Protective Effects Of PBX1 On HaCaT Cells Injury Induced By H₂O₂ And Its Related Mechanisms

Background:Skin,as the largest surface organ of human body,constitutes the first barrier between human body and environment.As air pollution increases,skin becomes more vulnerable to harmful substances in the environment.Reactive oxygen species(ROS)is a common product of skin cell damage.Excessive ROS leads to oxidative damage of DNA,proteins and lipids in body cells.Therefore,reducing excess ROS in cells is of great significance for reducing cell damage caused by oxidative stress.Pre-B-cell leukemia homeobox 1(PBX1)is a member of the homeobox gene family,which promotes cell proliferation.TAT is a group of short peptides with high efficiency of penetrating tissue barrier.It is connected with PBX1 to form fusion protein Tat-PBX1,which promotes PBX1 to enter cells and play biological roles.In this study,H2O2 was used as ROS inducer,lentivirus transduction was used to establish HaCaT cell lines overexpressing PBX1,and the fusion protein TAT-PBX1 was prepared in large quantities by the prokaryotic expression system of Escoli,so as to explore the protective effect of PBX1 on HaCaT cell damage induced by H2O2 and its mechanism.Objective:To study the damage effect of H2O2 on HaCaT cells.To study the biological effects of PBX1 expression on HaCaT cells.To investigate the protective effect of PBX1 on H2O2-induced HaCaT cell damage and its mechanism.To investigate the protective effect of fusion protein TAT-PBX1 on H2O2-induced HaCaT cell damage and its mechanism.Methods:1.HaCaT cells were treated with H2O2.Morphological changes of cells were observed by living cell imaging system,cell survival rate was detected by CCK8 method.Flow cytometry was used to detect cell apoptosis.ROS levels in cells were detected by living cell imaging system at different time points of H2O2 and at the same time points of different concentration of H2O2.2.PBX1 overexpression of HaCaT cells was established.PBX1 expression was detected by live cell imaging system and Western blot.The effect of PBX1 on HaCaT cell proliferation was detected by growth curve.The effect of PBX1 on the clonogenesis ability of HaCaT cells was detected by plate cloning assay.The effect of PBX1 on HaCaT cell migration was detected by cell scratch assay.The effects of PBX1 on the expression of DNA damage,repair and apoptosis-related proteins in HaCaT cells were detected by Western blot.3.The morphological changes of cells were observed by living cell imaging system after a certain dose of H2O2 was given to each group.Cell survival rate was detected by CCK8 method.Intracellular ROS levels were determined by flow cytometry.Flow cytometry was used to detect cell apoptosis.Western blot assay was used to detect the expression levels of DNA damage,repair and apoptosis related proteins.4.TAT-PBX1 protein was prepared by prokaryotic expression system,and the sample was purified by nickel column affinity chromatography.Pulse dilution;Concentrate and lyophilize into powder;Western blot assay was used to detect label and target proteins.The membrane penetration of TAT-PBX1 was verified by Western blot and immunofluorescence.The effect of different concentrations of TAT-PBX1 on the survival rate of HaCaT cells was detected by CCK8.Intracellular ROS levels were determined by flow cytometry.Flow cytometry was used to detect cell apoptosis.Western blot assay was used to detect the expression levels of DNA damage,repair and apoptosis related proteins.Results:1.H2O2 induced HaCaT cell damage(1)Morphological changes of HaCaT cells induced by H2O2:Microscopic observation results showed that with the increase of H2O2 concentration,the edge of the cells gradually blurred,the light transmittance gradually decreased,and finally the cells fell off and fragmented.(2)H2O2 decreased HaCaT cell survival rate:CCK8 test results showed that with the increase of H2O2 concentration,HaCaT cell survival rate decreased gradually.(3)H2O2 promoted apoptosis of HaCaT cells:Flow cytometry showed that with the increase of H2O2 concentration,the apoptosis rate of HaCaT cells increased gradually.(4)H2O2 increased the intracellular ROS level of HaCaT:fluorescence imaging results showed that the fluorescence intensity increased first and then decreased at different time of 1.5 mmol/L H2O2 treatment,and reached a peak around 4 h.When HaCaT cells were treated with different concentrations of H2O2 for 4 h,the fluorescence intensity showed a trend of increasing first and then decreasing,and reached a peak around the concentration of 1.5 mmol/L.2.The effect of PBX1 on HaCaT cell biology(1)Establishment of HaCaT cell lines overexpressing PBX1:The results of live cell imaging system and Western blot test showed that the HaCaT cells overexpressing PBX1 and its empty vector-pLVX-PBX1 and pLVX-Ctrl were successfully established.(2)PBX1 promoted the proliferation of HaCaT cells:The results of the growth curve showed that there were more cells in the pLVX-PBX1 group from the 4th day.(3)PBX1 promoted the formation of HaCaT cell cloning:The results of plate cloning experiment showed that the number of cell clones in pLVX-PBX1 group was higher.(4)PBX1 promoted HaCaT cell migration:The cell scratch test results showed that the cell mobility of pLVX-PBX1 group was higher after 24 hours of scratch.(5)PBX1 down-regulated DNA damage and repair related protein expression in HaCaT cells:The results of Western blot showed that the expression levels of KU 80,KU 70,Rad51 and γH2AX in pLVX-PBX1 group were significantly decreased.(6)PBX1 down-regulated apoptosis-related protein expression in HaCaT cells:The results of Western blot showed that the expression levels of PARP-1,PAR,AIF,Cyt C,Cleaved Caspase-3 protein in pLVX-PBX1 group decreased significantly.3.Protective effect of PBX1 on H2O2-induced HaCaT cell damage(1)PBX1 increased the survival rate of HaCaT cells after H2O2 injury:CCK8 assay showed that PBX1 increased the survival rate of HaCaT cells after H2O2 treatment.(2)PBX1 decreased the intracellular ROS level of HaCaT induced by H2O2:Flow cytometry results showed that PBX1 decreased the ROS level of HaCaT cells treated with H2O2.(3)PBX1 reduced H2O2-induced apoptosis of HaCaT cells:Flow cytometry showed that PBX1 reduced the apoptosis rate of HaCaT cells treated with H2O2.(4)PBX1 down-regulated the expression levels of H2O2-induced DNA damage and repair related protein expression in HaCaT cells:Western blot results showed that PBX1 down-regulated the expressions of KU 80,KU 70,Rad51 andγH2AX in HaCaT cells treated with H2O2.(5)PBX1 down-regulated HaCaT apoptosis-related protein expression induced by H2O2:Western blot results showed that PBX1 down-regulated HaCaT cells PARP-1,PAR,AIF,Cyt C,Cleaved Caspase-3 expression.4.Preparation of fusion protein TAT-PBX1 and its protective effect on HaCaT cell damage induced by H2O2(1)Strains with high expression of target protein were successfully screened out:seven monoclonal expression strains were selected and tested for HA,His and PBX1 by Western blot.The amount of protein expression was higher in Clone6 strain.(2)The fusion protein TAT-PBX1:SDS-PAGE was successfully obtained and identified.The results showed that the eluent contained relatively pure target protein.The proteins HA,His and PBX1 were identified by Western blot.(3)Fusion protein TAT-PBX1 penetrated into HaCaT cells:The results of live cell imaging system showed that the green fluorescence of HA in TAT-PBX1 group was stronger.Western blot results showed that the expression levels of HA,His and PBX1 were up-regulated in TAT-PBX1 treatment group.(4)TAT-PBX1 increased the survival rate of HaCaT cells after H2O2 injury:CCK8 assay showed that TAT-PBX1 increased the survival rate of HaCaT cells after H2O2 treatment.(5)TAT-PBX1 decreased the H2O2-induced intracellular ROS level of HaCaT:Flow cytometry results showed that TAT-PBX1 decreased the ROS level of HaCaT cells treated with H2O2.(6)TAT-PBX1 decreased the H2O2-induced apoptosis rate of HaCaT cells:Flow cytometry results showed that TAT-PBX1 decreased the apoptosis rate of HaCaT cells treated with H2O2.(7)TAT-PBX1 down-regulated H2O2-induced DNA damage and repair related protein expression in HaCaT cells:Western blot results showed that TAT-PBX1 downregulated the expression of KU 80,KU 70,Rad51 and γH2AX in HaCaT cells treated with H2O2.(8)TAT-PBX1 down-regulated HaCaT apoptosis-related protein expression induced by H2O2:Western blot results showed that TAT-PBX1 down-regulated HaCaT cells expressed PARP-1,PAR,AIF,Cyt C,Cleaved Caspase-3.Conclusions:1.H2O2 promotes ROS production and apoptosis in HaCaT cells.2.PBX1 promotes the proliferation of HaCaT cells.3.PBX1 can reduce ROS level and DNA damage to reduce H2O2 damage to HaCaT cells.4.TAT-PBX1 can reduce ROS level and DNA damage to reduce H2O2 damage to HaCaT cells.