Cytosolic Protection Against Ultraviolet Induced DNA Damage By Blueberry Anthocyanins In Hepatocarcinoma HepG2 Cells

DNA is to store and transmit genetic information from generation to generation, whose integrity is crucial for life activities. The excess of the ultraviolet (UV) radiation may cause cell injury because of DNA base mutation, DNA breakage, chromosome aberration, etc., if DNA damage cannot be repaired in time. With the increasingly serious environmental pollution, the ozone layer has been thinner, and UV reaching the earth’s surface increases. UV radiation causes the occurrence of photoaging of human skin, cancer, cataracts, immune suppression, and other diseases. To find efficient antioxidants for medication is important for the prevention and treatment of UV caused damage. This study is to find the cytosolic protection mechanism against UV induced DNA damage by blueberry anthocyanins in hepatocarcinoma HepG2 cells.In this study, the separation and purification of blueberry anthocyanins component were carried out. The result of the structural analysis shows that cyanidin and malvidin, cyaniding, cyaniding and delphinidin were obtained after the extraction of the blueberry by three solvents:methanol, ethanol or water, respectively.Initially, MTT cell proliferation assay were applied for the human hepatocarcinoma HepG2 cells with control group, UV irradiation group and blueberry anthocyanins protection group. For the UV irradiation with 20 s UV exposure on human hepatocarcinoma HepG2 cells was applied through the experiments. Following MTT assay cell morphology was observed by scanning electron microscopy (SEM); nuclear morphology by Hoechst 33258 staining was observed with ultraviolet microscopy; single cell gel electrophoresis (comet assay) was applied to detect DNA damage; FACS was used for scavenging effects of blueberry on reactive oxygen species (ROS), or free radicals and cell cycle analysis of DNA content; The laser scanning confocal fluorescence microscopy (LSCM) for the observation of change of mitochondrial transmembrane potential; For molecular mechanism of DNA damage by UV and repairing by blueberry extracts yH2AX with immunofluorescence staining was detected by LSCM; gene expression of p53, p21, by RT-PCR and protein expression of p53, p-p53, and p21 by western blotting were also performed.The study demonstrated that the anthocyanins protection group can decrease the mortality rate caused by UV, decrease the number of membrane vesicles; and cells tend to become round, reduce nuclei chromatin condensation, shorten the comet migration distances (tail moment) and alleviate the extent of the damage DNA. In addition, protection group also lowered the intracellular ROS levels, reduced the number of cells blocked in Gl phase, elevated the cell mitochondrial transmembrane potential. Particularly these changes were due to the triggering of cytosolic targets because anthocyanins significantly decreased the number of yH2AX foci and fluorescence intensity and the mRNA expression of p53, p21, Gadd45, MDM2, significantly lower the protein expression levels p53, p-p53, p21 Gadd45, MDM2. Therefore, blueberry anthocyanins have the function of protection and prevention against UV induced DNA damage in hepatocarcinoma HepG2 cells.