| Objective：To observe the protective effect of chlorogenic acid（CGA）to the hydrogenperoxide-induced oxidative stress in human retinal pigment epithelium cells（ARPE-19）and the changes of apoptosis-related molecules expression,explore thepossible molecular mechanism involved in this protection.Then we may provide atheoretical basis for the use of chlorogenic acid in treatment of ocular diseasesassociated with oxidative stress, such as age-related macular degeneration (ARMD).Methods：1. Reference to foreign literature, the ARPE-19cells were treated by500uMH2O2for24h to establish the model of oxidative stress．2. Cytotoxicity of chlorogenic acid to ARPE-19cells were measured by WST-1assay after ARPE-19cells were treated with CGA for24h.3. There were three groups:normal control group, H2O2injured group,chlorogenic acid protected group;the viability of ARPE-19cells was measured byWST-1assay.4. Apoptosis rates in each experimental group were detected by Flow cytometry.5. Bcl-2and Caspase-3gene expression were detected by RT-PCR in eachexperimental group.6. Bcl-2and Caspase-3protein expression were detected by Western blot in eachexperimental group.Results：1. ARPE-19cells were reated with chlorogenic acid (CGA) at12.5~200uM for24h.CGA did not significantly affect the cell viability of ARPE-19cells,the dataindicated that CGA(0~200uM) showed no apparent toxicity to ARPE-19cells.2. The relative cell viability in H2O2injured group was lower than that of controlgroup (P <0.05), while the relative cell viability in CGA protection group(50uM,100uM,200uM) was higher than that of H2O2injured group (P <0.05).The results showed that H2O2could induce oxidative stress in ARPE-19cells,and CGA protectedH2O2-induced oxidative stress in ARPE-19cells.3. Apoptosis rate（%）of experimental groups cells by flow cytometry：normalcontrol group：8.31±2.87,H2O2injured group:65.43±8.82,chlorogenic acid group：29.43±6.80.The results showed that apoptosis rate of H2O2injury group wassignificantly higher than that ofnormal control group(P <0.05),cell apoptosis of chlorogenic acid protectedgroup was significantly lower than H2O2injury group(P <0.05).The results indicatedthat H2O2can induce apoptosis in ARPE-19cells， and chlorogenic acid has asignificant function on anti-apoptosis．4． The result of RT-PCR showed the levels of Bcl-2mRNA expressed ofchlorogenic acid protection group were significantly higher than that of H2O2injuredgroup(P <0.05),the expression of Caspase-3mRNA was significantly lower than thatof H2O2injured group (P <0.05)．The results showed that when chlorogenic acidprotected ARPE-19cells,the expression of Bcl-2gene was increased,the expressionof Caspase-3gene was reduced.5. Westem blot showed that the expression of Bcl-2protein levels of chlorogenicacid protection group were significantly higher than that of H2O2injured group(P<0.05),Caspase-3protein expression was significantly lower than that of H2O2injuredgroup(P <0.05)．The resuIts indicated that when chlorogenic acid protected ARPE-19cells,the expression of Bcl-2protein was increased,the expression of Caspase-3protein was reduced.Conclusions:1. chlorogenic acid has a protection on ARPE-19cells against oxidative stresswhile showed no apparent toxicity to ARPE-19cells.2. chlorogenic acid has a protective effect against H2O2-induced ARPE-19cellapoptosis and its mechanism may be related to enhanced Bcl-2gene and proteinexpression and inhibition of Caspase-3gene and protein. |
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