Hydrogen sulphide and mild hypothermia activate the CREB signaling pathway and prevent ischemia-reperfusion injury

BACKGROUND Both hydrogen sulphide (H2S) and mild hypothermia have been reported to prevent brain damage caused by reperfusion assault through regulating the N-methyl-D-aspartate receptor (NMDAR). However, the relationship between the two treatments and how they exert neuro-protective effects through NMDARs remain to be elucidated. METHODS Transient cerebral ischemia was induced using the Pulsinelli four-vessel occlusion method. We used sodium hydrosulphide (NaHS) as the H2S donor. We randomly divided 100 Sprague-Dawley rats into five groups of 20: Sham operation group (Sh), normothermic (36-37 °C) ischemia group (NT), mild hypothermic (32-33 °C) ischemia group (mHT), normothermic ischemia combined with NaHS treatment group (NT + NaHS), and mild hypothermic ischemia combined with NaHS treatment group (mHT + NaHS). After 6 hrs of reperfusion, rats were decapitated and hippocampus samples were immediately collected. We measured NR2A (GluN1), NR2B (GluN2) and p-CREB protein levels using western blotting. We further analyzed BDNF mRNA expression by real-time PCR. Hematoxylin and eosin (HE) staining was used to examine pyramidal cell histology at the CA1 region. All statistical analyses were carried out by ANOVA and LSD t-test as implemented by the SPSS 13.0 software. RESULTS In the four test groups with ischemia-reperfusion, hippocampal H2S concentration increased following treatment, and administration of NaHS further increased H2S levels. Moreover, administration of both NaHS and mild hypothermia resulted in up-regulation of NR2A and NR2B protein expressions, as well as p-CREB protein and BDNF mRNA levels. At the cellular level, NaHS and mild hypothermia groups exhibited lower damage caused by ischemia-reperfusion in the CA1 region of the hippocampus. The strongest protective effect was observed in rats treated with combined NaHS and mild hypothermia, suggesting their effects were additive. CONCLUSION Our results support previous findings that hydrogen sulphide and mild hypothermia can prevent ischemia-reperfusion injury. Both treatments caused an up-regulation of NMDA receptors, as well as an elevation in p-CREB protein and BDNF mRNA levels. Thus, hydrogen sulphide and mild hypothermia may provide neuro-protective effect through activating the pro-survival CREB signaling pathway.