H2S protects hippocampal neurons against hypoxia-reoxygenation injury by promoting RhoA phosphorylation at Ser188

Abstract

Inhibition of RhoA-ROCK pathway is involved in the H₂S-induced cerebral vasodilatation and H₂S-mediated protection on endothelial cells against oxygen-glucose deprivation/reoxygenation injury. However, the inhibitory mechanism of H₂S on RhoA-ROCK pathway is still unclear. The aim of this study was to investigate the target and mechanism of H₂S in inhibition of RhoA/ROCK. GST-RhoA^wild and GST-RhoA^S188A proteins were constructed and expressed, and were used for phosphorylation assay in vitro. Recombinant RhoA^wild-pEGFP-N1 and RhoA^S188A-pEGFP-N1 plasmids were constructed and transfected into primary hippocampal nerve cells (HNCs) to evaluate the neuroprotective mechanism of endothelial H₂S by using transwell co-culture system with endothelial cells from cystathionine-γ-lyase knockout (CSE^−/−) mice and 3-mercaptopyruvate sulfurtransferase knockout (3-MST^−/−) rats, respectively. We found that NaHS, exogenous H₂S donor, promoted RhoA phosphorylation at Ser188 in the presence of cGMP-dependent protein kinase 1 (PKG1) in vitro. Besides, both exogenous and endothelial H₂S facilitated the RhoA phosphorylation at Ser188 in HNCs, which induced the reduction of RhoA activity and membrane transposition, as well as ROCK₂ activity and expression. To further investigate the role of endothelial H₂S on RhoA phosphorylation, we detected H₂S release from ECs of CSE^+/+ and CSE^−/− mice, and 3-MST^+/+ and 3-MST^−/− rats, respectively, and found that H₂S produced by ECs in the culture medium is mainly catalyzed by CSE synthase. Moreover, we revealed that both endothelial H₂S, mainly catalyzed by CSE, and exogenous H₂S protected the HNCs against hypoxia-reoxygenation injury via phosphorylating RhoA at Ser188.