The Protective Effect And Mechanism Of Exogenous Hydrogen Sulfide On Aging Cerebral Ischemia/Reperfusion Injury

Perioperative cerebral ischemia and reperfusion?ischemia/reperfusion,I/R?injury is an important cause of the aging patients mortality and cognitive dysfunction after operation.It's a serious threat to the safety and quality of elder patients'life,and imposes a heavy burden to the society and the family as well.However,the underlying mechamism has not been elucidated.Neuroprotective agents are among one of the most promising approaches to I/R injury and are applied to protect ischemic neurons in the brain from irreversible injury.However,although preclinical studies demonstrated numerous drugs are effective for treating I/R injury in rodents,subsequent clinical trials have been frustrating,and few of the agents have been proven effective.Hydrogen sulfide?H₂S?,as the third star gas signaling molecule,has a protective effect against I/R injury in the important organs such as heart,liver,kidney and lung.Previous studies have found that single administration of exogenous H₂S before cerebral ischemia can reduce cerebral infarction volume and improve survival rate.In addition,in the oxygen glucose deprivation?OGD?model of rat hippocampal neurons,administration of exogenous H₂S after the recovery of oxygen glucose can reduce the apoptosis of neurons and increase the activity of surviving neurons.However,there are few reports on the therapeutic effect of exogenous H₂S on central nervous system I/R injury in aged animals,and the mechanism is still unclear.On the basis of our preliminary studies,the project used the middle cerebral artery occlusion-reperfusion?MCAO?model and the OGD models of hippocampal neurons in aged SD rats to further probe the protective effect and underlying machanisms of exogenous H₂S on the cerebral I/R injury in the aged rats.This study was mainly carried out in three aspects.The first part,the MCAO model of SD rats was established to study the mechanism of neuroprotective effects of different concentrations of H₂S donor NaHS on brain I/R injury.The second part,the establishment of the youth and old age OGD model of SD rat hippocampal neurons,different concentration of H₂S donor NaHS elderly neurons to oxygen deprivation sugar injury protection,and compared with young group.The third part,on the basis of the second part,studies the potential mechanism and related molecular pathways of H₂S donor NaHS on the protective effects of damaged old hippocampal neurons.Part I Effects of different concentrations of exogenous hydrogen sulfide on cerebral ischemia/reperfusion injury in adult ratsObjective:To investigate the effects of different concentrations of exogenous H₂S donor NaHS on cerebral ischemia/reperfusion injury in adult rats and its possible mechanism.Methods:?1?The optimal therapeutic dose of NaHS:healthy adult SD rats were selected.The MCAO model was established by the method of internal carotid artery occlusion.After 2 hours of ischemia,reperfusion was performed.The rats were randomly divided into 5 groups:sham operation group,I/R group,low dose NaHS?1.25 mg/Kg?,middle dose NaHS?2.5 mg/Kg?and high dose NaHS?5 mg/Kg?treatment group,16in each group.Two hours after the start of reperfusion,the above three groups of experimental mice were injected intraperitoneally with corresponding concentrations of NaHS.Sham-operated group and I/R group were injected intraperitoneally with equal doses of normal saline.Seven days later,neurological severity scores?NSS?were performed on each group of rats.?2?Validation of the therapeutic effect of NaHS on cerebral ischemia-reperfusion injury:Rats were killed and observed using 2,3,5-three phenyl tetrazolium chloride?TTC?staining The volume and proportion of cerebral infarction in cerebral ischemia-reperfusion rats were calculated.The number and proportion of apoptotic neurons in ischemic peripheral area were detected by TUNEL staining.?3?Possible mechanism of NaHS intervention on cerebral ischemia-reperfusion injury:Oxidative stress in the homogenate supernatant of cerebral ischemic tissue was detected by spectrophotometry including malonaldehyde?MDA?,superoxide Superoxide dismutase?SOD?and glutathione peroxidase?GSH-Px?.Results:?1?Compared with untreated I/R group,the symptoms of neurological deficit in the three treatment groups of 1.25,2.5 and 5 mg/Kg NaHS intervention gradually reduced,but only 2.5 mg/Kg and 5 mg/Kg NaHS treatment group Compared with untreated I/R group,there was significant difference?all P<0.05?,5 mg/Kg NaHS treatment group effect is more significant.?2?The percentages of infarction volume of I/R rats in three treatment groups decreased gradually with 1.25,2.5 and 5 mg/Kg NaHS intervention.The percentages of apoptotic neurons?TUNEL-positive cells?But only 2.5 mg/Kg and 5 mg/Kg NaHS treatment group compared with untreated I/R group were significantly different?P<0.05?,5 mg/Kg NaHS treatment group effect is more significant.?3?The activities of superoxide dismutase?SOD?and glutathione peroxidase?GSH-Px?in the three NaHS groups were significantly increased compared with those in untreated I/R group,while three groups The content of malondialdehyde?MDA?in NaHS group was significantly lower than that in non-treated I/R group,but only three of 5 mg/Kg NaHS-treated group and untreated I/R group had Significant difference?all P<0.05?,indicating that NaHS intervention reduces the I/R induced rat brain oxidative stress levels increased.Conclusion:Exogenous H₂S donor NaHS has a protective effect on cerebral ischemia-reperfusion injury in adult rats,and the protective effect is most obvious when the dosage is 5 mg/Kg.This neuroprotective effect reduces the I/R ratio The level of oxidative stress is increased.Part II Effects of different concentrations of exogenous hydrogen sulfide on the cultured hippocampal neurons in old ratsObjective:To investigate the effects of different concentrations of exogenous H₂S donor NaHS on primary cultured hippocampal neurons in old rats with oxygen-glucose deprivation.Methods:?1?Discussion on optimal therapeutic dose of NaHS:Cultured hippocampal neurons were subjected to OGD first,and the primary culture medium of hippocampal neurons was replaced by a solution of pre-heated Earle's balanced salt solution?EBSS?95%N₂ and 5%CO₂ at 37°C for 2 hours.NaHS was added to cultured hippocampus of young and aged rats at concentrations of 10,50,250 and 1,000?M,respectively,for7 days.After that,the metabolic activity of neurons cultured in each group was measured by MTT method?3,4,5-Dimethyl-2-thiazolyl?-2,5-diphenyl-2-H-tetrazolium Bromide?Lactate Dehydrogenase,LDH?release assay to evaluate the cytotoxicity and compare the cell viability and cytotoxicity differences between the hippocampal neurons in the aged group and the young group.?2?Neuronal efficacy of NaHS on oxygen-glucose deprivation injury:neurons cultured in vitro for 7 days were stained with immunohistochemical MAP-2 antibody and examined by scanning confocal microscopy to examine neurite germination and growing situation.Then using Lipofectamine 3000,mCherry-actin plasmid was transfected into hippocampal neurons to further analyze the effect of NaHS on the dendrites of OGD-injured hippocampal neurons.Results:?1?Compared with untreated I/R group,the symptoms of neurological deficit in the three treatment groups of 10,50,250?M NaHS intervention gradually reduced,but only 50?M and 250?M NaHS treatment group and no compared with I/R group?all P<0.05?,250?M NaHS treatment group was more effective.The cell viability of hippocampal neurons from young rats and aged rats gradually increased with the increase of NaHS concentration from 0 to 250?M experimental groups at different concentrations.The cytotoxicity decreased gradually with the increase of NaHS concentration in each group.250?M NaHS showed the best protection,while 1,000?M NaHS showed significant cytotoxicity with a corresponding decrease in cell viability.?2?NaHS treatment?250 mM?protected both young and aged hippocampal neurons,as indicated by restoring number of primary dendrites by 43.9 and 68.7%,number of dendritic end tips by 59.8 and 101.1%,neurite length by 36.8 and 66.7%,and spine density by 38.0 and 58.5%in the OGD-damaged young and aged neurons,respectively.Although hippocampal neurons in older rats are more vulnerable than hippocampal neurons in young rats,our study found that NaHS intervention still provided significant neuroprotection in aged neurons.Conclusion:Our results demonstrated that exogenous H₂S donor NaHS has potent protective effects against neuron injury induced by OGD in both young and aged hippocampal neurons.Particularly,250 mM of NaHS provided the best protection among groups of different concentrations ranging from 0 to 250 mM,whereas 1,000mMof NaHS started to show cytotoxicity and reduced cell viability.Part III The mechanism of exogenous hydrogen sulfide on the cultured hippocampal neurons in old ratsObjective:To investigate the protective mechanism of 250?M exogenous H₂S donor NaHS on primary cultured hippocampal neurons in oxygen-deprived aged rats.Methods:?1?Change of growth-associated protein-43?GAP-43?:Western blotting was used to detect the effect of GAP-43 expression under 250?M NaHS on the hippocampal neurons of young and old rats cultured in vitro for 3 days and 7 days respectively.?2?Detection of change in activity associated with oxidative stress:SOD activity was measured using a glutathione peroxidase assay kit?Cayman Chemical,UK?.MDA levels were determined using a thiobarbituric acid reactive substances?TBARS?assay.NO levels were measured using Griess reagent in conjunction with sodium nitrite as a standard spectrophotometer.Glutathione?GSH?levels were determined by high performance liquid chromatography and fluorescence detection,and the reduced GSH was subtracted from the GSH value to obtain the level of oxidized glutathione?GSSG?.F2-isoprostanes were quantified by gas chromatography/mass spectrometry using a stable isotope dilution method.?3?To detect the changes of related protein levels in ERK signal pathway:The expression of phosphorylated ERK1/2 and PP2A and PKA in young and old hippocampal neurons damaged by OGD were detected by Western Blotting and quantified by GAPDH.Results:?1?After 3 days and 7 days in vitro,GAP-43 expression in hippocampal neurons of OGD-treated young and old rats were significantly reduced compared with the corresponding control group,especially in hippocampal neurons of aged rats.However,250?M NaHS significantly reversed the down-regulation of GAP-43 expression,and the effect of NaHS on GAP-43 expression was more obvious after 7 days of in vitro culture.?2?NaHS intervention at 250?M increased the relative SOD activity and GSH level in hippocampal neurons of OGD-treated aged rats and young rats,and inhibited the activities of MDA and NO in young and old hippocampal neurons induced by OGD Elevated levels of hydrogen peroxide,GSSG and F2-isoprostane.?3?250?M NaHS increased the phosphorylation of ERK1/2 in hippocampal neurons of young and old rats inhibited by OGD treatment,inhibited the upregulation of PP2A level induced by OGD treatment,while the level of PKA was not affected by OGD Or NaHS intervention.Conclusion:250?M NaHS significantly inhibited the down-regulation of GAP-43 and the increase of oxidative stress in hippocampal neurons of OGD-induced young and old rats.In addition,ERK signaling pathway may be involved in neuronal protection of NaHS.