The Neuroprotective Effects Of Hydrogen Sulfide On Mice Intracerebral Hemorrhage Model

Objective:Intracerebral hemorrhage（ICH）means non-traumatic parenchymal hemorrhage and is associated with extremely high rates of mortality and morbidity.ICH contribute to a substantial number of deaths and cases of permanent disability,which is caused by secondary brain injury.Protecting neurons is an important therapeutic target after brain injury.Hydrogen sulfide（H₂S）is an endogenous gaseous signaling molecule which plays an important physiological and pathological role in the central nervous system.In this study,we will explore the role and mechanisms of H₂S on neurological function after intracerebral hemorrhage in mice and whether the protective effect of H₂S is related to neuronal cell death（apoptosis and autophagy）.Methods:The procedure for inducing ICH by collagenase injection in mice has been described previously.In order to investigate the effect of H₂S on neurological function after intracerebral hemorrhage,water content of brain tissue was detected at different concentrations（1-100μmol/kg）of H₂S donor（NaHS）at different time points（pre-injury 30min,1 h,2 h,4 h,6 h after injury）.The expression of endogenous H₂S synthesis enzyme cystathionine-β-synthase（CBS）,3-mercaptopyruvat sulfur transferase（3-MST）,apoptosis-related proteins and autophagic related proteins in brain tissue were detected using Western blot.We use cerebral hemorrhage volume to evaluate histopathological changes.The behavior tests（motor function and Morris water maze test）were performed for detecting whether H₂S had a protective effect in the treatment with brain injury.To further explore the neuroprotective mechanism of H₂S after brain injury,loss of plasmalemma integrity was evaluated by intraperitoneal injection of propidium iodide（PI）1 h before sacrificing the animal.We investigated NaHS whether through endogenous hydrogen sulfide pathway in the brain tissue function using O-（Carboxymethyl）hydroxylamine hemihydrochloride（AOAA）,the inhibitor of CBS.Results:（1）Compared with sham group,the amount of water content of the injured peaked 1 d.Pretreatment 30 min before ICH by NaHS with dosage of 25μmol/kg showed the most obvious improvement in cerebral edema.Compared with ICH group,pretreatment with NaHS can significantly ameliorate motor deficits and reduce cerebral hemorrhage volume.（2）Comparison with ICH group,pretreatment with NaHS（25μmol/kg）influenced the expression of CBS and reduced the number of PI-positive cells at 1d after ICH.H₂S reverses ICH-induced caspase-3 cleavage and Bcl-2 decline.H₂S also inhibits ICH-induced autophagic activation,which suppressed ICH-induced the increase of P62 and reversed TBI-induced LC3II and Beclin-1 increase.（3）There were no significant differences between ICH group and ICH+NaHS+AOAA group in behavioral deficts,cerebral hemorrhage volume,PI-positive cells and the level of apoptosis-related proteins and apoptosis related proteins.Conclusion:（1）H₂S pretreatment had reduced brain edema,improved motor performance and ameliorated performance in Morris water maze test after ICH,and H₂S may have a therapeutic potential against neuron damage;（2）The protective effect and therapeutic potential of H₂S in the treatment of brain injury and the protective effect against ICH may be associated with suppressing cellular plasmalemma integrality and regulating apoptosis and autophagy.（3）H₂S played an important role in neuroprotection though CBS-H2S pathway.