The Role Of PEBP1 In Cerebral Ischemia-reperfusion Injury In Rats And Its Underly Mechanism

Part Ⅰ: Time course analysis of the protein levels of PEBP1 andphosphorylated PEBP1(p-PEBP1)after I/R.Objective: To investigate changes of PEBP1 and p-PEBP1 after different time points in the penumbra and cultured neurons after I/R.Methods: In vivo experiment,36 SD rats(41 SD rats were used,36 SD rats were survived after surgery)were randomly assigned to 6 groups of 6 SD rats each: a sham group and 5 experimental groups arranged by reperfusion time: 2 h,6 h,12 h,24 h and 48 h.The brain penumbra of 6 SD rats in each group was achieved at the indicated time point and used for western blot analysis and double immunofluorescence analysis.In vitro,experiment was performed to evaluate the effect of OGD/R treatment on the level of p-PEBP1 and PEBP1 in cultured neurons.Results: To detect the expression and phosphorylation of PEBP1 after MCAO/R,through western blot analysis of protein samples from penumbra tissue,we first tested the protein level of PEBP1 and p-PEBP1(ser153)in the penumbra tissue.The results demonstrated that,compared with the sham group,both the protein level of PEBP1 and p-PEBP1(ser153)in the penumbra tissue reached the highest point at 6h(p<0.01),and then rebounded gradually.Consistent with the in vivo data,western blot assay showed that the protein levels of PEBP1 and p-PEBP1(ser153)in cultured neurons were significantly increased by OGD/R at 2-12 h and peaked at 6h(p<0.01).Double immunofluorescence with the neuronal marker protein Neu N further verified the MCAO/R-induced increase in the protein level of PEBP1 in neurons.Immunofluorescence staining of PEBP1 on cultured neurons treated with OGD/R stimulus in vitro also showed an OGD/R-induced increase in PEBP1 when compared with control group.Conclusion: 1.The expression of PEBP1 and p-PEBP1 after the MCAO/R surgeryin SD Rats’ brain tissuereached the peak at 6h,and then rebounded gradually.2.The expression of PEBP1 and p-PEBP1 after the OGD/R in cultured neurons has the similar trends.Part Ⅱ: Effects of PEBP1 on cerebral I/R injuryObjective: Through site-directed mutagenesis and small RNA interference,to detect the effects of PEBP1 on cerebral I/R injury.Methods: In vivo,135 SD rats(146 rats were used,135 rats were survived)were randomly assigned to 9 groups(n=15 per group): sham group,MCAO/R group,MCAO/R+Vec group,MCAO/R+WT plasmid group,MCAO/R+mt group,MCAO/R+Ctr Si group,MCAO/R+Si RNA group,MCAO/R+Veh group and MCAO/R+rh PEBP1 group.The transfection of plasmids and Si RNAs was given at 48 h before surgery and the injection of rh PEBP1 was given immediately following reperfusion intracerebroventricularly.In vitro,cultured neurons were randomly assigned to 7 groups: Ctr group,OGD/R group,OGD/R+Vec group,OGD/R+WT plasmid group,OGD/R+mt group,OGD/R+Ctr Si group and OGD/R+Si RNA group.Through Hoechst 33258 staining and the level of LDH testing the level of cultured neurons apoptosis.Test cell viability by Sulforhodamine B(SRB)assay.Results: 1.The results demonstrated that both wild type GFP-PEBP1 and the mutant protein were strongly expressed in transfected penumbra tissue.In addition,compared with the sham group,a significant increase in the levels of PEBP1 and p-PEBP1(ser153)was observed in MCAO/R group,which was attenuated by PEBP1 knockdown and exacerbated by exogenous rh PEBP1 treatment(P<0.01).2.TTC staining displayed the benefit of PEBP1(S153A)overexpression and PEBP1 knockdown on the infarct area,while wild type GFP-PEBP1 overexpression and exogenous rh PEBP1 treatment exerted opposite effects(P<0.01).3.Clinical behavior scores assay showed the rescue effects of PEBP1(S153A)overexpression and PEBP1 knockdown on the neurobehavioral function of rats subjected to MCAO/R,while wild type GFP-PEBP1 overexpression and exogenous rh PEBP1 treatment exerted opposite effects.4.As compared with the OGD/R group,the apoptotic indexes,such as the number of Hoechst-positive apoptotic cells and the level of LDH,were significant exacerbated by wild type GFP-PEBP1 overexpression and attenuated by GFP-PEBP1(S153A)overexpression,suggesting phosphorylation at Ser153 may be a functional switch of PEBP1 in OGD/R-induced neuronal injury.Cell viability assay also identified the rescue effects of the Ser153 Ala mutation in PEBP1 in neurons under OGD/R stimulus.Consistently,the necrosis index showed the same trend with that in the apoptotic index.Conclusion: 1.PEBP1 has the rescue effects on brain tissue and cultured neurons after cerebral I/R injury.2.The phosphorylation of PEBP1 enhances the cerebral injury during I/R.Part Ⅲ : Mechanisms underlying PEBP1 actions during I/R.Objective: Through site-directed mutagenesis and small RNA interference,to detect the Mechanisms underlying PEBP1 actions during I/R.Methods: In vivo,135 SD rats(148 rats were used,135 rats were survived)were randomly assigned to 9 groups(n=15 per group): sham group,MCAO/R group,MCAO/R+Vec group,MCAO/R+WT plasmid group,MCAO/R+mt group,MCAO/R+Ctr Si group,MCAO/R+Si RNA group,MCAO/R+Veh group and MCAO/R+rh PEBP1 group.In vitro,cultured neurons were randomly assigned to 7 groups: Ctr group,OGD/R group,OGD/R+Vec group,OGD/R+WT plasmid group,OGD/R+mt group,OGD/R+Ctr Si group and OGD/R+Si RNA group.Test the level of inflammatory cytokines by ELISA,such as IL-1β,IL-6 and TNF-α.The effects of PEBP1 on apoptosis were examed by Western blot and FJB staining.Detect the expression level of Raf-1/MEK/ERK/NF-k B signaling pathway by Western blot.Exam the PC-PLC activity and test the level of LC3 II/LC3 I.Results: 1.The inflammatory cytokines,including IL-1β,IL-6 and TNF-α,were found to be significantly higher in the culture supernatant of the OGD/R group than that in the control group.Compared with OGD/R group,the mean inflammatory cytokine contents were higher in wild type GFP-PEBP1 overexpression group and lower in GFP-PEBP1(S153A)overexpression group(p<0.01).2.Compared to the sham group,FJB-positive cells were increased in the penumbra tissue in the MCAO/R group,which was significantly attenuated by PEBP1(S153A)overexpression and PEBP1 knockdown and enhanced by wild type GFP-PEBP1 overexpression and exogenous rh PEBP1 treatment(p<0.01).Consistently,western blot assay of caspase 3 activation showed the same trend.3.The results showed the phosphorylation level of ERK1/2 in neurons subjected to OGD/R to be significantly higher than that in the control group.Wild type GFP-PEBP1 overexpression and exogenous rh PEBP1 treatment significantly increased the level of ERK1/2 phosphorylation,while PEBP1(S153A)overexpression exerted opposite effects(p<0.01).Consistently,the nuclear content of NF-κB in MCAO/R group was significantly higher than that in the sham group,which was significantly attenuated by PEBP1(S153A)overexpression and enhanced by wild type GFP-PEBP1 overexpression and exogenous rh PEBP1 treatment(p<0.01).Notably,compared with OGD/R+Ctr Si group,PEBP1 knockdown did not induce significant changes in the phosphorylation of ERK1/2 and the nuclear transfer of NF-κB.4.PC-PLC activity was significantly higher in MCAO/R group than that in the sham group.Compared with MCAO/Rgroup,the PC-PLC activity was significantly enhanced in MCAO/R+WT group and MCAO/R+rh PEBP1 group,and weakened significantly in MCAO/R+mt group and MCAO/R+Si RNA group(p<0.01).4.The results also revealed increases in the LC3-II/LC3-I conversion in OGD/R group,which was inhibited by wild type GFP-PEBP1 overexpression and exogenous rh PEBP1 treatment,while PEBP1(S153A)overexpression and PEBP1 knockdown exerted opposite effects(p<0.01).Conclusion: Under normal condition,PEBP1 binds to Raf-1 and inhibits the Raf-1/MEK/ERK/NF-κB signaling pathway indirectly.Following MCAO/R,I/R injury stimulus induces an increase in the protein of PEBP1 and activates PKC,which in turn promotes the phosphorylation of PEBP1.The phosphorylation of PEBP1 inhibits the interaction between PEBP1 and Raf-1,and relieves the Raf-1/MEK/ERK/NF-κB signaling pathway.Subsequently,the released p-PEBP1 from Raf-1 binds to another binding partner PC-PLC.The phosphorylated PEBP1 promotes PC-PLC activation,which further enhances NF-κB signaling pathway and regulates autophagy negatively.Finally,excessive NF-κB signaling and abnormal autophagy enhance the inflammatory response and neuron apoptosis,and aggravates cerebral I/R injury.