| Hydrogen sulfide(H2S) is recently regarded as the third gaseous signal molecule following NO and CO. Relaxation of H2 S has been confirmed on peripheral arteries of multi-species in different types or different size, but there is no report pay attention to relaxation of H2 S on cerebral artery. Injury on endothelium of cerebral vessel is an important event in pathophysiological processes of cerebral ischemic disease. The protective effects of H2 S on cerebral vessel endothelial cells and the underlying mechanisms are unclear. Total flavones of rhododendra(TFR) has good protective effects against ischemic cerebral injury, but whether or not the action of TFR is involved with H2 S is unclear. By using si RNA gene silence and other techniques, we propose to observe the role of H2 S in endothelium-derived relaxation of cerebral artery and the mechanism. We also establish oxygen deprivation and reoxygenation model in rat cerebral micro-vascular endothelial cells to observe the protective effects of H2 S on cerebral vessel endothelium and to clarify the relationship between the action of H2 S and SIRT6. In this study, we also investigate the relationship between the protective effects of TFR against cerebral ischemic cerebral injury and H2 S in CSE-si RNA transfection in vivo rat model and CSE-KO mouse model.Purpose: 1. To observe the role of H2 S in endothelium-derived relaxation of cerebral artery and the ion mechanism.2. To observe the protective effects of H2 S on injury induced by oxygen deprivation and reoxygenation in rat cerebral vessel endothelial cell and to clarify the relationship between the action of H2 S and SIRT6. 3. To observe the relationship between protective effects of TFR against ischemic cerebral injury and H2 S.Methods: 1. Establishing rat CSE-si RNA transfection model in vivo, q-PCR was utilized to detect the expression of CSE-m RNA in blood vessels, and the automatic microplate reader was used to detect production of H2 S in cerebral vessel from rats. 2. Using angiotasis experiment technique, to observe the relaxation induced by ACh, H2 S substrate L-Cys or TFR on cerebral vessels from rats; to observe the contribution of endothelium removal or PPG(CSE inhibitors) on the vasodilation induced by them; to observe the relaxation induced by exogenous H2 S donor Na HS on cerebral vessels from rats; to observe the contribution of Apamin(SKCa channel blockers), Ch Tx(IKCa channel blockers), Iberiotoxin(BKCa channel blocker) and Glibenclamide(KATP channel blocker) on Na HS-induced vasodilator response. 3. Using angiotasis experimental technique, to observe the change in relaxation response induced by ACh, L-Cys and TFR on cerebral vessels from CSE-si RNA transfection rats in vivo; to observe the change in relaxation induced by TFR on the basilar arteries and aortas from CSE-KO mice. 4. Establishing focal cerebral ischemia-reperfusion injury(I/R) rat models transfected by CSE-si RNA in vivo, to observe the effects of TFR on brain function scores, percentage of infarct volume and H2 S production in cerebral vessels. 5. Establishing focal cerebral ischemia-reperfusion injury(I/R) model of CSE-KO mice, to observe the effects of TFR on brain function scores, percentage of infarct volume and H2 S production in cerebral vessels.6. Establishing oxygen deprivation and reperfusion(OGD/R)-stimulated b End.3 cells model, by detecting LDH, SOD, CAT, ROS and cell viability, to verify the protective effects of H2 S on OGD/R endothelial cells, and to determine expression of SIRT6 in OGD/R endothelial cells, and to observe the effects of H2 S or TFR on SIRT6 expression of OGD/R endothelial cells.Results: 1. When rats transfected by CSE-si RNA in vivo after 48 h, compare to Sham group, negative control group and the transfection reagent group, expression of CSE-m RNA in cerebral vessel decreased significantly and the production of H2 S in cerebral vessels and aortas also significantly reduced. 2. In the angiotasis experiment, ACh(10-8~10-5.5mol/L) induced concentration dependent relaxation on basilar artery(BA) or middle cerebral artery(MCA) from rats, the maximum relaxation rate(Emax) respectively were 72.88±5.02% and 69.84±7.45%; After application of PPG, Emax decreased significantly; after endothelium removal, the relaxation response disappeared. 3. In the angiotasis experiment, L-Cys(10-5~10-2.5mol/L) induced concentration dependent relaxation of BA and MCA from rats, Emax were 77.15±6.15% and 80.37±5.36% respectively; after application of PPG or endothelium removal, Emax decreased significantly. 4. In the angiotasis experiment, Na HS(10-5~10-2.5 mol/L) induced concentration dependent relaxation on BA and MCA from rats, Emax were 86.45±6.12% and 89.72±6.53% respectively; After endothelium removal, there was no change in the relaxation induced by Na HS; After application of Apamin(SKCa channel blocker), Ch Tx(IKCa channel blocker) and Glibenclamide(KATP channel blocker), Emax declined respectively with different degree; After application of Iberiotoxin(BKCa channel blocker), there was no change in the relaxation induced by Na HS.5. In the angiotasis experiment, ACh-induced concentration dependent relaxation of BA, MCA and aortas from CSE-si RNA transfection rats attenuated; L-Cys induced concentration dependent relaxation of BA and MCA from CSE-si RNA Transfection rats also weakened; 6. OGD/R decreased viability of endothelial cells and increased LDH; pretreatment with Na HS(25~100u M) concentration-dependently attenuated cell viability and LDH elevation; pretreatment with PPG aggravated cell viability and LDH elevation. 7. OGD/R decreased SOD and CAT in endothelial cells, as well as increased ROS in endothelial cells; Pretreatment with Na HS(50u M) attenuated OGD/R-induced SOD or CAT reduction and ROS elevation; pretreatment with PPG aggravated OGD/R-induced SOD or CAT reduction and ROS elevation. 8. OGD/R induced down-regulated expression of SIRT6 in endothelial cells; Pretreatment with Na HS(50u M) or TFR(900mg/L) up-regulated the expression of SIRT6 in endothelial cells. 9. In the angiotasis experiment, TFR(11~2700 mg/L) induced concentration dependent relaxation of BA and MCA from rats, Emax were 65.76±5.84% and 67.09±4.93% respectively; after application of PPG, Emax decreased significantly.TFR induced concentration dependent relaxation on BA and MCA from CSE-si RNA transfection rats also weakened. TFR induced concentration dependent relaxation on BA or MCA from CSE-KO mouse disappeared. 10. The amount of H2 S produced in cerebral vessel of rats which pre-incubated with TFR increased significantly, the effect of TFR inhibited by PPG significantly. 11. Ischemia-reperfusion(I/R) resulted in neurological function scores or percentage of infarct volume elevation and production of H2 S reduction in cerebral vessels; Pretreatment with TFR attenuated these changes triggered by I/R; CSE knockdown aggravated these changes triggered by I/R; In CSE-si RNA transfection rat model in vivo, the action of pretreatment with TFR improving neurological scores, percentage of infarct volume and production of H2 S weakened. 12. I/R leaded to neurological function scores or percentage of infarct volume elevation and production of H2 S reduction in cerebral vessels; Pretreatment with TFR attenuated these change triggered by I/R; CSE knockout aggravated these change triggered by I/R; In CSE-KO mouse model, the action of pretreatment with TFR improving neurological score, percentage of infarct volume and production of H2 S disappeared.Conclusion: 1. Endogenous H2 S was involved in ACh-induced endothelium-dependent relaxation response on BA or MCA from rats. 2. Exogenous H2 S dilatated BA or MCA from rats, SKCa, IKCa and KATP channels had been involved in the action of exogenous H2 S. 3. Exogenous H2 S had protective effects on injury induced by oxygen deprivation and reperfusion in cerebral vascular endothelium, which may be linked to the up-regulation of SIRT6. 4. TFR dilatated BA or MCA from rats, which may be related to the CSE/H2S/ SIRT6 pathway. 5. TFR had protective effects against cerebral ischemia-reperfusion injury, which may be linked to elevated production of H2 S in cerebral vessels. |
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