H2S protects rat cerebral ischemia-reperfusion injury by inhibiting expression and activation of hippocampal ROCK2 at the Thr436 and Ser575 sites

Background

H₂S is an endogenous gas signal molecule, which protects cerebral ischemia/reperfusion (I/R) injury by phosphorylating rho-associated coiled coil-containing protein kinase 2 (ROCK₂) at Tyr722, and inhibiting ROCK₂ protein expression and activities. We previously reported that H₂S protected rat neurons from hypoxia/reoxygenation injury in vitro through inhibiting phosphorylation of ROCK₂ at Thr436 and Ser575, but it is unclear whether these two sites are involved in protection of H₂S against cerebral I/R injury.

Method

Rats transfected with wild-type and mutant eukaryotic plasmids of ROCK₂ in hippocampus were used to establish I/R model by ligating bilateral common carotid artery. Rat behavioral deficit was detected by water maze assay, and ROCK₂, lactate dehydrogenase (LDH), nerve-specific enolase (NSE) and reactive oxygen species (ROS) were determined by ELISA. ROCK₂ expressions was examined by western-blot assay, and bcl-2 and Bax mRNAs were examined by RT-qPCR.

Results

NaHS (4.8 mg/kg) significantly inhibited the I/R-increased serum LDH, NSE and ROS in the ROCK₂^wild-pEGFP-N1-transfected rats, but had no obvious effect in the ROCK₂^T436A-pEGFP-N1- or the ROCK₂^S575F-pEGFP-N1-transfected rats; inhibitions of NaHS on the I/R-increased escape latency and the I/R-decreased percentage of target quadrant distance to total distance were markedly attenuated or abolished in the ROCK₂^T436A-pEGFP-N1- or the ROCK₂^S575F-pEGFP-N1-transfected rats compared with those in the ROCK₂^wild-pEGFP-N1-transfected rats; NaHS obviously inhibited the I/R-increased hippocampal ROCK₂ and GFP-ROCK₂ proteins, Bax mRNA, and ROCK₂ activity, as well as the I/R-decreased hippocampal bcl-2 mRNA in the hippocampus of the ROCK₂^wild-pEGFP-N1-transfected rats, but had no significant effect in the ROCK₂^T436A-pEGFP-N1- or the ROCK₂^S575F-pEGFP-N1-transfected rats.

Conclusion

H₂S protects cerebral I/R injury in rats by inhibiting expression and activation of hippocampal ROCK₂ via the Thr436 and Ser575 sites.