Total Flavones of <i>Rhododendron</i> Protect Against Ischemic Cerebral Injury by Regulating the Phosphorylation of the RhoA-ROCK<sub>2</sub> Pathway via Endothelial-Derived H<sub>2</sub>S
This study aims to investigate the mechanism by which the total flavones of <i>Rhododendron</i> (TFR) protect against cerebral ischemic injury through the endothelial-derived H<sub>2</sub>S-mediated regulation of RhoA phosphorylation at the Ser188 and Rho kinase 2 (ROCK<su...
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2025-07-01
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| author | Xiaoqing Sun Xingyu Zhang Yuwen Li Jiyue Wen Zhiwu Chen Shuo Chen |
| author_facet | Xiaoqing Sun Xingyu Zhang Yuwen Li Jiyue Wen Zhiwu Chen Shuo Chen |
| author_sort | Xiaoqing Sun |
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| description | This study aims to investigate the mechanism by which the total flavones of <i>Rhododendron</i> (TFR) protect against cerebral ischemic injury through the endothelial-derived H<sub>2</sub>S-mediated regulation of RhoA phosphorylation at the Ser188 and Rho kinase 2 (ROCK<sub>2</sub>) 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<sub>2</sub>, p-RhoA (RhoA phosphorylated at Ser188), and p-ROCK<sub>2</sub> (ROCK<sub>2</sub> phosphorylated at Thr436) were quantified. Additionally, the activities of RhoA and ROCK<sub>2</sub> were measured. Notably, TFR significantly inhibited H/R-induced H<sub>2</sub>S reduction and suppressed the increased expression and activity of RhoA and ROCK<sub>2</sub> in ECs, effects attenuated by CSE or 3-MST knockout. Moreover, TFR-mediated cerebrovascular dilation was reduced by RhoA or ROCK<sub>2</sub> inhibitors, while the protective effect of TFR against cerebral I/R injury in mice was markedly attenuated by the heterozygous knockout of ROCK<sub>2</sub>. In the ECs-co-cultured neurons, the inhibition of TFR on H/R-induced neuronal injury and decrease in H<sub>2</sub>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<sub>2</sub>, and p-ROCK<sub>2</sub> protein levels, as well as the activities of RhoA and ROCK<sub>2</sub>, 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<sub>2</sub>S level in the co-culture system, and increased RhoA activity and ROCK<sub>2</sub> expression in neurons. In summary, TFR protected against ischemic cerebral injury by endothelial-derived H<sub>2</sub>S promoting the phosphorylation of RhoA at Ser188 but inhibited the phosphorylation of ROCK<sub>2</sub> at Thr436 to inhibit the RhoA-ROCK<sub>2</sub> pathway in neurons. |
| format | Article |
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| institution | Kabale University |
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| spelling | doaj-art-3796e8fdc8fb4590b11520a170bc4a6d2025-08-20T03:58:26ZengMDPI AGCurrent Issues in Molecular Biology1467-30371467-30452025-07-0147751310.3390/cimb47070513Total Flavones of <i>Rhododendron</i> Protect Against Ischemic Cerebral Injury by Regulating the Phosphorylation of the RhoA-ROCK<sub>2</sub> Pathway via Endothelial-Derived H<sub>2</sub>SXiaoqing Sun0Xingyu Zhang1Yuwen Li2Jiyue Wen3Zhiwu Chen4Shuo Chen5School of Pharmaceutical Sciences, Anhui Medical University, Hefei 230032, ChinaSchool of Pharmaceutical Sciences, Anhui Medical University, Hefei 230032, ChinaSchool of Pharmaceutical Sciences, Anhui Medical University, Hefei 230032, ChinaSchool of Pharmaceutical Sciences, Anhui Medical University, Hefei 230032, ChinaSchool of Pharmaceutical Sciences, Anhui Medical University, Hefei 230032, ChinaThe Experimental Research Center, Anhui University of Chinese Medicine, Hefei 230038, ChinaThis study aims to investigate the mechanism by which the total flavones of <i>Rhododendron</i> (TFR) protect against cerebral ischemic injury through the endothelial-derived H<sub>2</sub>S-mediated regulation of RhoA phosphorylation at the Ser188 and Rho kinase 2 (ROCK<sub>2</sub>) 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<sub>2</sub>, p-RhoA (RhoA phosphorylated at Ser188), and p-ROCK<sub>2</sub> (ROCK<sub>2</sub> phosphorylated at Thr436) were quantified. Additionally, the activities of RhoA and ROCK<sub>2</sub> were measured. Notably, TFR significantly inhibited H/R-induced H<sub>2</sub>S reduction and suppressed the increased expression and activity of RhoA and ROCK<sub>2</sub> in ECs, effects attenuated by CSE or 3-MST knockout. Moreover, TFR-mediated cerebrovascular dilation was reduced by RhoA or ROCK<sub>2</sub> inhibitors, while the protective effect of TFR against cerebral I/R injury in mice was markedly attenuated by the heterozygous knockout of ROCK<sub>2</sub>. In the ECs-co-cultured neurons, the inhibition of TFR on H/R-induced neuronal injury and decrease in H<sub>2</sub>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<sub>2</sub>, and p-ROCK<sub>2</sub> protein levels, as well as the activities of RhoA and ROCK<sub>2</sub>, 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<sub>2</sub>S level in the co-culture system, and increased RhoA activity and ROCK<sub>2</sub> expression in neurons. In summary, TFR protected against ischemic cerebral injury by endothelial-derived H<sub>2</sub>S promoting the phosphorylation of RhoA at Ser188 but inhibited the phosphorylation of ROCK<sub>2</sub> at Thr436 to inhibit the RhoA-ROCK<sub>2</sub> pathway in neurons.https://www.mdpi.com/1467-3045/47/7/513total flavones of <i>Rhododendron</i>H<sub>2</sub>Sischemic cerebral injuryRhoA at Ser188ROCK<sub>2</sub> at Thr436 |
| spellingShingle | Xiaoqing Sun Xingyu Zhang Yuwen Li Jiyue Wen Zhiwu Chen Shuo Chen Total Flavones of <i>Rhododendron</i> Protect Against Ischemic Cerebral Injury by Regulating the Phosphorylation of the RhoA-ROCK<sub>2</sub> Pathway via Endothelial-Derived H<sub>2</sub>S Current Issues in Molecular Biology total flavones of <i>Rhododendron</i> H<sub>2</sub>S ischemic cerebral injury RhoA at Ser188 ROCK<sub>2</sub> at Thr436 |
| title | Total Flavones of <i>Rhododendron</i> Protect Against Ischemic Cerebral Injury by Regulating the Phosphorylation of the RhoA-ROCK<sub>2</sub> Pathway via Endothelial-Derived H<sub>2</sub>S |
| title_full | Total Flavones of <i>Rhododendron</i> Protect Against Ischemic Cerebral Injury by Regulating the Phosphorylation of the RhoA-ROCK<sub>2</sub> Pathway via Endothelial-Derived H<sub>2</sub>S |
| title_fullStr | Total Flavones of <i>Rhododendron</i> Protect Against Ischemic Cerebral Injury by Regulating the Phosphorylation of the RhoA-ROCK<sub>2</sub> Pathway via Endothelial-Derived H<sub>2</sub>S |
| title_full_unstemmed | Total Flavones of <i>Rhododendron</i> Protect Against Ischemic Cerebral Injury by Regulating the Phosphorylation of the RhoA-ROCK<sub>2</sub> Pathway via Endothelial-Derived H<sub>2</sub>S |
| title_short | Total Flavones of <i>Rhododendron</i> Protect Against Ischemic Cerebral Injury by Regulating the Phosphorylation of the RhoA-ROCK<sub>2</sub> Pathway via Endothelial-Derived H<sub>2</sub>S |
| title_sort | total flavones of i rhododendron i protect against ischemic cerebral injury by regulating the phosphorylation of the rhoa rock sub 2 sub pathway via endothelial derived h sub 2 sub s |
| topic | total flavones of <i>Rhododendron</i> H<sub>2</sub>S ischemic cerebral injury RhoA at Ser188 ROCK<sub>2</sub> at Thr436 |
| url | https://www.mdpi.com/1467-3045/47/7/513 |
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