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-RhoAwild and GST-RhoAS188A proteins were constructed and expressed, and were used for phosphorylation assay in vitro. Recombinant RhoAwild-pEGFP-N1 and RhoAS188A-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.
Highlights
Ischemic stroke is one of the main factors responsible for morbidity and death[1]
Endogenous H2S is mainly produced from L-cysteine by cystathionine-γ-lyase (CSE) and cystathionine-β-synthase, and from β-mercaptopyruvic acid catalyzed by 3-mercaptopyruvate sulfurtransferase (3-MST) in the mitochondria
RhoA requires cAMP-dependent protein kinase A or cGMP-dependent PKG30, we used PKG1 as the kinase to catalyze the phosphorylation of RhoA at Ser[188] in vitro
Summary
Ischemic stroke is one of the main factors responsible for morbidity and death[1]. The restoration of blood flow is a very important way for the treatment of ischemic injury. The restoration of blood flow will cause reperfusion injury. Cerebral ischemia-reperfusion (I/R) can cause neuronal damage in varying degrees, including inflammation, necrosis, and apoptosis[2]. Cerebral I/R injury is prone to induce apoptosis of vertebral neurons in the hippocampus area[3,4]. The protection of hippocampal nerve cells (HNCs) is Hydrogen sulfide (H2S) is a ubiquitous second messenger molecule. Endogenous H2S is mainly produced from L-cysteine by cystathionine-γ-lyase (CSE) and cystathionine-β-synthase, and from β-mercaptopyruvic acid catalyzed by 3-mercaptopyruvate sulfurtransferase (3-MST) in the mitochondria.
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