The Neuroprotective Effects Of PAQR3Overexpression Against Oxygen-glucose Deprivation Followed By Reperfusion And Its Underlying Mechanism

Chapter I Expression Pattern of PAQR3in Mouse N2a Cells upon OGD/R treatmentObjective:To elucidate alteration of cellular morphology, viability and apoptosis in mouse N2a cells upon oxygen-glucose deprivation followed by reperfusion (OGD/R), as well as expression pattern of PAQR3.Methods:We employed OGD/R model to mimic ischemic-like conditions in vitro. The morphologic change of N2a cells was determined by inverted optical microscope. Cellular viability and apoptosis were measured using the MTT assay and flow cytometry. PAQR3expression levels were determined by quantitative Real-Time PCR and Western blot.Results:1. Treatment with OGD/R markedly destroyed the cell morphology and reduced cellular viability in mouse N2a cells at12-hour and24-hour time points recovery from4-hour OGD (P<0.05), while increased apoptosis rate at4-hour,12-hour and24-hour time points recovery from4-hour OGD (P<0.05)2. The mRNA level of PAQR3was strongly downregulated in mouse N2a cells at0-hour,4-hour,12-hour and24-hour time points recovery from4-hour OGD (P<0.05).3. The protein level of PAQR3was strongly downregulated in mouse N2a cells at4-hour,12-hour and24-hour time points recovery from4-hour OGD (P<0.05)Conclusions:Cellular viability and apoptosis, as well as morphology of the cells damaged greatly in mouse N2a cells upon OGD/R treatment. Meanwhile, the expression pattern of PAQR3was downregulated upon OGD/R treatment.Chapter Ⅱ The construction of the plasmid of mouse PAQR3and its overexpression in N2a cellsObjective:To construct expression plasmid of PAQR3and discuss the feasibility of its expression in N2a cells.Methods:The gene sequences of mouse PAQR3was found from the genebank, artificially synthesized, and loaded in the expression vector CMV-MCS-EGFP-SV40-Neomycin using Restriction enzyme Xhol and BamH I, also PCR identificated after amplification. The correct plasmid was used to transfect N2a cells by using the transfection reagent Lipofectamine. The expression of the target gene was detected by Real-time PCR, and the expression of the target protein was detected by Western blot.Results:The eukaryotic plasmid of mouse PAQR3was successfully constructed and the N2a cells were successfully transfected by liposome transfecion reagent. We found that the transfection efficiency was the highest72hours after transfected. A high expression of the target gene was detected by Real-time PCR. Also a significant over expression was found by Western blot. Expression of PAQR3was confirmed by immunoblotting and its molecular weight was correct.Conclusion:We successfully constructed expression plasmid of PAQR3, and the eukaryotic plasmid was high expressed in N2a cells.Chapter Ⅲ The Neuroprotective Effects of PAQR3and its Underlying MechanismObjective:To elucidate the neuroprotective effects of PAQR3overexpression against oxygen-glucose deprivation followed by reperfusion (OGD/R) and its potential mechanism.Methods:Mouse N2a cells were transfected with pEGFP-PAQR3for72h, and then treated with0-hour,4-hour,12-hour and24-hour reperfusion following4-hour OGD. Cellular viability and apoptosis were measured using the MTT assay and flow cytometry. The morphologic change of N2a cells was determined by inverted optical microscope. The mRNA levels of ERK1, ERK2and AKT were determined by quantitative Real-time PCR after12-hour and24-hour reperfusion. The protein levels of ERK1/2, AKT and GSK3β and their phosphorylated proteins p-ERK1/2(Thr202/Tyr204), p-AKT(Ser473) and p-GSK3β(Ser9) were determined by Western blot after24-hour reperfusion.Results:1. Compared with the control, the cellular viability was significantly higher in N2a cells transfected with pEGFP-PAQR3at12-hour and 24-hour time points recovery from4-hour OGD (p<0.05). N2a cells transfected with pEGFP-PAQR3also displayed a significant decrease in the number of apoptotic cells upon OGD/R exposure (p<0.01). And also the morphology of N2a cells improved.2. The mRNA levels of ERK1, ERK2and AKT of N2a cells transfected with pEGFP-PAQR3exhibited no significant difference compared with the control (P>0.05)3. The normal N2a cells exhibited a marked increase in the protein expression of p-ERK1/2(Thr202/Tyr204) and p-AKT(Ser473), but an decrease expression of p-GSK3β(Ser9) compared with the non-OGD treated normal cells (p<0.05). The N2a cells transfected with pEGFP-PAQR3displayed a marked decrease in the protein expression of p-ERK1/2(Thr202/Tyr204) and an increase expression of p-AKT (Ser473) and p-GSK3β (Ser9)(p<0.05).Conclusions:1. PAQR3overexpression displayed neuroprotective effects against oxygen-glucose deprivation followed by reperfusion.2. Neuroprotective effects of PAQR3overexpression were probably mediated by inhibiting activation of the ERK signaling pathway.3. Neuroprotective effects of PAQR3overexpression were probably mediated through activating of the AKT/GSK3β signaling pathway.