| OBJECTIVE To repeat UVA-induced apoptotic model of HaCaT keratinocytes and research the function of Polypeptide from Chlamys farreri (PCF) stopping HaCaT cells from UVA-induced apoptosis by mitochondrial uncupling protein 2(UCP2) , reactive oxygen species(ROS) ,cytoC,Apaf-1and respiratory chain signaling pathway. METHODS The best apoptotic model of UVA-induced HaCaT cells was reproduced.Dviding the cells into four groups: control group, UVA group, PCF groups (5.69, 2.84, and 1.42 mmol·L-1PCF) and the NAC inhibitor groups (5mmol·L-1NAC). Using Hoechst 33258 fluorescent staining and agarose gel electrophoresis to investigate UVA influenced morphologic and biochemical alteration and the effects of PCF and ROS scavenger NAC on UVA-induced apoptosis. The effect of PCF on the production of ROS was inspected by 2', 7'-Dichlorofluorescin diacetate (DCFH-DA). Making using of western bloting inspected the expression of cytoplasmic superoxide dismutase (Cu,Zn-SOD),UCP2 and Apaf-1. The mRNA expression of glutathione peroxidase (GPx) and Catalase (CAT) were detected through inverted translation PCR. Using flow cytometry assayed the activity of caspase-3. RESULTS Successfully copyed the best apotosic model.UVA induced the emergence of the segment of DNA, 1.425.69 mmol·L-1 PCF significantly inhibited the emergence of the DNA ladder; Compared with the UVA model group ,the number of apoptosic cells in the PCF groups were reduced; ROS accumulation in PCF groups were evidently reduced(P<0.05); The expression of SOD,UCP2,cytoC and Apaf-1 in PCF groups were evidently mainfolded,contrarily there was a evidently reducing in the UVA model group;In the PCF groups the activity of caspase-3 could be reduced evidently. (P<0.05,P<0.01). CONCLUSION PCF can restrain the apoptosis of HaCaT cells through enhancing the expression of UCP2;Except that PCF also can protect HaCaT cells from UVA-induced apoptosis through reducing the production of ROS. |
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