Involvement Of Endoplasmic Reticulum Calcium In Paraquat-induced Apoptosis Of Human Type ? Alveolar Epithelial Like Cells A549

Objective:To investigate the role and mechanism of endoplasmic reticulum?ER?calcium(Ca²⁺)signal in Paraquat-induced apoptosis of human type II alveolar epithelial like cells A549 by establishing a paraquat?PQ?poisoning model.Methods:Based on the establishment of PQ induced A549 cell apoptosis model,the following six experimental groups was established:control group,the cells were added to the same amount of PBS;Heparin group,the cells were preconditioned with Heparin?200?g/mL?for 2h;Thapsigargin group,the cells were preconditioned with Thapsigargin?4umol/L?for 2h;PQ treatment group,the cells were treated with PQ?200?M?;Heparin+PQ group,the cells were preconditioned with Heparin?200?g/mL?for 2h and then treated with PQ?200?M?;Thapsigargin+PQ group,the cells were preconditioned with Thapsigargin?4umol/L?for 2h and then treated with PQ?200?M?;After the treatment of 48h,the cell survival rate was assessed by MTT assay;The morphology of the nucleus were observed by Hoechst 33258 staining;The changes in the intracellular Ca²⁺concentration were detected by Fluo-3-AM fluorescence probe;The apoptosis rate was detected by Annexin V-FITC/PI double staining;The activities of Caspase-7 and-12 were assayed by spectrophotometric method;The expression of endoplasmic reticulum stress protein,GRP78 and CHOP were detected by Western-Blot.Results:Compared with PQ group,in the Heparin+PQ group,Heparin pretreatment significantly reduced PQ induced cell cytotoxicity and cell nuclear morphologic changes,decreased intracellular Ca²⁺concentration and the rate of cell apoptosis,inhibited the activation level of Caspase-7 and Caspase-12 and the expression of GRP78 and CHOP;While the Thapsigargin+PQ group was compared with the PQ group,the Thapsigargin pretreatment increased the changes in all aspects.Compared with control group,both Heparin and Thapsigargin group showed there were no significant difference of detection indicators.Conclusion:This study used Ca²⁺-ATPase inhibitors,Thapsigargin and inositol1,4,5-triphosphate receptor inhibitors,heparin to prove that the Ca²⁺signal of endoplasmic reticulum was involved in PQ induced apoptosis in human lung type II alveolar epithelial like A549 cells.