The Study About Anti-HaCaT Apoptotic Agent Polypeptide From Chlamys Farreri (PCF) Modulate Apoptosis Related Gene

Object We established the irradiation damage models of HaCaT cells caused by 20mJ/cm2 UVB radiation in vitro to study the antiapoptotic mechanism of polypeptides from Chlamys farreri (PCF) on HaCaT cells damaged by UVB radiation. MATHODS Experiments were designed into 6groups xontrol, model,UVB+0.5%PCF,UVB-0.25%PCF,UVB+0.125%PCF, UVB+0.1 %Vc.HaCaT cells were incubated with PCF or VitC for 2h then irradiated with 20mJ/cm2. Except Caspase-3 the following assays were performed 18 h after irradiation: differenet dosis ( 5,10, 15, 20mJ/cm2) were used to determine the best radiation dose;HaCaT cells were also pretreated with or without ERK inhibitors ( PD98059) , JNK inhibitors (SP600125 ) , p38 inhibitors ( SB203580) and Caspase-3 inhibitors ( DEVD-FMK) prior to UVB irradiation and DNA fragmentation was assessed by agarose gel electrophoresis;flow cytometry was used in examining Caspase-3 production;The activation of ERK, JNK, p38 kinase were investigated by western-blot;Gene expression profile was identified by cDNA m icroarrays in HaCaT cells exposed to UVB(20 mJ/cm2) pretreated with PCF. RESULTS Agarose gel electrophoresis reveal exposure to 20mJ/cm2 UVB , HaCaT cells appear characteristic apoptic ladder;(0.125-0.5%)PCF could significantly inhibited the apoptosis of HaCaT cells induced by UVB. PCF attenuated UVB caused DNA fragmentation in HaCaT cells. While combined PD98059 yielded typical DNA fragmentation, co-treatment of HaCaT cells with PCF and SP600125 , SB203580 and DEVD-FMK blocked UVB-induced DNA fragmentation. PCF significantly abolished activation of JNKs and p38 kinase activation while enhanced ERKs activation. The result of flow cytometry reveal that (0.125-0.5%)PCF dose dependent depress HaCaT cells Caspase-3 contents (p<0.05,p<0.01) .The result of gene array indicate PCF inhibited p27 and pl5 gene that regulating cell cycle;enhanced NDRGl gene that regulating apoptosis;suppress apoptotic gene p8, STK3 and TP53INP1;potentize stree response gene GPX and p44, regulate genes about cell proliferation and energy metabolism. CONCLUSION With the parameter of UVB radiance of 20mJ/cm2, PCF had the antiapoptotic effects on damages of HaCaT cell caused by the irradiation of UVB. The antiapoptotic mechanisms of PCF might be related to inhibite the amount of Caspase-3 , increase p-ERK activity , inhibit p-JNK and p-p38 activity. Cell cycle controlling gene p27,p15, apoptosis related gene NDRG1,p8,STK3 and TP53INP1, stress response gene GPX,p44 , cell proliferation related gene and energy metabolism related gene all participated the antiapoptotic effect of PCF.