The Inflammatory Effect Of Prdx6-iPLA2 In Microglias On Neuron After Oxygen-glucose Deprivation/Reoxygenation Injury

Background: Inflammation was one of the main factors that aggravating cerebral ischemic reperfusion damage.And microglias, as the resident macrophages in the brain, played a crucial role in the inflammatory response caused by ischemic stroke.Peroxiredoxin-6(Prdx6) belonging to the endogenous antioxidant enzyme, had been proved to be protective in cell and animal models. However, the iPLA2 activity of Prdx6 has not been studied deeply, especially its role in cerebral ischemic-reperfusion injury and the mechanism hasn’t been reported.Objective: To explore the inflammatory effect of Prdx6-iPLA2 in microglias on neuron induced by OGD/R injury and the related mechanism.Methods:(1)Building four lentivirus SiRNA of Prdx6, then transfect to purified microglias.The transfection efficiency was observed under inverted fluorescence microscope, and the interference efficiency was detected by real-time qPCR and Western Blot analysis.(2)Building one lentivirus Prdx6-iPLA2 Si RNA without iPLA2 activity by gene mutation, and transfect to microglias with selected Prdx6 SiRNA.Then establish microglia-neuron co-culture OGD/R model.(3)Petreat MJ33 with microglias, and establish cell co-culture OGD/R model.(4)Measure the neuron viability and damage in co-culture system by MTS and LDH assays.(5)Using Elisa assay to detect the content of inflammatory cytokines IL-1β,IL-17 and IL-23 in the medium.(6)Using Western Blot to measure the expression of TLR2/4, NF-κBp65, iNOS and COX-2 protein.Results:(1) After microglias were transfected with lentivirus Prdx6 SiRNA, Y2091 interference efficiency was the most significant.(2) The MTS and LDH results showed that interference Prdx6-iPLA2 and petreat with MJ33 to microglias could increase neuron viability and reduce neuron damage.(3) The Elisa result showed that interference Prdx6-iPLA2 and petreat with MJ33 to microglias reduced the release of inflammatory cytokines IL-1β,IL-17 and IL-23 in the medium.(4) Western Blot result showed that the inflammatory protein TLR2/4, NF-κ Bp65, i NOS and COX-2 expression all decreased after inhibiting i PLA2 activity of Prdx6 in microglias.Conclusion:(1) Prdx6-iPLA2 in microglias induced the inflammatory damage to neuron after OGD/R injury, and the mechanism was related to the activation of TLR2/4 signaling pathway and the downstream inflammatory meditors in microglias.(2) MJ33 had the similar anti-inflammation effect with Prdx6-iPLA2 interference.