Role Of Activin And Its Signaling Pathway In Protective Effect Of Isoflurane Postconditioning Against Oxygen And Glucose Deprivation In Rats

Object: To evaluate the most suitable concentration(1.5%,3.0% and 4.5%)of isoflurane-induced postconditioning against oxygen and glucose deprivation insult of rat hippocampal slices;to study the effects of activin A and its downstream ERK1/2 in oxygen and glucose deprivation injury after isoflurane-induced postconditioning.Methods: A total of 80 healthy Sprague–Dawley rats weighing 50-70 g were randomly divided into 10 groups of 8: normal control,oxygen and glucose deprivation(OGD),1.5% ISPOC,3.0% ISPOC,4.5%ISPOC,blocker of activin A(SB431542),blocker of ERK1/2(U0126),3.0% ISPOC + SB431542,3.0%ISPOC + U0126,and vehicle(DMSO)group.Blockers(SB431542 and U0126)were used in each concentration of isoflurane before OGD.Hematoxylin–eosin(HE)staining,2,3,5-triphenyl tetrazolium chloride(TTC)staining,and propidium iodide(PI)staining were conducted to assess the reliability in the brain slices.Immunofluorescence(IF),Western blot,and quantitative real-time PCR(FQ-PCR)were performed to validate the protein expression levels of activin A,Smad2/3,p-Smad2/3,ERK1/2,and phosphorylation ERK1/2(p-ERK1/2).Results: The number of damaged neurons and mean fluorescence intensity(MFI)of PI staining increased,but formazan generation,expression levels of activin A and p-ERK1/2 protein,and m RNA synthesis level of activin A decreased in the OGD group compared with the normal control group(P<0.05).The number of damaged neurons and MFI of PI staining decreased,but formazan production,expression levels of activin A,p-Smad2/3,and p-ERK1/2,and m RNA synthesis level of activin A increased significantly in the 1.5%ISPOC and 3.0% ISPOC groups(P<0.05)compared with the OGD group.The result in the 4.5% ISPOC group,was completely opposite to the 1.5% ISPOC and 3.0% ISPOC groups.The number of damage neuron and MFI of PI staining increased,but formazan production,expression levels of activin A,p-Smad2/3,and p-ERK1/2,and m RNA synthesis level of activin A decreased in the 4.5% ISPOC group.However,the expression levels of activin A,p-Smad2/3,and p-ERK1/2,and m RNA synthesis level of activin A in the 4.5% ISPOC group were higher than the OGD group(P<0.05).The other results were compared between the SB431542 group/the U0126 group and 3.0% ISPOC group.The MFI of PI staining increased,but the expression levels of activin A,p-Smad2/3,and p-ERK1/2 decreased(P<0.05).The expression level of ERK1/2 protein in all groups exhibited no change(p > 0.05).Conclusion: Results of this study showed that 3.0% concentration of isoflurane postconditioning provided better neuroprotection than 1.5% and 4.5% concentrations of isoflurane.Activin A / Smad 2/3 and activin A/ ERK1/2 signaling pathway may be involved in ISPOC-induced neuroprotection against oxygen and glucose deprivation insult.