H(2)S protects hippocampal neurons against hypoxia-reoxygenation injury by promoting RhoA phosphorylation at Ser188

Inhibition of RhoA-ROCK pathway is involved in the H(2)S-induced cerebral vasodilatation and H(2)S-mediated protection on endothelial cells against oxygen-glucose deprivation/reoxygenation injury. However, the inhibitory mechanism of H(2)S on RhoA-ROCK pathway is still unclear. The aim of this study was to investigate the target and mechanism of H(2)S 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 H(2)S 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 H(2)S donor, promoted RhoA phosphorylation at Ser188 in the presence of cGMP-dependent protein kinase 1 (PKG1) in vitro. Besides, both exogenous and endothelial H(2)S facilitated the RhoA phosphorylation at Ser188 in HNCs, which induced the reduction of RhoA activity and membrane transposition, as well as ROCK(2) activity and expression. To further investigate the role of endothelial H(2)S on RhoA phosphorylation, we detected H(2)S release from ECs of CSE(+/+) and CSE(−/−) mice, and 3-MST(+/+) and 3-MST(−/−) rats, respectively, and found that H(2)S produced by ECs in the culture medium is mainly catalyzed by CSE synthase. Moreover, we revealed that both endothelial H(2)S, mainly catalyzed by CSE, and exogenous H(2)S protected the HNCs against hypoxia-reoxygenation injury via phosphorylating RhoA at Ser188.