Toxoplasma Gondii Excreted-secreted Antigens Induced Apoptosis Of Neural Stem Cells Via Endoplasmic Reticulum Stress Pathaway

Objective To explore the role of Toxoplasma gondii ESA on the apoptosis level ofNSCs in vitro and its underlying mechanisms.Methods (1)Isolation、culture and identification of NSCs.The6-8weeks old femaleICR mice were mated with male mice at a ratio of2:1.On GD13to GD15, embryoswere collected by caesarean section and the tissue of forebrain were isolated andmechanically disrupted into single cells,and re-seeded for following experiments.Nestinantibody was used to identify the NSCs by immunofluorescence staining.(2)Differentially expressed genes of NSCs of infected group were screened by MouseGene Expression profiling.The transwell co-culture system of T.gondii and NSCs wasbuilt,1×10~6RH tachyzoites were added into the upper chamber,NSCs were seeded intothe under chamber,after48h,the NSCs of control group and infected group werecollected and total RNA was extracted.The samples were analyzed by Mouse GeneExpression profiling,and the differentially expressed genes were identified.(3)Detectedthe apoptosis level of NSCs. The transwell co-culture system of T.gondii and NSCs was built, NSCs were treated with various quantity of tachyzoites (2×10~5、1×10~6and5×10~6)for12h、24h or48h, cells were collected, then detected by FCM and DNA Ladder.(4)The apoptosis level of NSCs and the expression of related molecules after ERSinhibitors pretreatment.NSCs were pretreated by inhibitors (0.5mmol/L TUDCA and4μmol/L Z-ATAD-FMK)for6h, the co-culture system was established,after48h,theNSCs were collected, the apoptosis level was detected by FCM and the expression levelof CHOP、caspase12、JNK、p-JNK were detected by Western blot.Results (1) Mouse embryonic NSCs were successfully set up,and subcultured invitro.The expression of nestin of NSCs was positive.（2）The results of Mouse GeneExpression profiling showed that there were973up-regulated genes and871down-regulated genes(≥2Fold change）compared with the control group. Go analysisdisplayed that the biological process of cell apoptosis was significantly affected,and15genes involved in this process (≥2Fold change).(3)When co-cultureed with1×10~6RHtachyzoites for12h,24h and48h,the apoptosis level of NSCs were26.21±0.78%,37.01±1.23%and45.70±0.56%,respectively,whereas in control group,theapoptosis rates were21.12±1.34%,25.13±2.07%and25.31±1.03%,Respectively.Significant differences were found between the infected group and control group(P＜0.01） at24h and48h.The apoptosis level of NSCs treated with differentquantity(2×10~5、1×10~6and5×10~6)tachyzoites for48h were36.72±4.52%,45.73±2.42%and56.94±1.35%,respectively,significantly higher than that in the controlgroup(27.52±1.80%).(4)The apoptosis level of NSCs pretreatmented with TUDCA andZ-ATAD-FMK were28.41±0.60%and27.63±0.82%respectively,significantly lowerthan that in infection group（P<0.01）.(5)The results of Western blot showed that theexpression of CHOP、caspase12and p-JNK were up-regulated. TUDCA pretreatmentcould inhibit the expression of these genes.Z-ATAD-FMK pretreatment could inhibit the expression of caspase12. However, the expression of JNK in infected group or inhibitorgroup appeared stable compare with control group.Conclusion (1)T.gondii ESA significantly elevated the apoptosis level in NSCs with adose-and time-dependent manner.(2)T.gondii ESA induce apoptosis of NSCs thoughERS signal pathway.