The Mechanisms Of Purple Sweet Potato Pigments Protect Murine Thymocytes From ⁶⁰co γ Ray Induced Damage

Objective: Establish the ⁶⁰Coγray-induced irradiation damage model in murine thymocytes. Investigate the protective effects of purple sweet potato(PSP)pigments on ⁶⁰Co-induced damage.Methods: After ⁶⁰Coγray-single dose-irradiation with various doses of 0, 2, 4, 6Gy, the murine thymocytes viabilities are detected by MTT at the post-irradiated time point ranged from 0 to 12h. Treated the thymocytes with indicated dose of irradiation in the present of different concentrations of PSP pigments, the cell viabilities are detected by MTT to investigate the effective and safe concentrations of PSP pigments against irradiation. After the model of PSP pigments against irradiaion is established, DNA ladder is preformed to test whether PSP pigments have the protective effect on late apoptosis. The early apoptosis and cell cycle of thymocytes are analyzed by flow cytometry with annexin V-FITC/PI staining and PI staining.The mechanisms of the radioprotective effects of PSP pigments are explored. The antioxidative effects of PSP pigments are detected. DCFH-DA fluorescent probe is applied to investigate the intra-cellular ROS. The activities of antioxidative enzymes SOD and GSH-px are detected by biochemistry methods. The effects of PSP pigments on mitochondrion-mediated apoptosis are analyzed. Mitochondrial membrane potential is detected by JC-1 fluorescent probe. The released cytochrome C, caspase 3, PARP, Bcl-2 and Bax proteins are investigated by western-blot. The enzyme activities of caspase 3 and caspase 9 are also detected. The effects of PSP pigments on death receptor-mediated apoptosis are determined by Fas mRNA RT-PCR analysis, FADD protein western-blot analysis and caspase 8 enzyme activity detection. The MAPK pathways including ERK, JNK and p38MAPK are analyzed by western-blot. The effects of PSP pigments on p53 are determined by p53 mRNA RT-PCR detection and p53, p21Cip1 and p27Kip1 protein western-blot analysis.Results: MTT showes that ⁶⁰Coγray decreases the cell viabilities and a sharp decline of cell viability starts from 4h post-irradiation. 4Gy irradiation causes approximate 50% cell viability loss at 4h after ⁶⁰Coγray irradiation. So the ⁶⁰Co-irradiating model should be established at 4h post-4Gy-irradiation. With 4Gy irradiation, the cell viablities are increased in the present of 0.625g/L～2.500g/L PSP pigments in a concentration-dependent manner and no cytotoxcity is observed in these PSP pigments concentrations. DNA ladders are smeared and weak in PSP pigments pretreated groups compared to that of the irradiated group. Flow cytometry results show that PSP pigments can decrease the early apoptosis and attenuate the cell cycle arrest induced by irradiation.The mechanisms of radioprotective effects of PSP pigments are explored and the results demonstrate that PSP pigments can decrease the intra-cellular ROS and increase the activities of SOD and GSH-px as well. PSP pigments stabilize the mitochondrial membrane potential, decrease the released cytochrome C, caspase 3 and PARP proteins. The activities of caspase 3 and caspase 9 are inhibited by PSP pigments. Bcl-2 protein is increased by PSP pigments while Bax is decreased to draw the cells towards antiapoptosis. PSP pigments show no significant effect on Fas mRNA and FADD protein but the activity of caspase 8 is increased in the present of medium and large dose of PSP pigments. MAPK pathway including JNK and p38MAPK is inhibited by PSP pigments but no changes happen to ERK. P53 mRNA and p53 protein are both depressed by PSP pigments and p27Kip1 is decreased consequently to attenuate the cell cycle arrest. PSP pigments have no obvious effects on p21Cip1 protein.Conclusion: PSP pigments can inhibit the ⁶⁰Coγray-induced damage in murine thymocytes. The cell viability is increased, apoptosis is decreased and cell cycle arrest induced by irradiation is attenuated in the present of PSP pigments. The radioprotective effects are related to the anti-oxidative effects of PSP pigments, which scavenging the intra-cellular ROS and enhancing the activities of anti-oxidative enymes to rebanlance the redox. The mitochondrial-mediated apoptosis pathway but not the death receptor-mediated apoptosis pathway is inhibited by PSP pigments. Bcl-2 and Bax are involved in the anti-apoptosis effects of PSP pigments. PSP pigments depress JNK and p38MAPK pathways but keep no influence on ERK. P53 mRNA is inactivated and p53 protein is decreased by PSP pigments pretreatment. Cell cycle arrest related p53 flare-up is cooling down by PSP pigments and p27Kip1 but not p21Cip1 is consequently decreased. Thus, the radio-protective effects of PSP pigments are via modulation of multiple genes and proteins to yield the anti-oxidation, anti-apoptosis and anti-cell cycle arrest. The PSP pigments might become a promising candidate for radio-protector.