Preventive And Therapeutic Effect Of Brozopine On Stroke In Dahl Salt-sensitive Hypertensive Rats

l-3-n-butylphthalide(NBP),as a new drug anti-cerebral ischemic agent for neuroprotective,which was extracted from the seeds of Apium graveolens Linn and approved by the State Food and Drug Administration of China at the end of 2002.It is limited to use in clinical due to its serious adverse reactions,such as the elevated blood glucose,abnormal liver function and gastrointestinal irritation.In order to developed a safer and better efficacy drug,Zhengzhou university synthesized Sodium(±)-5-Bromo-2-(α-hydroxypentyl)benzoate(brandname: Brozopine,BZP),an analogue of NBP.Moreover,BZP has been passed the Phase I clinical trial.Studies have shown that hypertension is one of the most important independently risk factor of stroke.Additionally,stroke is a common complication of hypertension.In this study,we established the model of stroke in Dahl-SS hypertensive rats to explore the preventive and therapeutic effect of BZP on stroke by observing the change of neuroethology,blood pressure and the important organs(such as brains,hearts and kidneys).Part I Establishment the model of cerebral stroke in Dahl salt-sensitive hypertensive ratsObjective Establish the model of stroke with high salt diet in Dahl salt-sensitive(Dahl-SS)hypertensive rats.Methods 12 male seven-week-old Dahl/SS rats and 12 male seven-week-old SS-13 BN rats were randomly divided in control group and model group.Each group contained 12 rats.Control group was SS-13 BN rat(0.3% salt 5L79 diet).Model group was Dahl-SS rats(8% salt AIN-76 a diet).Dahl-SS rats had adapted to the environment for one week and then given the high-salt diet(8% salt AIN-76 a diet).Monitoring body weight,blood pressure and neurological scores once every three days,mortality rates and incidence of stroke were additionally recorded.The content of nitric oxide,activity of catalase and glutathione peroxidase was detected in plasma after 48 days of high salt diet,respectively.Brain,kidney,heart and thoracic aorta were taken out to observe the pathological changes by hematoxylin-eosin staining.Results 1 Effect of high salt diet on body weight High salt diet at day 12,body weight of model group was obviously decreased comparing with controls(P < 0.01).2 Changes on blood pressure after high salt diet Systolic blood pressure(SBP),mean arterial pressure(MAP)and dystolic blood pressure(DBP)were significantly increased(P < 0.01)in model group as compared to the control after 12 days high salt diet.It showed that high salt diet could raise blood pressure in Dahl-SS rats.3 Effect of high salt diet on motor function 3.1 Effect of high salt diet on neurological scores Neurological scores of model group were significantly increased(P < 0.01)with high salt diet for 12 days.With the increasing intake of high salt diet,neurological function scores were markedly increased(P < 0.01)in model group.It showed that high salt diet could induce motor dysfunction in Dahl-SS hypertensive rats.3.2 Incidence of stroke after high salt diet We defined stroke as the comprehensive scores were equal or greater than three points according to the changes of motor function after high salt diet.91.7% rats were developed into cerebral stroke after high salt diet for 12 days.And after high salt diet for 18 days,rats were all developed into cerebral stroke,which illustrated that it was successfully to induce the formation of stroke in Dahl-SS hypertensive rats.4 Effect of high salt diet on fraction surviving A total of 4 rats died in the model group during 48 days high salt diet with a survival rate(66.7%),while survival rate of NBP-K 9.6mg/kg group was 91.7%.Stroke was the main cause of death with the existed of scattered infarction focus near the optic chiasm,together with the obviously neurobehavioral abnormalities.It was illustrated that high salt diet could successfully induce the formation of stroke in Dahl-SS hypertensive rats.5 Correlation of neurological scores with blood pressure It existed a significant correlation between neurological scores(Bederson,Prehensile traction test,Beam walking test and comprehensive evaluation)and blood pressure(P < 0.01)in model group.6 Effect of high salt diet on organs damage Nerve cells of brain were sparse,irregular and even disappeared with the interstitial edema,loose and nucleus shrinkage in model group;glomerular capillary was infiltrated with inflammatory cells,fibrosis,atrophy and disappearance;the intercellular space of cardio myocyte which arranging in disordered was markedly enlarged with the muscle fibers fracture.It suggested that high salt diet could cause damage to important organs in Dahl-SS hypertensive rats.7 Effects of high salt diet on oxidative stress Activities of GSH-PX and CAT were obviously decreased(P < 0.01),while the content of NO was significantly increased(P < 0.01)in model group.The results showed that high salt diet was able to induce oxidative stress.Conclusion High salt diet can induce the model of cerebral stroke in Dahl-SS hypertensive rats.Part II Preventive and therapeutic effect of brozopine on stroke in Dahl Salt-sensitive hypertensive ratsObjective To explore the preventive and therapeutic effect of BZP on stroke in Dahl-SS hypertensive rats,so as to provide relevant evidence for clinical application.Methods 72 male seven-week-old Dahl/SS rats and 12 male seven-week-old SS-13 BN rats were divided into seven experimental groups randomly: control group;model group;BZP-treated groups(0.75,3,12mg/kg);Br-NBP group(10.5mg/kg)and NBP-K group(9.6mg/kg).Each group contained 12 rats.Control group was SS-13 BN rat(0.3% salt 5L79 diet).Other groups were Dahl-SS rats(8% salt AIN-76 a diet).Dahl-SS rats had adapted to the environment for one week and then given the high-salt diet(8% salt AIN-76 a diet)for two weeks.Drug was administrated by caudal vein after two weeks two weeks once a day until 28 days.Monitoring of blood pressure and neurological scores were necessary.The occurrence of cerebral apoplexy symptoms indicated that the model was successfully established,then record the incidence of stroke.Results 1 BZP can increase the body weight Body weight of model group was obviously decreased(P < 0.01)after 3 weeks of high salt diet.While BZP 12 mg/kg increased body weight(P < 0.01)than model group after administrated for 14 days.Moreover,body weight of BZP(0.75,12 mg/kg)were significantly increased(P < 0.01)in dose-dependent manner after administrated for 28 days when compared with model group.2 BZP does not have a decreased effect on blood pressure Systolic blood pressure(SBP),mean arterial pressure(MAP)and dystolic blood pressure(DBP)were significantly higher than normal ranges(P < 0.01)in model group as compared to control group after 2 weeks high salt diet.Blood pressure of BZP 12 mg/kg group was slightly decreased(P < 0.05,P < 0.01)just at 21 days of administration comparing with model group.It meant BZP was not belong to anti-hypertensive drug.3 Effect of BZP on neurological scores 3.1 Neurological function scores Neurological scores of model group were significantly increased(P < 0.01)after high salt diet for 2 weeks.With the increasing intake of high salt diet,neurological function scores were markedly increased(P < 0.01)in model group.Compare with model group,only BZP 12 mg/kg group got lower scores(P < 0.01)after 7 days of administration.Moreover,BZP(0.75,3,12 mg/kg)groups can dose dependently improve the motor dysfunction after 14 days of administration(P < 0.01).It indicated that BZP could significantly improve motor dysfunction of stroke in Dahl-SS hypertensive rats.3.2 BZP can reduce the incidence of stroke With the intake of high salt diet,the incidence of stroke was gradually increased in model group,which tended to higher than other groups.The incidence of stroke was 83.3% in model group with apathetic,lethargy and motor dysfunction(such as circling behavior)after high salt diet for 7 days.Moreover,rats were all developed into cerebral stroke after high salt diet for 14 days.While BZP(0.75,3,12 mg/kg)groups got lower incidence of stroke(58.3%,58.3%,16.7%,respectively)after 7 days treatment.Additionally,BZP 12 mg/kg group has a better effect than NBP-K 9.6mg/kg group and Br-NBP 10.5 mg/kg group.It indicated that BZP could significantly reduce the incidence of stroke in Dahl-SS hypertensive rats.4 BZP can increase the fraction surviving of Dahl-SS hypertensive rats A total of 5 rats died in the model group during 42 days high salt diet with a fraction surviving(58.3%).The fraction surviving of BZP(0.75,3,12mg/kg)group was significantly increased(83.3%,91.7%,100%,respectively).Stroke was the main cause of death with the existed of scattered infarction focus near the optic chiasm,together with the obviously neurobehavioral abnormalities.Moreover,BZP(0.75,3,12mg/kg)group with a longer survival time than model group [(41.25±2.05)day,(41.25±2.60)day,(42±0)day vs(39.42±4.54)day].The results showed that BZP could significantly increase the fraction surviving and prolong the survival time.5 BZP can improve the activity of CAT and GSH-PX,while reduce the content of NO Activities of GSH-PX and CAT were obviously decreased(P < 0.01),but the content of NO was significantly increased(P < 0.01)in model group.Activities of GSH-PX and CAT of BZP 12mg/kg group were obviously increased(P < 0.05,P<0.01),while the content of NO in BZP(3,12mg/kg)groups were significantly decreased(P < 0.01)compare with model group.BZP 12mg/kg has a better effect than NBP-K 9.6mg/kg group and Br-NBP 10.5 mg/kg group(P < 0.05,P < 0.01).The results showed that stroke could reduce body antioxidant capacity and BZP could improve the oxidant damage. 6 Changes of histomorphology Nerve cells of brain were sparse,irregular and even disappeared with the interstitial edema,loose and nucleus shrinkage in model group.Glomerulus was obviously increased the hemorrhagic spot and capillary basement membrane with inflammatory cells infiltration of models.As well as the cardiomyocytes were arranged in a mess and the cell nucleus were arranged irregularly in model group.BZP(0.75,3,12 mg/kg)groups can dose dependently reduce swelling and necrosis of brain than those of model as well as can decrease lymphocyte infiltration of glomerular and can significantly reduce necrosis of cardiomyocytes.Which suggested that BZP had a protective effect on the injuries in brain,heart and kidney after stroke in Dahl-SS hypertensive rats.Conclusions BZP has a significant effect on preventive and therapeutic of stroke in Dahl-SS hypertensive rats.1.BZP can significantly improve motor dysfunction of stroke in Dahl-SS hypertensive rats.2.BZP has a protective effect on the injuries in brain,heart and kidney after stroke in Dahl-SS hypertensive rats.3.The mechanism of BZP may be related to the improvement of antioxidant capacity.