Protective Effect Of Cyanidin-3-glucoside Against Ultraviolet B Radiation Induced Cell Damage In Human HaCaT Keratinocytes Through The Regulation Of ROS, P53

Objective: By exploring the damage under UVB in the Ha Ca T cells activated ROS and p53 levels, to explore the relationship between the two and apoptosis induction by UVB; and using the "ROS--DNA damage--p53--mitochondrial apoptosis pathway" as the target, to explore the protective mechanism of cyanidin-3-glucoside(C3G) on photodamaged skin keratinocytes. So as to the clinical effective prevention and treatment of skin photodamage to provide new ideas.Methods: 1. Select the Ha Ca T cells to build the model of skin photo damage, and to explore the best protective concentration of cyanidin-3-glucoside(C3G). 2. Experimental groups: control group of normal cells, UVB irradiation group, UVB with different concentration of C3 G in the experimental group. 3. Detection of cell index: The cell activity: MTT assay was used to measure the cell proliferation. The cell apoptosis: used Annexin V-FITC/PI staining by flow cytometric analysis. The level of ROS: were evaluated by DCFH-DA fluorescence assay. The DNA damage: detected ATM, ATR by western blot analysis. p53: detected p53 phosphorylation level by western blot analysis. The mitochondrial membrane potential: JC-1 staining by flow cytometric analysis. The protein levels of apoptosis: Bcl-2 family protein(including pro-apoptotic protein Bax, anti-apoptosis protein Bcl-2) and cleaved caspase-3, used western blot analysis.Results: 1. UVB irradiation in a dose- and time-dependent manner inhibited Ha Ca T cell survival, of which 12 h cultured in 300 m J/cm2 dose, the survival rate of the cells began to appear significantly decreased. In addition of C3 G, cell survival rate were increased, and there is a dose dependence; compared with the UVB irradiation group, UVB + 80, 160 and 200 μmol/L of C3 G the three group can significantly improve the survival rate of cells. 2. Compared with normal control group, 300 m J/cm2 UVB irradiation after12 h, cell apoptosis rate increased, and the proportion of active cells decreased significantly; while in the join of C3 G, could significantly decrease the apoptosis. 3. With the increase of the UVB dose, ROS production gradually increased, with dose dependent; and after adding C3 G, along with the increase of anthocyanin concentration, scavenging effect of anthocyanin on UVB induced ROS gradually increased, and with the dose dependent. 4. Compared with normal control group, after 300 m J/cm2 UVB irradiation, the Δψm decreased(showed increased JC-1 green fluorescence); and after joining C3 G, can make the Δψm increased, and decreased the mitochondrial membrane permeability. 7. After 300 m J/cm2 UVB irradiation caused significant DNA damage, expressed in ATM, ATR phosphorylation up-regulated; while in the join of C3 G, are starting to protect DNA from damage, reduce the performance in the phosphorylation of ATM, ATR. 8. Cells in the normal state of phosphate p53 level is very low, but after 300 m J/cm2 UVB irradiation, the phosphorylation p53 increased significantly; while in the join of C3 G, p53 phosphorylation level became down. 9. After exposure to UVB significantly increased the expression of pro-apoptotic protein Bax, but decreased the expression of anti-apoptosis protein Bcl-2, and also increased the cleaved-caspase-3; while in the join of C3 G, the effect can be reversed, so that down-regulated the Bax expression, and up-regulated Bcl-2 expression, also reduced the cleavage of caspase-3.Conclusion: 1. UVB radiation reduced the survival rate of Ha Ca T cells, and increased the apoptosis of Ha Ca T cells.2. Apoptosis induced by UVB through over produced ROS, induced DNA damage, activation p53 expression, p53 effect on the mitochondrial membrane of Bcl-2 family proteins, the mitochondrial membrane potential changes, the release of a series of cytokines, eventually leading to the activated of caspases and start the apoptosis cascade reaction. 3. The antioxidant activity of C3 G is efficient, can raise the survival rate of Ha Ca T cells after UVB irradiation, and reduced apoptosis. 4. C3 G inhibited apoptosis by UVB induced, is through the removal of excess ROS, reduce DNA damage, reduce the p53 activation, regulation of Bcl-2 protein family expression at mitochondrial membrane, recovery of mitochondrial membrane potential, reduced the mitochondrial membrane permeability, then the apoptosis related cytokine release is reduced, thereby reducing the cleaved caspases activation, and inhibited apoptosis reaction.