Scallop Polypeptide Mediates Anti-UVB-induced Apoptosis Of HaCaT Cells Through Endoplasmic Reticulum Stress

Aim:With the established UVB-induced apoptosis model in HaCaT cell,to explore the roles of reactive oxygen specie(ROS),endoplasmic reticulum(ER)stress and their interaction in UVB induced apoptosis.Furthermore,to investigate the modulation of ROS and ER stress as a potential mechanism of PCF.Methods:In the established UVB-induced apoptosis model in HaCaT cell,the microstructure alterations of ER following UVB radiation was assessed by transmission electron microscope;the time-course changes of ER stress factors GRP78,GRP94,PERK,ATF6,IRE-1,p-eIF2a,CHOP,Bcl-2 were investigated with RT-PCR and western blot;siRNA interference was used to determine the interactions among these factors;To explore PCF's mechanism,the effects of NAC,4-PBA and PCF(1.42,2.84,5.69mmol·L-1)pretreatment in UVB-exposed HaCaT cells were evaluated with MTT,Hoechst 33258 and western blot for cell viability,apoptosis and ER stress factors expression,respectively.Results:1.UVB radiation induced ER stress:20mJ.cm-2 UVB irradiation followed by 18h incubation induced cell apoptosis(apoptotic rate:39.67%).ER expansion with degranulation was observed with transmission electron microscope.Time-dependently increased expression of the ER stress factors GRP78 and GRP94 were detected with RT-PCR and western blot during 0-12 h post UVB irradiation.2.PERK pathway was stimulated in UVB induced HaCaT cell apoptosis:The expression of PERK and its downstream molecule eIF2a increased from 3h post UVB radiation,and peaked at 24h.3.UVB induced HaCaT cell apoptosis is medicated via CHOP:20mJ.cm-2 UVB induced CHOP's expression at both mRNA and protein level.siRNA interference of CHOP resulted in increased expression of Bcl-2 and decreased expression of Bax,both at protein level(P<0.01).4.NAC and 4-PBA pretreatment increased cell viability as shown in MTT assay;decreased the expression of GRP78/CHOP/Bax protein while increased the expression of Bcl-2(P<0.05).5.PCF pretreatment dose-dependently improved cell survival rate over UVB model group(61.23±7.42;82.79±3.21;94.31 ±2.16 for 1.42,2.84,5.69 mmol·L-1 PCF,respectively).Apoptotic rate was also significantly decreased in PCF pretreated groups comparing to UVB model group(P<0.01)as shown by Hoechst 33258 staining.The protein expression of GRP78,CHOP/GADD153,p-PERK and p-eIF2a was found significantly lower relative to UVB model group(P<0.01).Conclusions:UVB radiation increased expression of GRP78/PERK/eIF2a/CHOP and thus stimulated ER stress in HaCaT cells;ROS is located upstream in UVB induced ER stress;PCF's anti-apoptotic effect in UVB-irradiated HaCaT cells involved its modulation of ERS signaling pathway.