{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,23]],"date-time":"2026-08-23T17:38:21Z","timestamp":1787506701103,"version":"build-2736575974"},"reference-count":33,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2023,1,31]],"date-time":"2023-01-31T00:00:00Z","timestamp":1675123200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Science Foundation of China","doi-asserted-by":"publisher","award":["81973510"],"award-info":[{"award-number":["81973510"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceuticals"],"abstract":"<jats:p>Background: H2S (hydrogen sulfide) protects cerebral vasodilatation and endothelial cells against oxygen-glucose deprivation\/reoxygenation injury via the inhibition of the RhoA-ROCK pathway and ROCK2 expression. However, the inhibitory mechanism of H2S on ROCK2 expression is still unclear. The study aimed to investigate the target and mechanism of H2S in inhibition of ROCK2. Methods: His-ROCK2wild protein was constructed, expressed, and was used for phosphorylation assay in vitro. Liquid chromatography\u2013tandem mass spectrometry (LC\u2013MS\/MS) was used to determine the potential phosphorylation sites of ROCK2. Recombinant ROCK2wild-pEGFP-N1, ROCK2T436A-pEGFP-N1, and ROCK2S575F-pEGFP-N1 plasmids were constructed and transfected into rat hippocampal neurons (RHNs). ROCK2 expression, cell viability, the release of lactate dehydrogenase (LDH), nerve-specific enolase (NSE), and Ca2+ were detected to evaluate the neuroprotective mechanism of H2S. Results: Phosphorylation at Thr436 and Ser575 of ROCK2 was observed by mass spectrometry when Polo-like kinase 1 (PLK1) and protein kinase A (PKA) were added in vitro, and NaHS significantly inhibited phosphorylation at Thr436 and Ser575. Additionally, NaHS significantly inhibited the expression of ROCK2 and recombinant proteins GFP-ROCK2, GFP-ROCK2T436A, and GFP-ROCK2S575F in transfected RHNs. Compared with empty plasmid, GFP-ROCK2T436A, and GFP-ROCK2S575F groups, NaHS significantly inhibited the release of LDH, NSE, and Ca2+ and promoted ROCK2 activity in the GFP-ROCK2wild group. Thr436 and Ser575 may be dominant sites that mediate NaHS inhibition of ROCK2 protein activity in RHNs. Compared with the empty plasmid, GFP-ROCK2T436A, and the GFP-ROCK2S575F group, NaHS had more significant inhibitory effects on hypoxia\/reoxygenation (H\/R) injury-induced cell viability reduction and increased LDH and NSE release in the GFP-ROCK2wild group. Conclusion: Exogenous H2S protected the RHNs against H\/R injury via Thr436 and Ser575 of ROCK2. These findings suggested that Thr436 and Ser575 may be the dominant sites that mediated the effect of NaHS on protecting RHNs against H\/R injury.<\/jats:p>","DOI":"10.3390\/ph16020218","type":"journal-article","created":{"date-parts":[[2023,2,1]],"date-time":"2023-02-01T02:06:44Z","timestamp":1675217204000},"page":"218","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Protection of H2S against Hypoxia\/Reoxygenation Injury in Rat Hippocampal Neurons through Inhibiting Phosphorylation of ROCK2 at Thr436 and Ser575"],"prefix":"10.3390","volume":"16","author":[{"given":"Fang","family":"Fang","sequence":"first","affiliation":[{"name":"Department of Pharmacology, School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ju","family":"Sheng","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yan","family":"Guo","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiyue","family":"Wen","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhiwu","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,1,31]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e279","DOI":"10.1212\/WNL.0000000000013115","article-title":"Global, Regional, and National Burden of Ischemic Stroke, 1990\u20132019","volume":"98","author":"Ding","year":"2021","journal-title":"Neurology"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1391","DOI":"10.1038\/nm.2507","article-title":"Ischemia and reperfusion\u2014From mechanism to translation","volume":"17","author":"Eltzschig","year":"2011","journal-title":"Nat. Med."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1038\/s41392-022-01064-1","article-title":"Signaling pathways involved in ischemic stroke: Molecular mechanisms and therapeutic interventions","volume":"7","author":"Qin","year":"2022","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"e364","DOI":"10.1161\/STR.0000000000000375","article-title":"2021 Guideline for the Prevention of Stroke in Patients With Stroke and Transient Ischemic Attack: A Guideline From the American Heart Association\/American Stroke Association","volume":"52","author":"Kleindorfer","year":"2021","journal-title":"Stroke"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"115122","DOI":"10.1016\/j.bcp.2022.115122","article-title":"NO, CO and H2S: A trinacrium of bioactive gases in the brain","volume":"202","author":"Siracusa","year":"2022","journal-title":"Biochem. Pharmacol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"114267","DOI":"10.1016\/j.bcp.2020.114267","article-title":"Mechanism of cystathionine-beta-synthase inhibition by disulfiram: The role of bis(N,N-diethyldithiocarbamate)-copper(II)","volume":"182","author":"Zuhra","year":"2020","journal-title":"Biochem. Pharmacol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"497","DOI":"10.1124\/pr.117.014050","article-title":"International Union of Basic and Clinical Pharmacology. CII: Pharmacological Modulation of H2S Levels: H2S Donors and H2S Biosynthesis Inhibitors","volume":"69","author":"Szabo","year":"2017","journal-title":"Pharmacol. Rev."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"6108","DOI":"10.1039\/C6CS00212A","article-title":"A practical guide to working with H2S at the interface of chemistry and biology","volume":"45","author":"Hartle","year":"2016","journal-title":"Chem. Soc. Rev."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Schmidt, S.I., Blaabjerg, M., Freude, K., and Meyer, M. (2022). RhoA Signaling in Neurodegenerative Diseases. Cells, 11.","DOI":"10.3390\/cells11091520"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1101","DOI":"10.1007\/s11011-021-00709-4","article-title":"RhoA\/ROCK signaling pathway and astrocytes in ischemic stroke","volume":"36","author":"Lu","year":"2021","journal-title":"Metab. Brain Dis."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Kimura, T., Horikoshi, Y., Kuriyagawa, C., and Niiyama, Y. (2021). Rho\/ROCK Pathway and Noncoding RNAs: Implications in Ischemic Stroke and Spinal Cord Injury. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms222111573"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1565","DOI":"10.1021\/acschemneuro.8b00533","article-title":"WRole of CSE-Produced H2S on Cerebrovascular Relaxation via RhoA-ROCK Inhibition and Cerebral Ischemia-Reperfusion Injury in Mice","volume":"10","author":"Wen","year":"2019","journal-title":"ACS Chem. Neurosci."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"779","DOI":"10.3389\/fneur.2018.00779","article-title":"Vascular Protection of Hydrogen Sulfide on Cerebral Ischemia\/Reperfusion Injury in Rats","volume":"9","author":"Wen","year":"2018","journal-title":"Front. Neurol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"174682","DOI":"10.1016\/j.ejphar.2021.174682","article-title":"Hydrogen sulphide protects mice against the mutual aggravation of cerebral ischaemia\/reperfusion injury and colitis","volume":"914","author":"Ding","year":"2022","journal-title":"Eur. J. Pharmacol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"18498","DOI":"10.1021\/acsomega.2c00996","article-title":"Roles of the RhoA-ROCK Signaling Pathway in the Endothelial H2S Production and Vasodilation in Rat Cerebral Arteries","volume":"7","author":"Chen","year":"2022","journal-title":"ACS Omega"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"2580","DOI":"10.1021\/acschemneuro.0c00674","article-title":"CSE-Derived H2S Inhibits Reactive Astrocytes Proliferation and Promotes Neural Functional Recovery after Cerebral Ischemia\/Reperfusion Injury in Mice Via Inhibition of RhoA\/ROCK2 Pathway","volume":"12","author":"Zhang","year":"2021","journal-title":"ACS Chem. Neurosci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"C720","DOI":"10.1152\/ajpcell.00014.2020","article-title":"3-Mercaptopyruvate sulfurtransferase\/hydrogen sulfide protects cerebral endothelial cells against oxygen-glucose deprivation\/reoxygenation-induced injury via mitoprotection and inhibition of the RhoA\/ROCK pathway","volume":"319","author":"Zhang","year":"2020","journal-title":"Am. J. Physiol. Physiol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"132","DOI":"10.1038\/s41420-021-00514-z","article-title":"H2S protects hippocampal neurons against hypoxia-reoxygenation injury by promoting RhoA phosphorylation at Ser188","volume":"7","author":"Chen","year":"2021","journal-title":"Cell Death Discov."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1016\/0014-5793(96)00811-3","article-title":"ROCK-I and ROCK-II, two isoforms of Rho-associated coiled-coil forming protein serine\/threonine kinase in mice","volume":"392","author":"Nakagawa","year":"1996","journal-title":"FEBS Lett."},{"key":"ref_20","first-page":"6145","article-title":"LncRNA ROR is involved in cerebral hypoxia\/reoxygenation-induced injury in PC12 cells via regulating miR-135a-5p\/ROCK1\/2","volume":"11","author":"Chen","year":"2019","journal-title":"Am. J. Transl. Res."},{"key":"ref_21","first-page":"3783","article-title":"Mechanism of H2S-mediated ROCK inhibition of total flavones of Rhododendra against myocardial ischemia injury","volume":"18","author":"Jiao","year":"2019","journal-title":"Exp. Ther. Med."},{"key":"ref_22","first-page":"137","article-title":"Metody analizy fosforylacji bia\u0142ek [Methods of an alysis of protein phosphorylation]","volume":"63","author":"Taracha","year":"2017","journal-title":"Postep. Biochem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"946","DOI":"10.1016\/j.ijbiomac.2021.06.149","article-title":"Phosphorylation of polysaccharides: A review on the synthesis and bioactivities","volume":"184","author":"Xia","year":"2021","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2339","DOI":"10.1002\/1873-3468.13797","article-title":"Importance of protein Ser\/Thr\/Tyr phosphorylation for bacterial pathogenesis","volume":"594","author":"Jers","year":"2020","journal-title":"FEBS Lett."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"141","DOI":"10.3389\/fmicb.2016.00141","article-title":"Mass Spectrometry Offers Insight into the Role of Ser\/Thr\/Tyr Phosphorylation in the Mycobacteria","volume":"7","author":"Calder","year":"2016","journal-title":"Front. Microbiol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2262","DOI":"10.1038\/sj.emboj.7601683","article-title":"Proteomic screen defines the Polo-box domain interactome and identifies Rock2 as a Plk1 substrate","volume":"26","author":"Lowery","year":"2007","journal-title":"EMBO J."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Xue, M., Chen, S., Xi, J., Guan, Q., Chen, W., Guo, Y., and Chen, Z. (2022). Protection against Hypoxia-Reoxygenation Injury of Hippocampal Neurons by H2S via Promoting Phosphorylation of ROCK2 at Tyr722 in Rat Model. Molecules, 27.","DOI":"10.3390\/molecules27144567"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"130","DOI":"10.1097\/FJC.0000000000000943","article-title":"Role of Hydrogen Sulfide in Myocardial Ischemia-Reperfusion Injury","volume":"77","author":"Zhang","year":"2021","journal-title":"J. Cardiovasc. Pharmacol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1074","DOI":"10.1124\/pr.115.010595","article-title":"Rho Kinases in Health and Disease: From Basic Science to Translational Research","volume":"67","author":"Loirand","year":"2015","journal-title":"Pharmacol. Rev."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"353","DOI":"10.3233\/CBM-160336","article-title":"Elevated lactate dehydrogenase (LDH) can be a marker of immune suppression in cancer: Interplay between hematologic and solid neoplastic clones and their microenvironments","volume":"19","author":"Ding","year":"2017","journal-title":"Cancer Biomark."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1007\/978-94-017-7215-0_9","article-title":"Neuron-Specific Enolase as a Biomarker:Biochemical and Clinical Aspects","volume":"867","author":"Bottoni","year":"2015","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_32","first-page":"1095","article-title":"Effects of anisodamine on altered [Ca2+]i and cerebral cortex ultrastructure following acute cerebral ischemia\/reperfusion injury in rabbits","volume":"3","author":"Zhou","year":"2008","journal-title":"Neural Regen. Res."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1016\/j.taap.2019.02.017","article-title":"Inhibition of RhoA\/ROCK signaling pathway ameliorates hypoxic pulmonary hypertension via HIF-1\u03b1-dependent functional TRPC channels","volume":"369","author":"Wang","year":"2019","journal-title":"Toxicol. Appl. Pharmacol."}],"container-title":["Pharmaceuticals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1424-8247\/16\/2\/218\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T18:19:30Z","timestamp":1760120370000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1424-8247\/16\/2\/218"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,1,31]]},"references-count":33,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2023,2]]}},"alternative-id":["ph16020218"],"URL":"https:\/\/doi.org\/10.3390\/ph16020218","relation":{},"ISSN":["1424-8247"],"issn-type":[{"value":"1424-8247","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,1,31]]}}}