Protective Effects Of Human Recombinant Neutrophil Inhibitory Factor And Hirulog Hybrid (TNHH) On Cerebral Ischemic Injury In Rats

ObjectiveCerebrovascular disease is one of the major three causes of death in human disease with high mutilated rate and fatality rate,threatening to people's health badly.Ischemic cerebrovascular disease(ICVD) is more usually in clinic,accouting for 60%～70%in whole patients suffering cerebrovascular disease.It can be divided into global brain ischemia and focal brain ischemia.The pathogenesy of cerebrovascular disease is not absolutely clear. Study shows that cerebral edema and excessive inflammatory reaction play important part in cerebral ischemia and postischemic reperfusion damage.After cerebral ischemia,plenty of thrombin activation in region is the major cause of brain edema.Large amount of neutrophil concentrating in capillary vessels and infiltrating into brain tissue can cause inflammatory cascade reaction.Among this process,oxygen free radical,cytokines,product of lipid metabolism and proteolytic enzyme are produced.Inflammatory reaction plays a key role in acute cerebral ischemia and secondary brain injury caused by postischemic reperfusion.It is important to prevent brain edema and excessive inflammatory reaction.By doing these we can depress the fatality rate of ischemic cerebrovascular diseases and improve the prognosis.Human recombinant neutrophil inhibitory factor and hirulog hybrid(TNHH),a chimeric protein of recombined human neutrophil inhibitory factor(NIF) and hirulog,possesses the effects of inhibiting adherence activation of neutrophilic leukocyte and inhibiting thrombin activity,planning to treat acute cerebral arterial thrombosis.Its mechanism of action may be, on one hand,inhibiting the adhesion,migration activation and release of neutrophil,to lessen the inflammatory reaction in acute cerebral arterial thrombosis;on the other,by combining to the inactive area of thrombin,inhibiting the brain edema caused by thrombin.In this experiment,the focal cerebral ischemia model and the global cerebral ischemia model were prepared to observe the effects of TNHH on brain ischemia by detecting the neurological deficit score,infarction area,brain water content,the levels of cytokine such as interleukin-1β(IL-1β),interleukin-10(IL-10)and tumor necrosis factor-α(TNF-α),and the anti-oxygenized indexes containing superoxide dismutase(SOD) and malonaldehyde (MDA)in rats.By doing these,we tried to approach the protective mechanism of TNHH to brain ischemia injury and broaden the spectrum of drugs which can be used in treating ischemic cerebrovascular disease.MethodsPersistent focal cerebral ischemia experiment.One hundred adult male Wistar rats(body weight:250～310g) were used in the experiment.Animals were randomly divided into five groups:blank group,sham group,ischemia group,TNHH group and vehicle groups.After one week feeding in laboratory condition for adaptability,middle cerebral artery occlusion (MCAO)was used to induce focal cerebral ischemia.One hour after the model preparation, drug or agent were administered throught vena caudalis injection.Eight hours after the occlusion,the administration was taken for the second time.For blank,sham and ischemia rats,normal sodium were given by intravenous injection at 0.5ml in each animal.TNHH solution was given to rats in TNHH group at dose of 6.75 mg/kg.Vehicle group rats received 4%mannit(10mM PB,pH7.0) 0.5ml which served as vehicle.Twelve hours after the model preparation,we evaluated the neurobehavioral score of rats,measured the infarction area of brain and observed the histopathologic changes of ischemic brain tissue.The expression of IL-10 in brain tissue and the levels of TNF-αand IL-1βin serum were measured.Global brain ischemia experiment.One hundred and fifty female and male Wistar rats (body weight:180～220g) were used in the experiment.Animals were randomly divided into five groups:blank group,sham group,ischemia group,TNHH group and vehicle groups. After one week feeding in laboratory condition for adaptability,modified four-vessel occlusion was used to induce global cerebral ischemia.The treatment for every group was same to that in experimentⅠ.The administrations were carried out four times after model preparation:the first time was one hour after reperfusion,then eight hour later taken the second injection.The third and fourth injections were carried out in the second day after the experiment with an 8-hour interval.After reperfusion for 48 hours,we observed the pathologic histological changes and calculated the brain water content in rats.The levels of MDA,SOD,MPO,TNF-αand IL-10 in brain,as well as contents of TNF-αand IL-1βin serum were measured.ResultsFor persistent focal cerebral ischemia,TNHH at dose of 6.75 mg/kg via intravenous injection could significantly decrease neurological deficit scores,reduce the percentage of infarction area,and alleviate the histopathological changes in the ischemic cerebral hemisphere.The expression of IL-10 in brain tissue and the levels of TNF-αand IL-1βin serum were also down-regulated.For global cerebral ischemia,TNHH at dose of 6.75 mg/kg via intravenous injection could significantly reduce brain edema,decrease the levels of MDA,MPO and TNF-αin brain tissue as well as the levels of TNF-αand IL-1βin serum.Furthermore,it could elevate the SOD activity in brain and had no influence on IL-10 expression in brain tissue.ConclusionThe finding suggests that TNHH might provide neuroprotective effects on rats which encountered persistent focal cerebral ischemia and global cerebral ischemia through its anti-inflammatory effect,antioxidant activity and anti-brain edema action.