{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,18]],"date-time":"2026-03-18T17:39:05Z","timestamp":1773855545985,"version":"3.50.1"},"reference-count":44,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2022,7,18]],"date-time":"2022-07-18T00:00:00Z","timestamp":1658102400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Science Foundation of China","award":["81973510"],"award-info":[{"award-number":["81973510"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>The RhoA-ROCK signaling pathway is associated with the protective effects of hydrogen sulfide (H2S) against cerebral ischemia. H2S protects rat hippocampal neurons (RHNs) against hypoxia-reoxygenation (H\/R) injury by promoting phosphorylation of RhoA at Ser188. However, effect of H2S on the phosphorylation of ROCK2-related sites is unclear. The present study was designed to investigate whether H2S can play a role in the phosphorylation of ROCK2 at Tyr722, and explore whether this role mediates the protective effect of H\/R injury in RHNs. Prokaryotic recombinant plasmids ROCK2wild-pGEX-6P-1 and ROCK2Y722F-pGEX-6P-1 were constructed and transfected into E. coli in vitro, and the expressed protein, GST-ROCK2wild and GST-ROCK2Y722F were used for phosphorylation assay in vitro. Eukaryotic recombinant plasmids ROCK2Y722-pEGFP-N1 and ROCK2Y722F-pEGFP-N1 as well as empty plasmid were transfected into the RHNs. Western blot assay and whole-cell patch-clamp technique were used to detect phosphorylation of ROCK2 at Tyr722 and BKCa channel current in the RHNs, respectively. Cell viability, leakages of intracellular enzymes lactate dehydrogenase (LDH), and nerve-specific enolase (NSE) were measured. The H\/R injury was indicated by decrease of cell viability and leakages of intracellular LDH and NSE. The results of Western blot have shown that NaHS, a H2S donor, significantly promoted phosphorylation of GST-ROCK2wild at Tyr722, while no phosphorylation of GST-ROCK2Y722F was detected. The phosphorylation of ROCK2wild promoted by NaHS was also observed in RHNs. NaHS induced more potent effects on protection against H\/R injury, phosphorylation of ROCK2 at Tyr722, inhibition of ROCK2 activity, as well as increase of the BKCa current in the ROCK2Y722-pEGFP-N1-transfected RHNs. Our results revealed that H2S protects the RHNs from H\/R injury through promoting phosphorylation of ROCK2 at Tyr722 to inhibit ROCK2 activity and potentially by opening channel currents.<\/jats:p>","DOI":"10.3390\/molecules27144567","type":"journal-article","created":{"date-parts":[[2022,7,18]],"date-time":"2022-07-18T11:32:23Z","timestamp":1658143943000},"page":"4567","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Protection against Hypoxia-Reoxygenation Injury of Hippocampal Neurons by H2S via Promoting Phosphorylation of ROCK2 at Tyr722 in Rat Model"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5925-5337","authenticated-orcid":false,"given":"Meng","family":"Xue","sequence":"first","affiliation":[{"name":"Department of Pharmacology, School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shuo","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiaojiao","family":"Xi","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qianjun","family":"Guan","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wei","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yan","family":"Guo","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zhiwu","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,7,18]]},"reference":[{"key":"ref_1","unstructured":"Mandalaneni, K., Rayi, A., and Jillella, D.V. 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