Effect Of Folic Acid Inhibits Astrocyte Apoptosis By Alleviating Telomere Attrition In Vitro

Objective Folic acid(the form of folate,is used therapeutically),is an important coenzyme in the one carbon unit metabolism,which is necessary for deoxythymidine synthesis,purine synthesis and DNA methylation.Folic acid deficiency can lead to homocysteine(Hcy)accumulation and increases the oxidative stress level.Telomeres are DNA-protein complex structures covering the linear ends of chromosomes in eukaryotic cells.Telomeric DNA consists of repetitive sequences of "TTAGGG",the guanine-rich structure is susceptible to oxidation.Astrocytes are the most widely distributed cells in the brain,the apoptosis of astrocytes is associated with a variety of diseases in the nervous system.Therefore,the purpose of this study was to investigate the effect of folic acid on the level of astrocyte apoptosis in vitro,and to explore whether folic acid can regulate the telomere attrition and astrocytes apoptosis by affecting oxidative stress,so as to lay a foundation for understanding the mechanism of folic acid on astrocyte apoptosis.Methods Purification of the primary cerebral cortex astrocytes of SD rats in vitro,and then devided equally into four groups,the folic acid deficiency group(0 μmol/L),normal folic acid group(10 μmol/L),low-dose folic acid supplement(20 μmol/L)and high-dose folic acid supplement group(40 μmol/L)according to folic acid dose.After incubated in different folic acid concentration for 12 days,MTS method is used to measure cell proliferation activity.Flow cytometry and Hoechst 33342 staining was used to measure cell apoptosis rate,chemical immune luminescence test was used to detect intracellular folic acid content.Hcy was determined by enzyme linked immunosorbent assay.The content of reactive oxygen species(ROS)in cells was detected by flow cytometry.The incorporation rate of 8-oxoguanine(8-Oxo G)into telomeric DNA was determined by RT-q PCR.Telomere length was determined by RT-q PCR and Southern Blot.The telomere-dysfunction induced foci were determined by immuno-fluorescence fluorescent in situ hybridization.After extracted intracellular protein,the expression levels of ATM,p-ATM,Chk2,p-Chk2,p53 and p-p53 were determined by Western blot.Results We incubated the primary astrocytes with different concentrations of folic acid for 12 days in vitro.Compared to the folic acid normal group(10 μmol/L),intracellular folic acid concentration was lower in the folic acid deficiency group(0μmol/L),but there was no statistically significant difference(P>0.05),while intracellular folic acid concentration was higher in the high-dose of folic acid supplement group(40 μmol/L)than in the normal folic acid group(10 μmol/L),the difference was statistically significant(P<0.05).Compared with the folic acid normal group(10 μmol/L),folic acid deficiency had lower cell proliferation activity and higher cell apoptosis rate,the difference was statistically significant(P<0.05).In addition,folic acid deficiency had higher intracellular ROS level,higher Hcy level,higher level of 8-Oxo G incorporated into telomeric DNA,higher level of telomere-dysfunction induced foci,shorter telomere length and higher phosphorylation level of ATM-Chk2-p53 pathway,the differences were statistically significant(P<0.05).Within the folic acid dose range in this study,we found that folic acid could increase astrocytes proliferation activity in a dose-dependent manner(r=0.756,P=0.004).Flow cytometry data showed a dose-dependent reduction in the apoptotic rate(r =-0.799,P = 0.002)and Hoechst 33342 staining showed the similar trend(r =-0.928,P < 0.001).However,folic acid supplementation reduced the incorporation of8-Oxo G into telomeric DNA in a dose-dependent manner(r =-0.907,P < 0.001)and reduced TIF-positive cells of astrocytes in a dose-dependent manner(r =-0.950,P <0.001).Folic acid deficiency shortened the relative telomere length,while folic acid supplementation increased telomere length in dose-dependent manner(r = 0.885,P <0.001).Conclusion This study showed that folic acid could promote astrocyte proliferation,inhibit cell apoptosis,reduce ROS mediated oxidative stress,reduce telomeric DNA oxidative damage,inhibit telomere shortening and reduce telomeric DNA double-strand breakage.The possible mechanism is that folic acid can inhibit oxidative stress-induced telomere attrition,maintain telomeres stability,then inhibit astrocyte apoptosis in vitro.