Total Flavones of Rhododendron Protect Against Ischemic Cerebral Injury by Regulating the Phosphorylation of the RhoA-ROCK(2) Pathway via Endothelial-Derived H(2)S.

This study aims to investigate the mechanism by which the total flavones of Rhododendron (TFR) protect against cerebral ischemic injury through the endothelial-derived H(2)S-mediated regulation of RhoA phosphorylation at the Ser188 and Rho kinase 2 (ROCK(2)) phosphorylation at Thr436. For experimental design, mouse or rat cerebrovascular endothelial cells (ECs) were cultured with or without neurons and subjected to hypoxia/reoxygenation (H/R) injury. The vasodilation of the cerebral basilar artery was assessed. Cerebral ischemia/reperfusion (I/R) injury was induced in mice by bilateral carotid artery ligation, followed by Morris water maze and open field behavioral assessments. The protein levels of cystathionine-γ-lyase (CSE), 3-mercaptopyruvate sulfurtransferase (3-MST), RhoA, ROCK(2), p-RhoA (RhoA phosphorylated at Ser188), and p-ROCK(2) (ROCK(2) phosphorylated at Thr436) were quantified. Additionally, the activities of RhoA and ROCK(2) were measured. Notably, TFR significantly inhibited H/R-induced H(2)S reduction and suppressed the increased expression and activity of RhoA and ROCK(2) in ECs, effects attenuated by CSE or 3-MST knockout. Moreover, TFR-mediated cerebrovascular dilation was reduced by RhoA or ROCK(2) inhibitors, while the protective effect of TFR against cerebral I/R injury in mice was markedly attenuated by the heterozygous knockout of ROCK(2). In the ECs-co-cultured neurons, the inhibition of TFR on H/R-induced neuronal injury and decrease in H(2)S level in the co-culture was attenuated by the knockout of CSE or 3-MST in the ECs. TFR notably inhibited the H/R-induced upregulation of neuronal RhoA, ROCK(2), and p-ROCK(2) protein levels, as well as the activities of RhoA and ROCK(2), while reversing the decrease in p-RhoA. However, the knockout of CSE or 3-MST in the ECs significantly attenuated the inhibition of TFR on these increases. Furthermore, 3-MST knockout in ECs attenuated the TFR-mediated suppression of p-RhoA reduction. Additionally, CSE or 3-MST knockout in ECs exacerbated H/R-induced neuronal injury, reduced H(2)S level in the co-culture system, and increased RhoA activity and ROCK(2) expression in neurons. In summary, TFR protected against ischemic cerebral injury by endothelial-derived H(2)S promoting the phosphorylation of RhoA at Ser188 but inhibited the phosphorylation of ROCK(2) at Thr436 to inhibit the RhoA-ROCK(2) pathway in neurons.