Effect And Mechanism Of Prdx1 Knockdown On Apoptosis Of N2a Cells After OGD/R

ObjectiveCerebral ischemia-reperfusion injury(CIRI)is a serious complication in the treatment of ischemic stroke.Reducing neuronal apoptosis is the key to improve the life quality of patients.Peroxirodoxin1(Prdx1),as a member of the antioxidant enzyme family,plays an important role in the pathophysiological processes of many diseases,but its role in the neuronal apoptosis in CIRI is unclear.This study aimed to mechanistically investigate the effect of knocking down Prdx1 on the apoptosis of mouse brain neuroma N2 a cells after oxygen glucose deprivation/reoxygenation(OGD/R)injury.MethodsIn this study,RNA interference(RNAi)technology was used to downregulate Prdx1 in N2 a cells,and the OGD/R injury model was used to simulate CIRI.The study is divided into four parts.The first part is dedicated to the assessment of the efficiency of the si RNA transfection and Prdx1 knock down in N2 a cells.The experiment in this part was divided into the following three groups: normal control group,negative control group(si NC group),and interference group(si Prdx1 group).Prdx1 si RNA and NC si RNA were designed and synthesized.N2 a cells were transfected with either si RNA by using the ribofect CP transfer kit.The fluorescence of the transfected cells was observed 48 h after the transfection via fluorescence microscopy.The Prdx1 m RNA and protein levels in each group were measured using reverse transcription–quantitative polymerase chain reaction and western blot analyses,respectively.The second part explores the effect of Prdx1 knockdown on the post-OGD/R injury and the activity of the JNK/Caspase-3 pathway in N2 a cells.The experiment in this part was divided into the following four groups: normal control group,OGD/R group,si NC+OGD/R group,and si Prdx1+OGD/R group.Except the control group,the N2 a cells in each group were subjected to OGD for 8 h,followed by reoxygenation for 24 h.CCK-8 colorimetry,Lactate dehydrogenase(LDH)release rate,and TUNEL staining were used to evaluate cell viability,cytotoxicity,and apoptosis,respectively.Western blot analysis was used to measure the levels of Prdx1,JNK,and phosphorylated JNK(p-JNK)proteins and the cleaved amount of the apoptosis executive protein Caspase-3.The third part explores the relationship between N2 a cell apoptosis and the JNK/caspase-3 pathway upon OGD/R.The experiment in this part was divided into the following three groups: normal control group,OGD/R group,and OGD/R+JNK activation inhibitor(SP600125)group(OGD/R+SP600125 group).Apoptosis and levels of JNK,p-JNK,and Cleaved caspase-3 were evaluated as described above.The fourth part explores whether Prdx1 knockdown induces apoptosis in N2 a cells upon OGD/R through the JNK/Caspase-3 pathway.The experiment in this part was divided into the following five groups: normal control group,OGD/R group,si NC+OGD/R group,si Prdx1+OGD/R group,and si Prdx1+OGD/R+SP600125 group.Apoptosis and levels of JNK,p-JNK,and Cleaved caspase-3 were evaluated as described above.Results1.Under the fluorescence microscope,the transfected cells emitted bright red fluorescence,indicating that the N2 a cells were successfully transfected with the si RNA constructs.Prdx1 m RNA and protein levels were significantly lower in si Prdx1 group than in the normal group(P<0.001).2.OGD/R group had significantly decreased cellular activity(P<0.001),a significantly increased LDH release rate(P<0.001),significantly increased apoptosis(P<0.001),and significantly upregulated Prdx1,p-JNK,and Cleaved caspase-3(P<0.001)levels than the normal group.Compared with the levels in OGD/R group,si Prdx1+OGD/R group displayed reduced cell viability(P<0.001),increased LDH release rate(P<0.001),increased apoptosis(P<0.01),decreased level of Prdx1 protein,and increased levels of p-JNK and Cleaved caspase-3 protein(P<0.001),indicating that Prdx1 knockdown promoted N2 a cell apoptosis after OGD/R presumably through the activation of the JNK/Caspase-3 pathway.3.Compared with the level in OGD/R group,apoptosis was significantly decreased in OGD/R+SP600125 group(P<0.01),and the levels of p-JNK and Cleaved caspase-3 were significantly decreased(P<0.01),indicating that the JNK/Caspase-3 pathway is involved in mediating the apoptosis of N2 a cells upon OGD/ R.4.Compared with the level in si Prdx1+OGD/R group,apoptosis was significantly decreased in siprdx1+OGD/R+SP600125 group(P< 0.01),and the levels of p-JNK and Cleaved caspase-3 were significantly decreased(P<0.01),indicating that SP600125 inhibited the stimulatory effect of Prdx1 knockdown on the apoptosis of N2 a cells subjected to OGD/R and that the JNK/Caspase-3 pathway is involved in the apoptosis of N2 a cells after OGD/R.ConclusionsThe Prdx1 expression in N2 a cells is upregulated upon OGD/R.Prdx1 knockdown can aggravate the OGD/R-induced apoptosis of N2 a cells and further activate the JNK/Caspase-3pathway.The JNK activation inhibitor SP600125 partially reversed the stimulatory effect of Prdx1 knockdown on the apoptosis of N2 a cells subjected to OGD/R,Taken together,Prdx1 may reduce the apoptosis of N2 a cells subjected to OGD/R by inhibiting the activation of JNK/Caspase-3 pathway.