| Part I The protective Effect of Yulangsan Plysaccharide on Acute Convulsion Induced by Pentaerytetrazole in MiceObjective: To explore the protective effect of Yulangsan Plysaccharide(YLSP)on acute convulsion induced by Pentaerytetrazole(PTZ)in mice.Methods: PTZ was used to establish acute convulsive mice model.60 male Kunming mice were randomly divided into control group,PTZ model group,valproate sodium group(250mg/kg),low(300mg/kg),medium(600mg/kg)and high(1200mg/kg)YLSP group.Animals were fed for 3 days before experiment.Then drug treatment mice were intragastrically administered drugs for 7 days(at8:00 in the morning)and control group mice were received saline.After administration 1 hour on day 7,except the control group mice,all group mice were given PTZ by intraperitoneal injection(ip.,56mg/kg)to establish convulsive model.After PTZ injection,mice were placed in a transparent box and observed for 30 minutes.The paroxysnal grade,delitensence and times of epileptic seizure were recorded.After the behavioral observation,the Morriswater maze(MWM)experiment was performed.Before formal experiment,mice were allowed to swimming for 60 s in order to adaptive environment.The test including navigation phage with a duration of 5-day training and 1-day probe phage.The escape latency,swimming distance and crossing times were recorded.Samples were collected after the experiment for further detection.Results: The experimental results showed that compared with the control group,the mice in the model group all showed convulsions with different degrees,suggesting that the model was successfully established.Compared with the model group,the high and medium doses of YLSP could significantly reduce paroxysnal grade(P﹤0.05 or P﹤0.01)and prolong delitesence(P﹤0.05 or P﹤0.01).High and low doses of YLSP can significantly reduce times of epileptic seizure(P﹤ 0.05 or P﹤ 0.01).However,there was no significant difference in low YLSP dose group(P>0.05).Morris water maze test showed that compared with the control group,the mice in the model group mice showed significantly longer time to find the platform(P ﹤ 0.01)and more total swimming distance(P<0.01).Compared with model group,there were significant differences in escape latency and total swimming distance in the high dose group of YLSP(P<0.01).The escape latency and total swimming distance in the middle and low dose groups of YLSP were also showed significance(P<0.05),and the low dose group of YLSP had significantly different(P <0.01).In the probe test,there was not statistically significance in the result of crossing times among groups(P>0.05).Conclusion: YLSP can inhibit acute convulsion induced by PTZ,reduce paroxysnal grade,prolong delitesence,reduce times of epileptic seizure and obviously improve spatial learning ability of mice.Part II The effect and mechanism of Yulangsan on pentylenetertazole-induced epilepsy model of mice.Objective: To explore the effect of Yulangsan polysaccharide(YLSP)on pentylenetertazole(PTZ)-induced epilepsy model of mice.Methods: PTZ was used to establish a kindling model.60 male Kunming mice were randomly divided into the control group、PTZ model group、valproate sodium group(250mg/kg)、 low(300mg/kg)、 medium(600mg/kg)and high(1200mg/kg)dose YLSP group.They were fed for 3 days before experiment.Model mice were received PTZ intragastrically(37.5mg/kg,i.p.)with one-day interval manner for a total of 20 days(total administration for 10 times).Drug treatment groups were additionally intragastrically administered drugs and control group were treated with saline.Then placed mice into transparent box to observe for 30 min and recorded paroxysnal grade,as well as delitensence and times of epileptic seizure.After anesthesia by intraperitoneal injection of pentobarbital(dose of 50 mg/kg),left ventricular perfusion was carried out.We quickly opened the thoracic cavity,inserted the needle of the infusion set into the ascending aorta through the left ventricle at the apex of the mouse,cut off the right atrial appendage at the same time,perfused 0.9% normal saline,quickly flushed until the outflow liquid from the right atrial appendage was colorless,and then perfused with 4% paraformaldehyde at 4℃.We cut off the brain of mice and immersed the brain tissue in 4% paraformaldehyde solution for immobilization.The brain tissue was embedded in paraffin and sliced(including cortex and hippocampus).Hematoxylin and eosin(HE)staining and Nissl staining were used to observe the morphological changes ofhippocampus.The Bax,Bcl-2 and caspase-3 protein expressions were detected by immuohistochemical method.Results: The effects of YLSP on the PTZ-kindling model mice: PTZkindling epilepsy mice were successfully established.Compared with the model group,the high and medium doses of YLSP could significantly delay the occurrence of epilepsy,reduce paroxysnal grade,prolong delitensence and reduce the times of epileptic seizure,the(P<0.05 or P<0.01).Low dose of YLSP also showed certain protective effect(P<0.05 or P<0.01).HE staining showed that YLSP had significant protective effect on hippocampus against to PTZ detriment.The neuronal loss in the model group were higher than control group in the hippocampus and there were presented a significant decreasing number of neuronal loss among positive drug and YLSP groups.Nissl staining also showed the similar condition.Further we detected apoptotic-associated protein caspase-3,Bax and Bcl-2 expression in hippocampus.In the model group,the repression of Bax and Caspase-3 were obviously higher and Bcl-2 was significantly lower than control group.Compared with model mice,positive drug and YLSP groups mice showed significant increasing Bcl-2 expression and descending Bax and caspase-3 expression.Conclusion: YLSP could prolong the progression of chronic epilepsy induced by PTZ in mice.The protective mechanisms may linked to protective hippocampal neuron structure,alleviate neuronal impairment and inhibit neuronal apoptosis via decreasing casapse-3,Bax expression and increasing Bcl-2 expression. |
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