Protection against Hypoxia-Reoxygenation Injury of Hippocampal Neurons by HS via Promoting Phosphorylation of ROCK at Tyr722 in Rat Model.

The RhoA-ROCK signaling pathway is associated with the protective effects of hydrogen sulfide (HS) against cerebral ischemia. HS protects rat hippocampal neurons (RHNs) against hypoxia-reoxygenation (H/R) injury by promoting phosphorylation of RhoA at Ser188. However, effect of HS on the phosphorylation of ROCK-related sites is unclear. The present study was designed to investigate whether HS can play a role in the phosphorylation of ROCK at Tyr722, and explore whether this role mediates the protective effect of H/R injury in RHNs. Prokaryotic recombinant plasmids ROCK-pGEX-6P-1 and ROCK-pGEX-6P-1 were constructed and transfected into in vitro, and the expressed protein, GST-ROCK and GST-ROCK were used for phosphorylation assay in vitro. Eukaryotic recombinant plasmids ROCK-pEGFP-N1 and ROCK-pEGFP-N1 as well as empty plasmid were transfected into the RHNs. Western blot assay and whole-cell patch-clamp technique were used to detect phosphorylation of ROCK at Tyr722 and BK channel current in the RHNs, respectively. Cell viability, leakages of intracellular enzymes lactate dehydrogenase (LDH), and nerve-specific enolase (NSE) were measured. The H/R injury was indicated by decrease of cell viability and leakages of intracellular LDH and NSE. The results of Western blot have shown that NaHS, a HS donor, significantly promoted phosphorylation of GST-ROCK at Tyr722, while no phosphorylation of GST-ROCK was detected. The phosphorylation of ROCK promoted by NaHS was also observed in RHNs. NaHS induced more potent effects on protection against H/R injury, phosphorylation of ROCK at Tyr722, inhibition of ROCK activity, as well as increase of the BK current in the ROCK-pEGFP-N1-transfected RHNs. Our results revealed that HS protects the RHNs from H/R injury through promoting phosphorylation of ROCK at Tyr722 to inhibit ROCK activity and potentially by opening channel currents.