Protection of H₂S against Hypoxia/Reoxygenation Injury in Rat Hippocampal Neurons through Inhibiting Phosphorylation...

H₂S (hydrogen sulfide) protects cerebral vasodilatation and endothelial cells against oxygen-glucose deprivation/reoxygenation injury via the inhibition of the RhoA-ROCK pathway and ROCK₂ expression. However, the inhibitory mechanism of H₂S on ROCK₂ expression is still unclear. The study aimed to investigate the target and mechanism of H₂S in inhibition of ROCK₂.

His-ROCK₂^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 ROCK₂. Recombinant ROCK₂^wild-pEGFP-N1, ROCK₂^T436A-pEGFP-N1, and ROCK₂^S575F-pEGFP-N1 plasmids were constructed and transfected into rat hippocampal neurons (RHNs). ROCK₂ expression, cell viability, the release of lactate dehydrogenase (LDH), nerve-specific enolase (NSE), and Ca²⁺ were detected to evaluate the neuroprotective mechanism of H₂S.

Phosphorylation at Thr436 and Ser575 of ROCK₂ 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 ROCK₂ and recombinant proteins GFP-ROCK₂, GFP-ROCK₂^T436A, and GFP-ROCK₂^S575F in transfected RHNs. Compared with empty plasmid, GFP-ROCK₂^T436A, and GFP-ROCK₂^S575F groups, NaHS significantly inhibited the release of LDH, NSE, and Ca²⁺ and promoted ROCK₂ activity in the GFP-ROCK₂^wild group. Thr436 and Ser575 may be dominant sites that mediate NaHS inhibition of ROCK₂ protein activity in RHNs. Compared with the empty plasmid, GFP-ROCK₂^T436A, and the GFP-ROCK₂^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-ROCK₂^wild group.

Exogenous H₂S protected the RHNs against H/R injury via Thr436 and Ser575 of ROCK₂. 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.