Role Of H₂S-RhoA-ROCK Pathway In Anoxic Injury Of Rat Cerebrovascular Endothelial Cells And H₂S-mediated Protection Of Total Flavonoids Of Rhododendra Against Cerebral Ischemia

Stroke is a serious global public health problem with high mortality and disability.However,there is no effective treatment.Endogenous hydrogen sulfide?H₂S?and RhoA-ROCK pathways are promising new targets for cardiovascular drugs,but their interactions,especially in ischemic brain injury,has not been reported.Total flavones of rhododendron?TFR?is an effective component of flavonoids extracted from azalea.It has a protective effect on cerebral ischemic injury,but its mechanism of action is still unclear.In this paper,a hypoxia model was established to observe the dynamic changes of H₂S/CSE?NO/eNOS and RhoA/ROCK channels through the primary cultured rat cerebral vascular endothelial cells.Secondly,the interaction of H₂S and RhoA-ROCK pathways in endothelial cells was observed.Build small mouse cerebra ischemia model by ligation of bilateral common carotid artery,using laser speckle imaging system to observe the cerebral blood flow before and after building.We observe the effect of on cerebral blood flow,and discuss whether the possible protective mechanism is involved in H₂S.Purpose:1.Dynamic changes of H₂S-CSE and RhoA-ROCK pathways in cerebral hypoxic injury;2.The interaction of H₂S and RhoA-ROCK pathways in rat brain vascular endothelial cells;3.To study the relationship between the effect of TFR anti cerebral ischemia with H₂S.Methods:1.Primary cultured rat vascular endothelial cells were digested with collagenase and identified by immunofluorescence method:2.The CCK-8 assay showed changes in the vitality of rat brain vascular endothelial cells at different time of hypoxia.3.G-LISA method to detect RhoA activity;4.Methylene blue spectrophotometric to detect the content of H₂S;5.Nitrate reductase assay NO content;6.Western blot assay was used to detect the expression of CSE?eNOS?ROCK₁ and ROCK₂ protein in rat vascular endothelial cells.7.Bilateral common carotid artery ligation establish mouse acute cerebral ischemia model;8.Laser speckle blood flow detection system detectd the changes in cerebral cortex blood flow before and after ligation of bilateral common carotid arteries;9.LDH activity was measured,MDA content was observed in mice with cerebral ischemic injury.Results:1.The CCK-8 assay showed that the activity of rat vascular endothelial cells gradually decreased with the prolongation of hypoxia time.The cell viability of hypoxia 8 h decreased by 39.2±1.6%,and the cell viability after hypoxia 24 h decreased by 60.7±2.2%.2.Rat brain vascular endothelial cells were hypoxic for 0?1?2?4?8 and 24 hours respectively.H2 S content changed first after 1h of hypoxia,and the expression level of CSE protein decreased significantly after hypoxia for 4h;hypoxia 4 h.After the NO content was significantly reduced,the expression level of e NOS protein was significantly down-regulated after 8 h of hypoxia.On the other hand,compared with the gradual decrease of H2 S and NO content,the Rho A activity increased significantly with the prolongation of hypoxia time after 8 h of hypoxia.And after 8 hours of hypoxia,ROCK1 and ROCK2 protein expression increased significantly.3.Adding exogenous H2 S donor Na HS?0-100 ?M,30min?to rat brain vascular endothelial cells can significantly inhibit Rho A activity of endothelial cells,suggesting that exogenous H2 S can inhibit Rho A activity;otherwise,add CSE inhibitor PPG?10m M?after 4h,Rho A activity was significantly increased,suggesting that endogenous H2 S can inhibit Rho A activity;4.The content of H2 S increased after adding Rho A activity specific inhibitor C3 transferase?25 ?g/ml,8h?to rat brain vascular endothelial cells,whereas the content of H2 S decreased after adding U46619?10-8 mol/L for 30min?.Compared with the normal group,the CSE protein level was significantly up-regulated after C3 transferase inhibited Rho A activity.5.Cerebral blood flow,brain LDH activity decreased significantly,and MDA content increased significantly after cerebral ischemic injury;TFR 40 and 80 mg/kg can inhibit the cerebral blood flow and cerebral ischemia caused by ischemic injury in different degrees.LDH activity decreased and MDA content increased in tissue;6.After CSE enzyme inhibitor PPG was pretreated in mice,TFR 40 and 80 mg/kg could not significantly inhibit cerebral blood flow,LDH activity and MDA content in brain tissue caused by ischemia injury.Conclusions: 1.H2S/CSE pathway changes first after endothelial cell hypoxia;2.H2 S and Rho A/ROCK pathway in endothelial cells have mutual inhibitory effect;3.TFR has anti-mouse cerebral ischemia injury and its possible mechanism is related to H2 S.