulum stress,reduce the ability of cell migration,and increase the level of apoptosis;2.The combined treatment of 1,25?OH?₂D₃ and TAK-242 to reduce intracellular ROS production is better than the single treatment;3.1,25?OH?₂D₃ and TAK-242 pretreatment can reduce cell damage caused by UVB and improve cell migration ability;4.After pretreatment with 1,25?OH?₂D₃ and TAK-242,the damage of HaCaT cells can be reduced by inhibiting the activation of NF-?B;5.The combined treatment of 1,25?OH?₂D₃ and TAK-242 can reduce the apoptosis of Cytc,Caspase-8 and Caspase-3 protein,which is better than the treatment alone.