Effect Of Hydrogen Sulfide On Early Brain Injury After Subarachnoid Hemorrhage And Its Mechanism

Part Ⅰ: Biosynthesis of endogenous hydrogen sulfide and its effect on early brain injury after subarachnoid hemorrhage in ratsObjective:To investigate the biosynthesis of endogenous hydrogensulfide(H2S) and its effects on the early brain injury(EBI) after subarachnoid hemorrhage(SAH) in rats.Methods: The total of 96 adult male SD rats were randomly allocated into a normal group,a SAH group,a low dose Na HS group and a high dose Na HS group,The rat model of SAH was induced by injecting autologous blood into prechiasmatic cistern,We detected H2 S content in plasma and brain tissue,located cystathionine-β-synthase(CBS) and 3-merecaptopyruvate sulfurtransferase(3-MST) by immunofluorescence,detected the expression of CBS and 3-MST by western blot,evaluated by measuring MDA,ROS content and SOD,GSH-PX activity in the fresh brain tissue,and then we observed neurologic scoring,brain water content,blood brain barrier permeability by Evans blue staining,apoptosis cells by TUNEL.Results: CBS and 3-MST colocated in Neurons. In the SAH group,H2 S content was obviously reduced,CBS expression was markedly down-regulated,3-MST was slightly up-regulated.Brain edema,BBB damage,apoptosis cells,oxidative stress and neurological score strikingly deteriorated.Na HS can obviously make up the deficiency of endogenous H2 S and up-regulate CBS and 3-MST expression.Na HS can markedly resist the adverse effects induced by SAH in a certain dose-dependent manner.Conclusion:The CBS /H2 S and 3-MST/H2 S pathways are the biosynthesis of endogenous H2 S,H2S plays a neuroprotective function in a dose-dependent manner throughalleviating EBI.Part II: Effect of hydrogen sulfide on acute cerebral vasospasm induced by subarachnoid hemorrhage in ratsObjective: To study the effects of hydrogen sulfide(H2S) on acute cerebral vasospasm(a CVS) after subarachnoid hemorrhage(SAH) in rats and explore its possible mechanism.Methods: The total of 48 adult male SD rats were randomly allocated into a normal group,a SAH group,a low dose Na HS group and a high dose Na HS group.The rat model of SAH was induced by injecting autologous blood into prechiasmatic cistern.Vasospasm was evaluated by H&E staining,endothelial cell apoptosis was measured by TUNEL,expression of Caspase 3 was detected by western blot at 48 h after surgery.Results:Animal experiment showed that A2 segment of the anterior cerebral artery and M1 segment of the middle cerebral artery obviously contracted after SAH,apoptosis index of cerebral artery endothelial cells significantly increased.Na HS can dilate cerebral artery,inhibit endothelial cell apoptosis.Meanwhile,the effect of high dose is better than the low.Conclusion: H2 S can effectively expand cerebral vasospasm after SAH,its protective mechanism may be through inhibiting vascular endothelial cell apoptosis.Part III: Effect of hydrogen sulfide on subarachnoid hemorrhage induced by oxygenated hemoglobin in vitroObjective: To study the effect of hydrogen sulfide(H2S) on subarachnoid hemorrhage(SAH) induced by oxygenated hemoglobin(Oxy Hb) and its possiblemechanism in vitro.Methods: The vitro model of SAH was imitated by simulating brain microvascular endothelial cells(BMVECs) and Neurons respectively with 10μM Oxy Hb. We observed the survival rate of the cells,reactive oxygen species(ROS) content and Caspase 3 expression by western blot after Na HS(15μM or 30μM) pretreatment about 24 h.Results: The survival BMVECs and Neurons were obviously decreased,the ROS content was significantly increased,and the expression of Caspase 3 was remarkedly upregulated in Oxy Hb treated group.With the Na HS pretreatment, the survival BMVECs and Neurons were obviously increased, the ROS content was significantly decreased,the expression of Caspase 3 was remarkedly decreased.Conclusion: H2 S can inhibit ROS content induced by Oxy Hb, which can aggravate the apoptosis of BMVECs and Neurons.