| OBJECTIVE To establish UVA-induced apoptotic model of HaCaT cells and investigate themechanism of Polypeptide from Chlamys farreri (PCF) protecting HaCaT cells from UVA-inducedapoptosis through ROS, p38 mitogen activated protein kinase (p38MAPK) pathway and caspase-3.METHODS UVA-induced apoptotic model of HaCaT cells was established by orthogonal design andapoptotic rate was determined by flow cytometry PI staining. Cells were divided into six groups:control group, UVA model group, UVA+5.68 mmol·L-1 vitamine C positive control group, UVA+5.69mmol·L-1 PCF group, UVA+2.84 mmol·L-1 PCF group, UVA+1.42 mmol·L-1 PCF group. Effect ofPCF on UVA-induced production of ROS was detected by 2', 7'-Dichlorofluorescin diacetate(DCFH-DA). Using agarose gel electrophoresis, the effects of PCE p38MAPK inhibitor SB203580and caspase-3 inhibitor Ac-DEVD-CHO on UVA-induced apoptosis were investigated. Morphologyalteration caused by H2O2 was observed by Hoechst 33258 fluorescent staing. Protein expressionlevels of p38MAPK, phosphorylated p38MAPK and c-fos were determined by Western blot analysts.Expression of c-fos mRNA was examined by Real-Time PCR. Caspase-3 activity was assayed byflow cytometry. RESULTS Results of orthogonal experiment suggest that 8J·cm-2 UVA and 18 hoursincubation after radiation make up the best apoptotic model. 1.42～5.69 mmol.L-1 PCF significantlyinhibited UVA-induced apoptosis and ROS accumulation (P＜0.05). PCF attenuated apoptoticmorphology alteration caused by H2O2. SB203580 and Ac-DEVD-CHO had inhibitory effects onUVA-induced apoptosis of HaCaT cells. 1.42～5.69 mmol·L-1 PCF dose-dependently attenuatedUVA-induced p38MAPK activation, caspase-3 activation and expression of c-fos (P＜0.05, P＜0.01).CONCLUSION PCF could protect HaCaT cells from UVA-induced apoptosis. Its inhibitory effect onapoptosis may attributes to inhibition of production of ROS, activation of p38MAPK pathway andcaspase-3. |
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