Isoflurane postconditioning (ISPOC) plays a neuroprotection role in the brain. Previous studies confirmed that isoflurane postconditioning can provide better protection than preconditioning in acute hypoxic–ischemic brain damage, such as acute craniocerebral trauma and ischemic stroke. Numerous studies have reported that activin A can protect rat’s brain from cell injury. However, whether activin A and its downstream ERK1/2 were involved in isoflurane postconditioning-induced neuroprotection is unknown.

A total of 80 healthy Sprague–Dawley rats weighing 50–70g 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 (dimethyl sulfoxide(DMSO)) group. Blockers (SB431542 and U0126) were used in each concentration of isoflurane before OGD. Hematoxylin–eosin staining, 2,3,5-triphenyl tetrazolium chloride staining, and propidium iodide (PI) staining were conducted to assess the reliability in the brain slices. Immunofluorescence, Western blot, and quantitative real-time PCR(Q-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).

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 mRNA 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 mRNA 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 mRNA 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 mRNA 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).

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.