Protection of H(2)S against Hypoxia/Reoxygenation Injury in Rat Hippocampal Neurons through Inhibiting Phosphorylation of ROCK(2) at Thr436 and Ser575

Background: H(2)S (hydrogen sulfide) protects cerebral vasodilatation and endothelial cells against oxygen-glucose deprivation/reoxygenation injury via the inhibition of the RhoA-ROCK pathway and ROCK(2) expression. However, the inhibitory mechanism of H(2)S on ROCK(2) expression is still unclear. The study aimed to investigate the target and mechanism of H(2)S in inhibition of ROCK(2). Methods: His-ROCK(2)(wild) protein was constructed, expressed, and was used for phosphorylation assay in vitro. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) was used to determine the potential phosphorylation sites of ROCK(2). Recombinant ROCK(2)(wild)-pEGFP-N1, ROCK(2)(T436A)-pEGFP-N1, and ROCK(2)(S575F)-pEGFP-N1 plasmids were constructed and transfected into rat hippocampal neurons (RHNs). ROCK(2) expression, cell viability, the release of lactate dehydrogenase (LDH), nerve-specific enolase (NSE), and Ca(2+) were detected to evaluate the neuroprotective mechanism of H(2)S. Results: Phosphorylation at Thr436 and Ser575 of ROCK(2) was observed by mass spectrometry when Polo-like kinase 1 (PLK1) and protein kinase A (PKA) were added in vitro, and NaHS significantly inhibited phosphorylation at Thr436 and Ser575. Additionally, NaHS significantly inhibited the expression of ROCK(2) and recombinant proteins GFP-ROCK(2), GFP-ROCK(2)(T436A), and GFP-ROCK(2)(S575F) in transfected RHNs. Compared with empty plasmid, GFP-ROCK(2)(T436A), and GFP-ROCK(2)(S575F) groups, NaHS significantly inhibited the release of LDH, NSE, and Ca(2+) and promoted ROCK(2) activity in the GFP-ROCK(2)(wild) group. Thr436 and Ser575 may be dominant sites that mediate NaHS inhibition of ROCK(2) protein activity in RHNs. Compared with the empty plasmid, GFP-ROCK(2)(T436A,) and the GFP-ROCK(2)(S575F) group, NaHS had more significant inhibitory effects on hypoxia/reoxygenation (H/R) injury-induced cell viability reduction and increased LDH and NSE release in the GFP-ROCK(2)(wild) group. Conclusion: Exogenous H(2)S protected the RHNs against H/R injury via Thr436 and Ser575 of ROCK(2). These findings suggested that Thr436 and Ser575 may be the dominant sites that mediated the effect of NaHS on protecting RHNs against H/R injury.