| Objective To investigate the effect and apoptotic mechanism of C17.2induced byTgCtwh3，a representative Toxoplasma strain of ChineseⅠthat prevalent in China.Methods (1) The cultivation and identification of C17.2cells, and the co-culture withGFP-RH tachyzoites. Thawing C17.2cells, routine culture, and indentify the cells withneural stem cells’ specific protein Nestin.Using a transwell system, conuted C17.2neural stem cells were added to the lower chamber, while GFP-RH tachyzoites wereadded to the upper chamber. Observe whether there were GFP-RH tachyzoites in thelower chamber to detect the filtration efficiency of the co-culture system afterco-cultured for several hours.(2) The detection of neural stem cells apoptosis inducedby Toxoplasma. According to the experimental plan,2×104、1×105、5×105TgCtwh3tachyzoites were added to the upper chamber, meanwhile the control group, theheat-inactivation group and the positive control group were set up. C17.2cells in thelower chamber were harvested after co-culture for6h,12h and24h, the Annexin V-FITC/PI staining and western blot were used to detect the apoptosis level and theexpression of caspase–3, respectively.(3) To detect the apoptosis level and the expression of related proteins involved in the endoplasmic reticulum stress pathwayafter inhibitors pretreatment. C17.2cells were pretreated by1mmol/L TUDCA for6h,then5×105TgCtwh3tachyzoites were added to the upper transwell chamber.After24hcultivation, cells in lower chamber were harvested to detect apoptosis level by flowcytometry and related protein expression involved in ERS by Western blot.Results (1) The expression of NSCs specific protein marker nestin was positive inC17.2cells.In the co-culture system,GFP-RH tachyzoites didn’t penetrate into the lowerchamber.(2) After co-culture with TgCtwh3tachyzoites for6h,12h and24h, theapoptosis rate of C17.2cells were9.62±1.02%,12.73±0.99%and15.6±1.35%,respectively, while the apoptosis level in negative control were4.54±1.72%,5.25±1.27%and6.98±1.11%, respectively. The apoptosis rates of heat-inactivationgroup were4.99±0.54%,6.29±2.08%and7.58±1.16%, respectively. Significantdifferences were found between tachyzoites co-clture group and control group or heat-inactivation group (P <0.05). No significant difference was found between heat-inactivation group and control group.(3) After co-culture with2×104、1×105、5×105TgCtwh3tachyzoites for24h, the apoptosis rates of C17.2were7.87±2.01%,11.05±0.77%and14.87±0.96%, respectively, and statistical differences were foundwhen compared with the control group of4.8±1.32%(P <0.05). Western Blot revealedthat caspase-3of the Toxoplasma infection group was activated.(4)After co-culturewith5×105TgCtwh3and RH tachyzoites for24h, the apoptosis rates of C17.2were12.52±0.11%,17.47±0.46%, respectively, significant difference was found between thetwo groups (P <0.05).(5) After the pretreatment of TUDCA, a inhibitor of endoplasmicreticulum stress signaling pathway, the apoptosis rates of C17.2co-cultured withTgCtwh3tachyzoites or RH tachyzoites were7.23±0.45%,9.93±0.16%, respectively,significantly lower that in the TUDCA untreated TgCtwh3(12.52±0.11%) group and theTUDCA untreated RH (17.47±0.46%) accordingly (P <0.05).(6) The Western Blotresults data displayed that, after co-cultured with TgCtwh3and RH tachyzoites, the expression level of caspase-12, CHOP and p-JNK in C17.2were increased significantlywhen compared with control group.When pretreated with TUDCA inhibitors, theexpression levels of those proteins were significantly decreased (P <0.05).Conclusion(1)TgCtwh3, a representative Toxoplasma strain of ChineseⅠthat prevalent in China,can induce the apoptosis of neural stem cells C17.2through its excretory-secretoryantigen in a time dependent and dose dependent manner. Significant differences ofC17.2apoptosis level were found between TgCtwh3co-culture and RH co-culturegroup.(2)TgCtwh3can induce the apoptosis of C17.2by the up-regulation of caspase-12,CHOP and p-JNK which are associated with endoplasmic reticulum stress signalingpathways. |
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