Inhibitory Effects Of Apigenin On Unfolded Protein Response And CXCL16 Release Induced By Oxidative Stress In Human Keratinocytes

1.Establish the oxidative stress model of human keratinocytes induced by hydrogen peroxide.2.To explore the effect and possible mechanism of apigenin（Apigenin,4’,5,7-trihydroxyflavone）on unfolded protein response（UPR）and CXCL16 release induced by oxidative stress.3.To explore the application prospect of apigenin in the treatment of vitiligo.Methods:1.MTT assay was used to detect the cell viability of Hacat cells treated with different concentrations of H₂O₂（100,150,200,250,300,350μmol/L）,different concentrations of apigenin（5,10,25,50,100,200μmol/L）or pretreated with different concentrations of apigenin（6.25,12.5,25μmol/L）before being treated with H₂O₂（250μmol/L）.2.Flow cytometry was used to detect the effects of ROS generation in Hacat cells which pretreated with different concentrations of apigenin（6.25,12.5,25μmol/L）before being treated with H₂O₂.3.Real-time quantitative PCR was used to detect the mRNA expression level of totalXBP1（XBP1t）,spliced XBP1（XBP1s）and unspliced XBP1（XBP1u）in Hacat cells which pretreated with different concentrations of apigenin（6.25,12.5,25μmol/L）before being treated with H₂O₂.4.Western Blot was used to detect the protein expression level of p-eIF2α,eIF2α,p-NF-κB and NF-κB in Hacat cells which pretreated with different concentrations of apigenin（6.25,12.5,25μmol/L）before being treated with H₂O₂.5.ELISA method was used to detect the level of CXCL16 in Hacat cells which pretreated with different concentrations of apigenin（6.25,12.5,25μmol/L）before being treated with H₂O₂.Results:1.H₂O₂ could inhibit the cell viability of Hacat cells in a dose dependant manner（p<0.01）.When Hacat cells were treated with 250μmol/L H₂O₂,the cell viability was 58.94%±4.11%,which was close to IC50,so this concentration was used in subsequent experiments.Apigenin at the concentration of 25μmol/L or less showed no significant effect on the cell viability of Hacat cells（p>0.05）.When the concentration is greater or equal to50μmol/L,apigenin inhibited cell viability in a dose dependant manner（p<0.01）.Apigenin could significantly reduce the cell damage induced by H₂O₂ in a dose dependant manner（p<0.01）.2.H₂O₂ could significantly increase ROS content in Hacat cells（p<0.01）,and apigenin could significantly inhibit ROS generation triggered by H₂O₂ in a dose dependant manner（p<0.01）.There was no significant difference between treated with apigenin alone and blank control group in ROS content（p>0.05）,it showed that apigenin did not increase the ROS content in Hacat cells.3.H₂O₂ could significantly increase the mRNA expression level of XBP1t,XBP1s and XBP1u in Hacat cells（p<0.01）,and apigenin could significantly inhibit it in a dose dependant manner（p<0.01）.4.H₂O₂ could significantly increase the protein expression level of p-eIF2α,eIF2α,p-NF-κB and NF-κB in Hacat cells（p<0.01）,and apigenin could significantly down-regulate it in a dose dependant manner（p<0.01）.5.H₂O₂ could significantly increase the level of CXCL16secretion in Hacat cells（p<0.01）,and apigenin could significantly decrease it in a dose dependant manner（p<0.01）.conclusion:1.Apigenin can inhibit ROS production of human keratinocytes induced by H₂O₂.2.Apigenin can inhibit H₂O₂-induced unfolded protein response and CXCL 16 release in human keratinocytes.Apigenin may have the potential to be developed as a therapeutic drug for vitiligo.