| Hydrogen sulfide (H2S), which has extensive physiological functions, is considered as the third gaseous signal molecule besides nitric oxide (NO) and carbon monoxide (CO). H2S is involved in the regulation of vascular relaxation and contraction, however, its vasodilating mechanism is not clear. The small G protein RhoA and its downstream effectors of Rho associated coiled coil-forming kinase (ROCK) are involved in various diseases including cerebral ischemia. RhoA-ROCK pathway is responsible for smooth muscle cells contraction in various vascular beds. Ischemic cerebrovascular disease, which is very common in clinic, cause serious damage to hunman health and social development. Our previous studies have shown that hyperin (Hyp), a flavonol compound extracted from Abelmoschus manihot, has significant protective effects against cerebral ischemia/reperfusion (I/R) injury in mice. However, its mechanism of cerebral protection remains poorly understood. In vitro vasomotor function experiments show that Hyp can relax rat cerebral basilar arteries (BAs) and the relaxtion effects associated with endogenous H2S.In this study, the model of cystathionine y-lyase (CSE, a generating enzyme of H2S) knockout (CSE-KO) mice and the Wire Myograph System were used to observe the role of H2S and RhoA-ROCK pathway in the vasomotor function in mice. Cerebral I/R models in mice were established to study the role of H2S in the protective action of Hyp on cerebral I/R injury in mice.Purpose:1. To observe the role of RhoA-ROCK pathway in H2S induced relaxation of BAs iin mice.2. To study the role of H2S in the protective action of Hyp on cerebral I/R injury in mice.Methods:1. The changes of BAs tension in mice were recorded by Wire Myograph System. test The effects of RhoA agonist LPA and U46619 on the tone of BAs both in WT and CSE-KO mice were observed. Effects of ROCK inhibitor Y27632 on the tone of endothelium-intact or endothelium-denuded BAs both from WT and CSE-KO mice were detected. The effects of Y27632 on NaHS induced relaxationof BAs were explored.2. BAs smooth muscle cells were cultivated with the primary culture method and identified by immunofluorescence.3. Effect of H2S on RhoA activity in mice BAs smooth muscle cells was measured by G-LISA RhoA activation assay biochem kit.4. Western blot method was used to explore the influences of H2S on the expressions of ROCK1 and ROCK2 protein in BAs smooth muscle cells.5. Cerebral I/R models were established by fastening bilateral common carotid artery in CSE-KO and wide-type (WT) mice and the models were established to study the role of H2S in the protective action of Hyp on cerebral I/R injury in mice. The mice brain tissue injury was observed by detecting the lactate dehydrogenase (LDH) activity and malondialdehyde (MDA) content. The learning and memory abilities of mice were measured by the step down test and Morris water maze test.Results:1. Application of LPA (10～160μmol/L, a agonist of RhoA) and U46619(10-9～10-6 mol/L, another agonist of RhoA) caused dose-dependent constriction in BAs of WT mice.2. The contraction effects of LPA (10～160μmol/L) and U46619 (10-9～10-6 mol/L) were elevated by knocking out the expression of CSE.3. A ROCK inhibitor, Y27632 (10-7～10-5 mol/L), induced a concentration-dependent relaxation of BAs in WT mice, however, the relaxation effect was weakened by removing the vascular endothelial cells.4. Y27632 (10-7～10-5 mol/L) induced relaxation was significantly decreased by knocking out CSE.5. The relaxation effects induced by NaHS (10-6～10-3 mol/L, a donor of H2S) were attenuated in the presence of 3μmol/L Y27632.6. The RhoA activity was increased in BAs smooth muscle cells by application of LPA (10μmol/L) or knocking out CSE gene. NaHS 100μmol/L,200μmol/L can remarkably inhibit the RhoA activity in BAs smooth muscle cells of CSE-KO mice.7. The expressions of ROCK1 and ROCK2 protein in BAs smooth muscle cells were elevated by application of LPA or knocking out CSE. NaHS 100μmol/L,200μmol/L can significantly inhibit the expressions of ROCK1 and ROCK2 in BAs smooth muscle cells of CSE-KO mice.8. After cerebral I/R, reductions of LDH activities and elevations of MDA contents in brain tissue were more obvious in CSE-KO mice than that in WT mice. Reductions of LDH activities and elevations of MDA contents were markedly inhibited by Hyp 50, 100 mg/kg in WT mice rather than that in CSE-KO mice.9. In the step down test, the learning Latency was extended, memory latency was shorted, Number of errors of learning and memory were increased in WT mice after cerebral I/R. The above indicators were changed more significantly in CSE-KO mice. Hyp 50 mg/kg,100 mg/kg can inhibited the changes of WT mice, but have no effect on CSE-KO mice.10. In the Morris water maze test, the escape lantecy was extened, the number of entries, and the proportion of time and swim distance were all decreased in WT mice after cerebral I/R. The above indicators were changed more significantly in CSE-KO mice. Hyp 50 mg/kg,100 mg/kg can inhibited the changes of WT mice, but have no effect on CSE-KO mice.After cerebral I/R, the impairments of learning and memory abilities in mice were more obvious in CSE-KO mice than that in WT mice. Hyp 50 mg/kg,100 mg/kg markedly inhibited the impairments of learning and memory abilities in WT mice, but have no effect on CSE-KO mice.11. NaHS 2.4 mg/kg,4.8 mg/kg markedly inhibited the decrease of LDH activity and the increase of MDA content in the brain tissue of WT mice after cerebral I/R. It also improved the impairments of learning and memory abilities of WT mice after cerebral I/R.Conclusions1. H2S induced relaxation of BAs is related to inhibiting of RhoA-ROCK pathway.2. H2S, which produced by CSE, is involved in the protective effect of Hyp on cerebral I/R injuries in mice. |
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