The Antioxidative And Antiapoptotic Effects Of Polypeptide From Chlamys Farreri On UVB-Damaged Keratinocytes

Objectives to establish animal damage model on Kunming hairless mice with 103 mJ/ cm2 UVB and cellular damage model on HaCaT cells with 30 mJ/cm2. Based on these models, the antioxidative and antiapoptotic effects of polypeptide from chlamys farreri (PCF) are investigated.Methods the research was divided into animal and cellular parts. Groups designed in animal experiments are control, model, UVB+5% PCF, UVB + 20% PCF, UVB+10% VC. The dorsal skin of hairless mice was irradiated with 103 mj/cm2 UVB 1 h after painted with drugs. Immunohistochemistry was performed to measure p53, EGFR, substance P in keratinocytes and dermal collagens. Cellular experiments were designed into 7 groups (control, model, UVB+0. 0625% PCF, UVB+0. 125% PCF, UVB+0. 25% PCF, UVB + 0. 5% PCF). HaCaT cells were incubated with PCF or VC for 1 h then irradiated with 30mJ/cm2. Except comet assay the following assays were performed 18 h after irradiation: MTT assay evaluated effect of PCF on cell viability; Technology in enzymatic chemistry was adopted to measure superoxide dismutase (SOD) , glutathione peroxidase (GSH-PX) , catalase (CAT), total antioxidative capacity (T-AOC) and malondindehyde (MDA) in cells; flow cytometry was used in examining apoptotic rate, mitochondrial membrane potential and intracellular free Ca2+ ; Cellular ultrastructure was observed through transmission electron microscope; In Situ hybridization was performed to measure p21 mRNA.Results immunohistochemistry revealed that p53 proteins are more expressed after the skin of the hairless mice is irradiated with UVB. p53 proteins in keratinocytes of UVB-irradiated murine skin that is treated with 5%, 20% PCF and 10% VC are fewer than that in model group. The inhibitory effect of 20% PCF on p53 proteins expression is statistically better than that of 5% PCF. UVB irradiation at 103 mj/cm2 induces much type I collagen which intricately distributes in dermis. Compared to model group, the dermal type I collagen expression decreases after the murine skin pre-painted with PCF and VC is exposed to UVB. PCF treatment makes configuration in UVB-irradiated dermis nearly normal. Single UVB irradiation, however, did not alter p53 protein, EGFR and type IV collagen in this study. In UVB-damaged HaCaT cells, PCF is able to elevate antioxidative en-zymes (SOD, GSH-PX and CAT) activities, enhance T-AOC and reduce lipid peroxidation product - MDA. Results from flow cytometry show that PCF ranged from 0. 0625% to 0.5% dose-dependently decreases UVB-induced apoptotic rate. Meanwhile PCF reduces intracellular free Ca2+ and blocks the loss of mitochondrial membrane potential in dose-dependent manner. Observed through transmission electron microscope, HaCaT cell cytoplasm that has numerous vesiculations is filled with many myeloid figures. Cytoplasmic or-ganells such as RER, Golgi complex, mitochondria and so on apparently decrease. In a-bout one third cells agglomerate chromatin localizes at the edge of nucleus, which shows apoptotic traits. The ultrastructral damage in PCF-treated HaCaT cells is reduced, for example, more normal organelle and fewer vesiculations in cytoplasm, apoptotic cells hardly seen, no apoptotic bodies. In comet assay the tail moments of UVB-irradiated groups are statistically bigger than that of model group. Tail moments of PCF-treated cells in which tail moments decrease in dose-dependent manner are smaller than that of model group, so is VC-treated group. Results from In Situ hybridization suggest that all UVB-irradiated groups express p21 mRNA. Data of both 0. 0625% and 0. 125% PCF groups do not differ from that of both control and model groups statistically. The p21 mRNA level of the rest PCF groups is lower than that of model group while close to that of control group. PCF dose-dependently inhibits UVB-induced transcription of p21 mRNA.Conclusions With the parameter of UVB radiance of 103 and 30 mj/cm2, we have succeeded in establishing animal and cellular damage models on hairless mice and HaCaT cells. PCF has antioxidative effects, which are embodied in e...